Miguel A Basante-Bedoya

Stéphanie Bogliolo

Rocio Garcia-Rodas

Oscar Zaragoza

Robert A Arkowitz

Martine Bassilana
email: mbassila@unice.fr

Keywords: Candida albicans, invasive growth, polarized growth, lipid transporters, flippases, phospholipids

Candida albicans is a human opportunistic fungal pathogen that can cause superficial or systemic infections; its ability to change from an ovoid to a filamentous form is associated with its virulence. During this highly polarized filamentous growth, an accumulation of vesicles (Spitzenkörper), characteristic of filamentous fungi, as well as a polarized distribution of lipids, such as ergosterol, phosphorylated derivatives of phosphatidylinositol (PI(4)P, PI(4,5)P2) and phosphatidylserine (PS) is observed at the apex of filaments. However, the importance of the asymmetry of these lipids in the membrane bilayer is not completely understood. Flippases (P4-ATPases) transport lipids across the membrane bilayer to generate and maintain its asymmetry.

C. albicans has 5 flippases, including Drs2 which is critical for filamentous growth and phosphatidylserine (PS) distribution. Furthermore, a drs2 deletion mutant is hypersensitive to fluconazole and copper. We show here that such a mutant is also critical to virulence in a mouse model of systemic infection. To clarify the role of Drs2 during C. albicans filamentous growth, we studied the distribution of this ATPase, as well as that of key lipids and regulators, during the initiation and maintenance of this growth process. We also characterized point mutants of Drs2, analogous to those altered for PS transport in S. cerevisiae. In addition, we examined the importance of other flippases, such as Dnf1-3, in invasive growth and the role of lipid transporters belonging to the oxysterol binding protein (Osh) family. Our results indicate in particular that Drs2 plays a unique role in the maintenance of invasive filamentous growth of C. albicans, which appears to be more critical after the first septum formation, and that an interaction between Drs2 and Osh4, via PI(4)P, plays an essential role during invasive filamentous growth.

i RÉSUMÉ Candida albicans est un champignon pathogène opportuniste de l'homme qui peut causer des infections superficielles ou systémiques; sa capacité à passer d'une forme ovoïde à une forme filamenteuse est associée à sa virulence. Pendant cette croissance filamenteuse hautement polarisée, une accumulation de vésicules (Spitzenkörper), caractéristique des champignons filamenteux, ainsi qu'une distribution enrichie de lipides, tels que l'ergostérol, les dérivés phosphorylés du phosphatidylinositol (PI(4)P, PI(4,5)P2) et la phosphatidylsérine (PS) est observée à l'apex des filaments. Cependant, l'importance de l'asymétrie de ces lipides dans la bicouche membranaire est méconnue. Les flippases (P4-ATPases) transportent les lipides à travers la bicouche membranaire pour générer et maintenir son asymétrie. C. albicans a 5 flippases, incluant Drs2 qui est critique pour la croissance filamenteuse et la distribution de phosphatidylsérine (PS). De plus, un mutant de délétion drs2 est hypersensible au fluconazole et au cuivre et nous montrons ici qu'un tel mutant est aussi critique à la virulence dans un modèle murin d'infection systémique. Pour préciser le rôle de Drs2 pendant la croissance filamenteuse de C. albicans, nous avons étudié la distribution de cette ATPase, ainsi que celle de lipides et régulateurs clés, pendant l'initiation et le maintien de ce processus de croissance.

Nous avons également caractérisé des mutants ponctuels de Drs2, analogues à ceux altérés pour le transport de PS chez S. cerevisiae. De plus, nous avons examiné l'importance d'autres flippases, telles que Dnf1-3, dans la croissance filamenteuse invasive ainsi que le rôle de transporteurs de lipides appartenant à la famille des « oxysterol binding protein » (Osh). Nos résultats indiquent que Drs2 joue un rôle unique dans le maintien de la croissance filamenteuse de C. albicans, qui paraît particulièrement critique après la formation du premier septum, et indiquent qu'une interaction entre Drs2 et Osh4, via PI(4)P, joue un rôle essentiel pour la croissance filamenteuse.

Mots clés : Candida albicans, croissance invasive, croissance polarisée, transporteurs lipidiques, flippases, phospholipides.

Ça fait six ans (déjà !) que j'ai rencontré ce labo. Je suis vraiment reconnaissant que ce soit dans ce labo que j'ai acquis, dans la pratique, mes premières connaissances en recherche. La bonne ambiance, le travail d'équipe et le soutien de tous les membres ont fait paraitre ce boulot plus facile de ce qu'il l'est vraiment. Je tiens donc à vous remercier, Rob et Martine, de m'avoir donnée la chance, pas seulement une fois, de rejoindre l'équipe Arkowitz pour ma formation, et de m'avoir aidé à participer dans différents congrès et formations. Martine, je voulais t'adresser un gros MERCI pour toute la confiance déposée en moi, pour avoir été toujours très franche et directe quand je pouvais faire mieux de ce que j'étais en train de faire, tu m'as poussé de la bonne manière à arriver plus loin pendant ces années que j'ai été à ta charge.

Cette équipe a changé tellement de taille, quand je suis arrivé en était presque quinze personnes maintenant beaucoup de gens sont parti et des nouveaux sont arrivés. Cela peut paraître un peu triste, mais en même temps j'ai eu la chance de rencontrer des excellent.e.s scientifiques et encore meilleures personnes. Rocío, je dois commencer par toi, pas seulement parce que tu as été la première scientifique avec qui j'ai travaillé côte à côte, mais parce que tu as contribué énormément à ma formation, tu m'as appris beaucoup pendant si peu de temps, et grâce à toi, pas seulement j'ai eu une excellente fin de licence à Madrid, mais aussi, tu m'as servi d'exemple de persévérance pour continuer dans ce boulot et tu m'as toujours soutenu, tu as eu plus de confiance en moi que je n'en ai eu en moi-même. Muchísimas gracias, de corazón.

Celui qui dit que le travail d'équipe n'est pas indispensable pour faire une thèse n'a pas apprécié la meilleure partie de ce boulot. J'ai eu la meilleure équipe et toi, Steph, tu as été mon compagnon de guerre pendant toutes ces années, tu as été là pour m'écouter et me conseiller, pour me remonter le moral, pour les fous rire. Merci d'avoir contribué autant à ce travail et d'avoir fait en sorte que l'on soit tous une grande famille qui travaille ensemble. v I think I am very lucky; I came to France because I wanted to learn French and at the same time, I wanted to continue my career. This lab was the best I could have found, not only because of the scientific skill I gained, but also because I learned how to communicate in so many languages… Hayet, Rohan, Pat, Nadia, Charlitos, thank you for sharing with me a bit of your culture, for making me a better speaker and for all the international laughs and great moments together! Charlitos, I am very thankful for everything you taught me (believe me, it was more than what you think), in and out of the lab, you and Maggy made me an English speaker and a snowboard beginner! "CON TACONES!!!!".

There are so many people I would like to thank; Antonio, last arriver but already your support has been very important! Ma petite Marine, j'ai l'impression de te connaitre depuis des années, tu as toujours eu un gros sourire pour moi, et je te remercie énormément que même depuis la distance tu aies toujours un petit mot au bon moment pour m'encourager.

Le soutien et la bonne compagnie ne venaient pas seulement des gens de ce bâtiment ou des personnes liées au travail. Après mon arrivée en France, il a fallu très peu de temps pour me faire une place dans un groupe très chaleureux qui a joué le rôle d'une deuxième famille et des profs de français. Merci ONEP d'avoir été là pour moi. Et merci aussi à tous mes collègues de promo et grand.e.s ami.e.s (ça va même pour toi, Baptiste), on a survécu les gars ! Tengo que decir, que la suerte me ha llovido de todos lados. Desde el principio ha habido mucha gente que aportaron su granito de arena para que hoy pueda mirarlos y decirles con orgullo, gracias. A mis profesores de instituto de los que heredé el gusto por la biología, Lidia, ¡espero haber conseguido el diez! Graciela, creo que los golpes de borrador en la pizarra me sentaron bien al final, ¡gracias! Il y a aussi bien évidement celle qui m'a fait adorer cette langue et qui m'a toujours encouragé pour venir en France et continuer à apprendre, Pilar, merci de m'avoir donné la clé pour ouvrir la première porte, celle qui m'a amené à cet endroit où j'ai tant grandi.

Marina, Nadia, después de tanto tiempo me cuesta hablar de vosotras como mis mejores amigas, creo que ya hace un buen rato que sois parte de mi familia, siempre estáis ahí, siempre he contado con vosotras, y siempre os encuentro cuando vuelvo a casa. Gracias por haberme apoyado durante este tiempo y por haberme dado fuerzas como habéis podido durante estos años de lejanía. Nadia, ha sido un honor haber compartido tantos años de carrera juntos, siento haberte abandonado en la última etapa, pero bueno, ¡no podía quedarme atrás! Siempre te he vi admirado muchísimo, espero que lo sepas. Marina, vete pensando en cómo vamos a grabar este logro con tinta, ¡quién mejor que tú para tal tarea! Michele, ciao, lo sai che ti meriti 1000 pagine. Sai anche molto bene che questo lavoro mi è piaciuto soprattutto perché l'ho condiviso con te in questi due anni, mi hai sempre aiutato a vederlo da un'altra prospettiva, e che mi hai sempre dato la forza necessaria per continuare, ancora e ancora. Grazie, grazie mille per tutto. Bau.

A mi padre, porque siempre fuiste un ejemplo de trabajo duro, adaptación y cuidado de los tuyos. A mi madre, quien me enseñó lo importante que es estar siempre ahí para los tuyos, que nunca hay que olvidarse a sí mismo. Vosotros dos me habéis enseñado que hay que dar siempre lo mejor de sí en todo. Nano, aunque no lo creas, también me has enseñado mucho, eres todo un luchador, y siempre he admirado eso. Sé que nunca fui un amante del balón, pero este gol os lo dedico a vosotros. Este trabajo es vuestro, fruto de vuestro trabajo, y de vuestra educación.

Gracias, infinitamente.

I know I haven't mentioned everyone, but that doesn't mean I haven't thought of you. All of you who have worked with me, who will be watching me online on the D-day or coming here to see how this story ends, all of you who were there when I needed you and will be there to celebrate, thank you!

Lipid classification

The functions of lipids are numerous. Key to the structuring of biological membranes, they are also involved in energy storage and in cell signaling. Biomolecules such as lipids and proteins can interact in a non-covalent and highly dynamic way. The physical properties of cell membranes are based on the various molecular characteristics of the lipids, such as the length and saturation of the carbon chains, the charges present in the polar heads and the asymmetric distribution of lipids between the two leaflets of the membrane. As an example of protein-lipid crosstalk, it was shown that, whether through direct lipid-protein interactions or strong lipidlipid interactions, lipids can regulate protein localization and activity [START_REF] Janmey | Biophysical properties of lipids and dynamic membranes[END_REF]. The numerous functions of lipids are based on their great diversity and their ubiquitous distribution, as lipids have hydrophobic or amphipathic properties. Technologies such as liquid chromatography and mass spectrometry, which are used in Lipidomics, have enabled the classification of lipids according to their structure (Table 1).

By the classification in LIPID MAPS® Lipidomics Gateway, lipids are separated in (i) lipids synthetized by the condensation of thioesters derived from carbanions (molecules of an organic origin with negative charge), such as acetyls (e.g., acetyl-CoA), and (ii) lipids synthesized by condensation of carbocations derived from isoprenes (e.g., Isopentenil-PP) [START_REF] Fahy | Lipid classification, structures and tools[END_REF][START_REF] Fahy | Update of the LIPID MAPS comprehensive classification system for lipids[END_REF][START_REF] Fahy | A comprehensive classification system for lipids[END_REF].

In the first group, there are lipids such fatty acyls, glycerides, sphingolipids, saccharolipids and polyketides, while sterols and prenol lipids are in the second group (Table 1).

-Fatty acyls constitute the most abundant group of biological lipids in cells. Among them, there are fatty acids, carboxylic acids with a carbon chain of 4 to 48 carbon atoms, which can be saturated or unsaturated. These fatty acids can also be "conjugated". The fatty acyls include for instance alcohols or fatty esters.

-Glycerides, glycerolipids or acylglycerols are based on a glycerol molecule esterified with one, two or three fatty acids. Triglycerides, the most abundant glycerides, are found in all living organisms, and are part of animal and vegetable fats.

-Glycerophospholipids or phospholipids have a glycerol molecule in their composition.

This glycerol has one of the hydroxyl groups esterified with a phosphate, originating a phosphatidic acid. The phosphate groups of the phosphatidic are esterified with several types of molecules, such as inositol, and amino acids, such as ethanolamine, choline, and serine. The phospholipids constitute the minimum structure of the biological membranes, with (i) a strongly hydrophilic polar head and (ii) chains of esterified fatty acids as the hydrophobic part.

-Sphingolipids are complex lipids with a common structural axis, sphingosine, an amino alcohol derived from serine, and a fatty acid with a long carbonic chain. The polar part of sphingosine may be associated with a substituent such as hydrogen, phosphocholine or a monosaccharide, giving respectively a ceramide, a sphingomyelin, or a glycosphingolipid.

-Sterols are steroids with 27-29 carbon atoms that derive from sterane. Sterols are grouped according to their function: (i) cholesterol and derivatives, essential components of biomembranes; notably, ergosterol is found in fungi; (ii) steroids, which are considered hormones because of their roles in cell signaling; (iii) secosteroids, which include vitamin D.

Membrane composition: the importance of charged lipids

Most lipids are hydrophobic and composed mainly of hydrocarbon chains, such as triglycerides, non-polar lipids involved in energy storage. Polar lipids, which have positive or negative charges associated with hydrophobic or non-polar parts, are fatty acids, phospholipids, and sphingolipids. Fatty acids have a polar head composed of a carboxylic group and a carbon chain whose degree of saturation and length confer the physical properties to form lipid bilayers. Unsaturated acyl chains are curved and reduce the level of membrane compaction. In contrast, the saturated chains are tightly bound to each other [START_REF] Lodish | Molecular cell biology[END_REF]. Fatty acids, together with phospholipids and sphingolipids, are the main lipids that composed cell membranes.

Phospholipids are composed of a diglyceride, a phosphate group that constitutes the negatively charged fraction, and an organic fraction. Depending on the composition of the polar head, there are zwitterionic phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), or negatively charged lipids such as phosphatidylserine (PS) and phosphatidylinositol (PI). Apart from their polar heads, these phospholipids are also distinguished by the length and saturation of the aliphatic chains [START_REF] Fahy | A comprehensive classification system for lipids[END_REF][START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF] ▪

Lipid interactions

The membrane fluidity is determined by the lipids and their interactions [START_REF] Janmey | Biophysical properties of lipids and dynamic membranes[END_REF]. Similar to fatty acids, phospholipids promote the grouping of aliphatic chains, but the presence of a polar head and a two-chain aliphatic part can modify the morphology of the membrane. Phospholipids such as PI form structures with a positive curvature, such as micelleforming lipids. PC, which represents 50% of phospholipids in eukaryotes, form cylindrical structure with a tendency to form a flat, stable, and fluid bilayer. Other phospholipids, such as PE, have a conical shape, but inverted from that of PI, which leads to negative curvatures (Figure 1) [START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF][START_REF] Janmey | Biophysical properties of lipids and dynamic membranes[END_REF][START_REF] Mcmahon | Membrane curvature at a glance[END_REF]. These lipid properties give membranes the level of curvature necessary to regulate cell shape, organelles formation, vesicle transport and protein activation [START_REF] Mcmahon | Membrane curvature at a glance[END_REF].

The properties of membranes such as order, elasticity and fluidity are dependent on lipid conformation and their interactions [START_REF] Pöyry | Role of charged lipids in membrane structures -Insight given by simulations[END_REF]. The dynamics of each lipid is different, e.g., the lateral diffusion of the phosphatidylinositol biphosphate (PIP2) is slower compared to other lipids, which is most likely due to the interactions between the basic Cterminal of proteins around and the anionic head of this lipid [START_REF] Pöyry | Role of charged lipids in membrane structures -Insight given by simulations[END_REF].

Membrane fluidity is regulated by cholesterol, which also has a high affinity for cylindrical anionic lipids, such as sphingolipids (e.g., sphingomyelin (SM)), or PE [START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF]. Saturated lipids tend to make compacted zones in the membrane, while the unsaturated lipid chains confer fluidity by inhibiting a solid gel formation. Membrane thickness is associated with the acyl-chain length and the presence of sterol. Surface charge is based on the content in anionic lipids, such as PS or PI.

The curvature is determined by lipid shape, as lipid's area ratio (polar head/ acyl chain) result in different shapes: a cone locally induces a negative curvature, while a cylinder and an inverted cone induce neutral and positive curvatures, respectively. From [START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF] ▪ The interactions between lipids and proteins:

Lipids and proteins are both involved in the organization and function of the plasma membrane.

One role of lipids is purely structural: the wide range of lipids and physico-chemical properties form lipid bilayers favorable for the distribution, the function and the regulation of proteins [START_REF] Harayama | Understanding the diversity of membrane lipid composition[END_REF][START_REF] Simons | Membrane organization and lipid rafts[END_REF][START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF]. However, lipids are more than a fluid scaffold, as protein-lipid interaction is also essential for the activity of proteins and, ultimately, their biological functions [START_REF] Harayama | Understanding the diversity of membrane lipid composition[END_REF].

As reviewed in [START_REF] Sezgin | The mystery of membrane organization: composition, regulation and roles of lipid rafts[END_REF], to avoid the hydrophobic tails of lipids to be exposed to polar environments, lipids diffuse laterally according to their acyl chain length.

Thus, lipids with a similar acyl chain length will group, generating zones of the membranes with different transversal thickness. These zones can generate hydrophobic mismatches and result in protein segregation (see lipid raft section). Moreover, it has been shown that the presence of charged lipids in the membrane impacts the level of maturation of transmembrane proteins. For example, (i) Interactions with lipids can alter the dimerization of protein complexes, such as rhodopsin. This protein must interact with a 16-carbon aliphatic chain PC for a correct monomer organization, as an interaction with PC having longer or shorter chains results in a tendency to form dimers and a decrease in diffusion through the bilayer [START_REF] Kusumi | Spin-label saturation-transfer electron spin resonance detection of transient association of rhodopsin in reconstituted membranes[END_REF][START_REF] Nicolson | The Fluid-Mosaic Model of Membrane Structure: Still relevant to understanding the structure, function and dynamics of biological membranes after more than 40years[END_REF]. ii) Other studies show the involvement of PS, PI and phosphorylated derivatives, or PA as recognition patterns for proteins that harbor domains such C2, PH (Pleckstrin homology) and FFAT (two phenylalanines (FF) in an acidic tract), e.g., an

Oxysterol Binding Protein function is dependent on the concentration of PI4P [START_REF] Stefan | Membrane dynamics and organelle biogenesis-lipid pipelines and vesicular carriers[END_REF][START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF] (see section lipid transporters). PS negative charges also contribute to stabilize Rho GTPases at the plasma membrane in yeast (Haupt and Minc, 2017;[START_REF] Meca | Mlc1 plays a role in regulating cell robustness necessary for invasive growth (Puerner et al[END_REF].

In summary, biological functions are not exclusively dependent on proteins, as lipid physical properties are essential for protein distribution and generation of suitable microenvironment.

Fluidity and organization of the mosaic:

▪ Lipid movements:

The fluid mosaic model is based on the dogma "everything can move" [START_REF] Singer | The Fluid Mosaic Model of the Structure of Cell Membranes[END_REF]. The amphipathic properties of lipids and their differences in size, charge and form make biomembranes a malleable and versatile matrix in which proteins are embedded. Several studies indicate that lipids move in three main directions: (i) rotation on their own axis, (ii) lateral diffusion, with these two movements occurring inside one of the monolayers, or (iii) transverse movement, or "flip-flop", by which lipids move from one side of the membrane to the other [START_REF] Berg | Lipids and Many Membrane Proteins Diffuse Rapidly in the Plane of the Membrane[END_REF][START_REF] Jacobson | The Lateral Organization and Mobility of Plasma Membrane Components[END_REF][START_REF] Pomorski | Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping[END_REF]. Rotation and lateral diffusion are very fast lipid movements, for example, one lipid rotates in its own axis in ~1ns, or laterally diffuses ~0.01nm/ns [START_REF] Pomorski | Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping[END_REF]. In contrast, flip-flop implies that a single amphipathic molecule traverses the hydrophobic core of the plasma membrane (PM)

and, compared to the lateral diffusion where lipid movement is in 2D, flip-flop must break a stronger energy barrier. The principal consequence of this energy requirement is a slower rate (one phospholipid molecule flip-flops once in several hours) compared to the diffusion coefficient of lipids of 2μm in 1s. The first analysis of lipid spontaneous flip-flopping was done in 1971 on synthetic vesicles [START_REF] Mcconnell | Inside-outside transitions of phospholipids in vesicle membranes[END_REF]. The translocation was monitored by measuring the appearance of unreduced lipids on an outer bilayer that was previously reduced with ascorbate, and reported a frequency of ~10 -5 s -1 at 30°C (reviewed in [START_REF] Pomorski | Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping[END_REF]. With this approach at different temperatures, they established that an initially asymmetric bilayer loses asymmetry very slowly, with each lipid translocating in ~100h. "Thus, a phospholipid molecule takes about 10 9 times as long to flip-flop across a membrane as it takes to diffuse a distance of 50 Å in the lateral direction" [START_REF] Berg | Lipids and Many Membrane Proteins Diffuse Rapidly in the Plane of the Membrane[END_REF][START_REF] Contreras | Transbilayer (flipflop) lipid motion and lipid scrambling in membranes[END_REF][START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF].

The hydrolysis of sphingolipids and glycerophospholipids produces lipid second messenger like Diacylglycerol (DAG) that promotes the recruitment of distinct proteins. The trans-bilayer movement of fluorescent nitrobenzoxadiazole-labeled DAG (NBD-DAG), observed in artificial lipid vesicles, is 63s -1 [START_REF] Berg | Lipids and Many Membrane Proteins Diffuse Rapidly in the Plane of the Membrane[END_REF][START_REF] Contreras | Transbilayer (flipflop) lipid motion and lipid scrambling in membranes[END_REF][START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF]. Other lipids, such as cholesterol in animal PMs, show flip-flop rates below milliseconds and this rate is important to regulate the Sphingomyelin (SM)-high-concentrated membrane fluidity;

reciprocally, interaction with SM restricts cholesterol flip-flop rate [START_REF] Gu | Cholesterol Flip-Flop in Heterogeneous Membranes[END_REF].

Lateral movements and lipid-lipid interactions are based on the lipid tail length and compaction, and are key for organization into lipid domains or lipid rafts that are rich in saturated lipids, sterols and SM [START_REF] Sezgin | The mystery of membrane organization: composition, regulation and roles of lipid rafts[END_REF].

▪ Lipid rafts

There are more than a thousand different lipids present in eukaryotic cells [START_REF] Fahy | Update of the LIPID MAPS comprehensive classification system for lipids[END_REF][START_REF] Fahy | A comprehensive classification system for lipids[END_REF][START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF]. This diversity results in countless different interactions allowing lipid compartmentalization within the same membrane, where the boundaries are drawn by the higher affinity between certain lipids. According to the following definition, "Membrane rafts are small (10-200 nm), heterogeneous, highly dynamic, sterol-and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions" [START_REF] Pike | Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function[END_REF].

Lipid rafts form a platform where proteins can be selectively included or excluded by their "raftophilic" properties, based on the specific length and charge mismatches between lipid aliphatic chains and protein transmembrane domains [START_REF] Diaz-Rohrer | Membrane raft association is a determinant of plasma membrane localization[END_REF][START_REF] Sezgin | The mystery of membrane organization: composition, regulation and roles of lipid rafts[END_REF][START_REF] Simons | Functional rafts in cell membranes[END_REF]. Alpha helices with hydrophobic amino-acid side chains are present in most of transmembrane proteins. For these proteins to be stably anchored in the PM, a hydrophobic match with the surrounding lipids is critical and lipid rafts, with longer and saturated chains, can provide such an environment. The actin cytoskeleton plays an important role in the diffusion and stabilization of lipids, by anchoring part of the membrane to specific sites, e.g., GPI-anchored proteins interact with lipids such as PS and the actin cytoskeleton asters through scaffold proteins. These interactions favor the clustering of the GPI-anchored proteins in the PM [START_REF] Sezgin | The mystery of membrane organization: composition, regulation and roles of lipid rafts[END_REF]. Segregation of different membrane components to optimize their interaction and activity is the most axiomatic function of these lipid domains. As an example, specific interactions with raft-enriched lipids such as cholesterol may change protein conformation [START_REF] Bogdanov | Lipids in the Assembly of Membrane Proteins and Organization of Protein Supercomplexes[END_REF][START_REF] Kusumi | Spin-label saturation-transfer electron spin resonance detection of transient association of rhodopsin in reconstituted membranes[END_REF][START_REF] Lingwood | Cholesterol modulates glycolipid conformation and receptor activity[END_REF].

In fungi, lipids are also organized into domains. Several fungi have SL-enriched domains (SLEDs), which are quite rigid, sterol-depleted, and distinct from lipid rafts. These domains play a role in membrane maturation events in endosomes, lysosomes and vacuoles in yeast [START_REF] Hurst | Lipid Rafts, Sphingolipids, and Ergosterol in Yeast Vacuole Fusion and Maturation[END_REF][START_REF] Santos | Sphingolipidenriched domains in fungi[END_REF]. However, lipid rafts, which are rich in ergosterol and sphingolipids, are an important topic in research; ergosterol lipids are absent in mammalian cells, thus making them potential targets for antifungal drugs. Ergosterol levels are different in specialized microdomains such as the Membrane Compartment of Can1 (MCC)/eisosomes, which facilitate a spatially confine endocytosis, and the MCP (containing Pma1) [START_REF] Grossmann | Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast[END_REF][START_REF] Lanze | Plasma Membrane MCC/Eisosome Domains Promote Stress Resistance in Fungi[END_REF][START_REF] Malinsky | Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi[END_REF]. In fungal PMs, sterol-rich domains (SRDs) are observed at the filamentous cell tip in Candida albicans or Aspergillus nidulans, as well as at the septum and mating projection (shmoos) in Saccharomyces cerevisiae or Schizosaccharomyces pombe [START_REF] Alvarez | Sterol-Rich Plasma Membrane Domains in Fungi[END_REF]Martin and Konopka, 2004).

Chapter II. How lipid composition is established and maintained.

Eukaryotic cells are defined by the presence of endomembranes that allow a compartmentalization of cell functions and are associated with specific lipid compositions that depend on where lipids are synthesized, modified and degraded, and on how they reach their final destination. Most of the lipids are produced in the endoplasmic reticulum (ER), yet not much of the lipids remain in this organelle. The Golgi is also site of lipid production, in particular for the synthesis of Sphingolipids [START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF][START_REF] Van Meer | Dynamic Transbilayer Lipid Asymmetry[END_REF].

In the secretory pathway, the vesicles that are generated by budding in early organelles will fuse with later ones and one of the main differences between these early and late organelles is the lipid composition. For example, PS and PI are more concentrated in the trans Golgi and PM inner leaflets, while in the cis-Golgi and the ER, these phospholipids face the lumen [START_REF] Kobayashi | Transbilayer lipid asymmetry[END_REF]. Although synthesized in the ER, sterol is limited in this organelle (5 mol% of lipids), compared to the PM (30-40 mol%) [START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF].

The PM is enriched in SM, yet most of the sphingolipids are synthesized at the Golgi.

Furthermore, ceramides, which are precursors of Sphingolipids, are supplied by the ER, making lipid transport essential for maintaining lipid composition [START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF][START_REF] Van Meer | Dynamic Transbilayer Lipid Asymmetry[END_REF][START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF].

Fine-tuning of lipid distribution is critical for membrane specific properties. The plasma membrane is characterized by an enrichment in anionic and unsaturated lipids in the inner leaflet [START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF][START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF]. Even once lipids reach the membrane they were synthetized for, they can be modified in order to maintain their correct distribution. For example, the concentration of PI is regulated by its phosphorylation into PI(4)P and PI(4,5)P2, which takes place at the Golgi and the PM [START_REF] Del Bel | Sac1, a lipid phosphatase at the interface of vesicular and nonvesicular transport[END_REF][START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF][START_REF] Klug | Yeast lipid metabolism at a glance[END_REF][START_REF] Van Meer | Dynamic Transbilayer Lipid Asymmetry[END_REF]. Other authors suggest that modifications such as the acyl chain length of PM lipids take place directly at the PM [START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF][START_REF] Makarova | Asymmetry across the membrane[END_REF].

In the following sections I will discuss studies on the role of lipid and protein transport in cell growth through three main sections: how lipids and proteins reach the targeted membrane; how specific lipid composition is generated; and how it is maintained.

Vesicular transport:

New material is continuously needed for membrane biogenesis to occur and understanding this process during cell development and growth has been the aim of a number of research groups during the last decades. The baker's yeast Saccharomyces cerevisiae is a useful model to study the intracellular organization, including the secretory pathway, which is key for membrane biogenesis [START_REF] Feyder | Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model[END_REF].

In the endocytic pathway, vesicles budding off the PM form early endosomes that mature ultimately into lysosomes in mammalian cells [START_REF] Mahmutefendić | Rapid Endosomal Recycling, Peripheral Membrane Proteins[END_REF], or vacuoles in yeast, where the molecules internalized from the external medium and the plasma membrane are degraded. Organelles, such as mitochondria, chloroplasts and peroxisomes do not appear to contribute substantially to these vesicular transports, and other ways of communication connect these organelles, such as direct contacts between their membranes or lipid pipelines with specific lipid transporters [START_REF] Horvath | Lipids of mitochondria[END_REF][START_REF] Prinz | Lipid trafficking sans Vesicles: where, why, how?[END_REF].

The vesicular pathway plays an important role in the remodeling of cell membranes. Aside from this role, the biological importance of this pathway is reflected also in processes such as neural synapsis formation [START_REF] Binotti | An overview of the synaptic vesicle lipid composition[END_REF][START_REF] Cooper | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF], pheromone communication (Martin, 2019a(Martin, , 2019b;;[START_REF] Yashiroda | Intraspecies cell-cell communication in yeast[END_REF], host-pathogen interactions (Naglik et al., 2019) or polarized growth [START_REF] Campanale | Development and dynamics of cell polarity at a glance[END_REF][START_REF] Chiou | Cell Polarity in Yeast[END_REF][START_REF] Martin | Cell polarization in budding and fission yeasts[END_REF].

In other words, vesicular traffic serves to deliver specific materials to different compartments and thus enables an organelle to have an identity and carry out its function.

▪ Anterograde transport:

In the anterograde vesicle transport, there are distinct steps. First, COPII-type coated vesicles bud from the ER and are delivered to the Golgi. Some areas of the reticulum are favorable for COPII assembly, with specific lipid composition and localized dynamic remodeling of PIs [START_REF] Schink | Phosphoinositides in Control of Membrane Dynamics[END_REF]. Proteins transported via these vesicles display sorting signals to differentiate proteins destinated to the ER or other membranes such as the PM [START_REF] Feyder | Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model[END_REF]. Moreover, the COPII machinery, as well as COPI and clathrin, cooperates in the formation of vesicles by creating tensions and folding the membrane and selects the proteins to be at the specific ER exit sites (ERES) [START_REF] Gomez-Navarro | Protein sorting at the ER-Golgi interface[END_REF][START_REF] Has | Recent developments in membrane curvature sensing and induction by proteins[END_REF].

From the trans-Golgi Network (TGN), vesicles are directed to the plasma membrane or to the late endosome-lysosomes/vacuoles. Vesicle budding mechanisms are different as a function of the vesicle final destination and content. For example, material is delivered to the plasma membrane through SEC vesicles that are transported by tropomyosin on actin cables and tethered at the PM via the exocyst complex, composed of Sec3/5/10/6/8/15 and Exo70/84 in yeast [START_REF] Feyder | Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model[END_REF][START_REF] Mei | The exocyst complex[END_REF]. Moreover, as recently demonstrated in HeLa cells, Exo70 would be critical not only for tethering but also for defining whether the vesicle will partially or completely fuse with the PM (An et al., 2021).

Lipid composition and shape are critical for vesicular budding. While cargoes leaving the TGN for endosomes are sorted by coat-dependent mechanisms [START_REF] Kim | Cargo trafficking from the trans-Golgi network towards the endosome[END_REF], cargo proteins destined to the plasma membrane are sorted at TGN into exocytic vesicles that are rich in SL and sterol lipid raft which select specific cargos [START_REF] Surma | Lipid-dependent protein sorting at the trans-Golgi network[END_REF].

▪ Retrograde transport:

To maintain the cell shape during membrane remodeling, retrograde vesicular transport is critical to recycle as well as to internalize material. Vesicles bud off of PM regions where clathrin is concentrated, via adaptor proteins, and high concentration of PI [START_REF] Haupt | Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast[END_REF][START_REF] Kay | Distribution, dynamics and functional roles of phosphatidylserine within the cell[END_REF]P2 facilitates these adaptor proteins interactions [START_REF] Feyder | Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model[END_REF][START_REF] Li | Phosphatidylinositol 4,5-bisphosphate in the Control of Membrane Trafficking[END_REF][START_REF] Mahmutefendić | Rapid Endosomal Recycling, Peripheral Membrane Proteins[END_REF]. Retrograde transport, which also returns resident molecules from the source compartment, is mediated by a class of coating proteins, called COPI, that will form vesicles and select molecules to be returned to the ER.

Retrograde and anterograde vesicular-transported cargos are selected at different levels, (i)

vesicle coats populate the vesicle with specific cargo proteins by sequence-based recognition, as an example, the yeast heteropentameric complex called retromer, together with ESCRT (endosomal sorting complex required for transport) and the sorting nexins, is necessary for discriminating PM-recycled and Golgi retrograde-transported proteins at the endosomes [START_REF] Ma | Retrograde trafficking and plasma membrane recycling pathways of the budding yeast Saccharomyces cerevisiae[END_REF]. (ii) Lipids facilitate membrane protein segregation in and out of vesicles [START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF]. The first evidence of this lipid-dependent sorting is based on the study of an artificial lipid raft protein FusMid that allowed the quantification of lipids in the secretory vesicles [START_REF] Klemm | Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network[END_REF]. Other authors demonstrated that plasma membrane proteins, such as Pma1 and GPI-anchor proteins, like Gas1, are transported in these SL-and sterol-rich vesicles [START_REF] Surma | Generic sorting of raft lipids into secretory vesicles in yeast[END_REF]. In clathrin-independent endocytic pathways, where endocytosis depends on BAR domain-containing proteins (e.g., endophilin) that mediate the membrane curvature, lipids such as PI [START_REF] Meca | Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions[END_REF][START_REF] Haupt | Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast[END_REF]P2 are necessary for endophilin recruitment at the PM of mammalian migrating cells [START_REF] Shafaq-Zadah | Clathrin-independent endocytosis, retrograde trafficking, and cell polarity[END_REF].

In summary, factors, such as sequence-based recognition, lipid raft affinities, as well as retrograde vesicular transport, are efficient mechanisms to retain proteins in specific organelles.

As described in the next section, maintaining lipids at specific locations is also critical to maintain membrane identity.

Non-vesicular transport: Lipid transporter proteins (LTPs)

One of the main challenges of having such dynamic vesicular transport is to maintain the membrane composition necessary for a proper compartmentalization of function. Lipids provide the membrane with specific thickness, fluidity and landmarks and membranes provide in turn the microenvironment necessary for correct protein function [START_REF] Harayama | Understanding the diversity of membrane lipid composition[END_REF][START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF]Lenoir et al., 2021) .

As a result, synthesis, degradation, modification of lipids must be constantly regulated in time and space. A process such as vesicular transport tends to equilibrate lipid composition of different compartments by mixing membranes while apporting new material [START_REF] Delfosse | Structural and Functional Specialization of OSBP-Related Proteins[END_REF]Lenoir et al., 2021). Moreover, some organelles such as mitochondria or chloroplasts do not substantially participate in vesicular transport, so alternative mechanisms are necessary to control the lipid composition in these compartments [START_REF] Horvath | Lipids of mitochondria[END_REF][START_REF] Prinz | Lipid trafficking sans Vesicles: where, why, how?[END_REF].

Lipid transfer proteins (LTPs) have been described for more than 40 years as capable of overcoming the hydrophilic barrier that lipids face to get from one endomembrane to the other. This is possible by the presence of a hydrophobic cavity where lipids bind and are transported between two membranes [START_REF] Delfosse | Structural and Functional Specialization of OSBP-Related Proteins[END_REF][START_REF] Lipp | Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism[END_REF]. LTPs fine-tune lipid transport and bypass vesicular transport [START_REF] Delfosse | Structural and Functional Specialization of OSBP-Related Proteins[END_REF][START_REF] Lipp | Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism[END_REF]. LTPs participate in a variety of processes, as the lipids they transport can serve as cofactors for other enzymes, activate transcription factors or regulate other lipid transporters [START_REF] Chiapparino | The orchestra of lipid-transfer proteins at the crossroads between metabolism and signaling[END_REF].

The variety of functions of these transporters is accompanied by a high diversity of LTPs.

Indeed, in humans, there are about 125 genes encoding different LTPs. All of them contain a specific Lipid Transfer Domain (LTD) that confers specificity for the transported lipids, and most of them have domains necessary for their localization [START_REF] Chiapparino | The orchestra of lipid-transfer proteins at the crossroads between metabolism and signaling[END_REF]Lipp et al., 2020). These characteristics are common for all Eukaryotic LTPs; for instance, Ceramide transporters acting between the Golgi and the ER (CERT), and the OxySterol Binding Proteins (OSBP) are well conserved in yeast and other fungi (Figure 2). CERTs have distinct domains:

a START (Steroidogenic Acute Regulatory Transfer) domain able to recognize Ceramides with a specific acyl chain length [START_REF] Bohnert | Tether Me, Tether Me Not-Dynamic Organelle Contact Sites in Metabolic Rewiring[END_REF][START_REF] Kudo | Structural basis for specific lipid recognition by CERT responsible for nonvesicular trafficking of ceramide[END_REF], a FFAT (two phenylalanine in an acidic tract) and a PH (Pleckstrin Homology) domain, which allow specific recognition of proteins in the ER and PI(4)P in the Golgi, respectively, establishing contact sites between these two organelles [START_REF] Bohnert | Tether Me, Tether Me Not-Dynamic Organelle Contact Sites in Metabolic Rewiring[END_REF][START_REF] Lev | Nonvesicular lipid transfer from the endoplasmic reticulum[END_REF]. Similarly, in S. cerevisiae, OSBP function is regulated by specific domains: it localizes at the Golgi by interaction with PI(4)P and GTP-Arf1 through its PH domain and interacts at the ER with the VAP-A protein through a FFAT domain (Figure 2) [START_REF] Lipp | Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism[END_REF].

Domains similar to the ligand-binding domain of OSBP are termed ORD (OSBP Related Domain); they contain a conserved EQVSHHPP motif and are found in all eukaryotes [START_REF] Delfosse | Structural and Functional Specialization of OSBP-Related Proteins[END_REF] . Humans have 12 OSBP-related proteins (ORP), compared to yeast, where only 7

ORP genes were identified and termed OSBP homologous (Osh) proteins. The PH and FFAT domains are only present in Osh1-3 and other members, such as Osh4-7, contain only the ORD domain, suggesting that the localization of these LTPs are dependent on its substrates (Figure 2) [START_REF] Lipp | Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism[END_REF][START_REF] Tong | Structural insights into nonvesicular lipid transport by the oxysterol binding protein homologue family[END_REF].

Blocking the secretory pathway in yeast has almost no effect on sterol transport [START_REF] Baumann | Transport of Newly Synthesized Sterol to the Sterol-Enriched Plasma Membrane Occurs via Nonvesicular Equilibration[END_REF][START_REF] Heino | Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface[END_REF][START_REF] Urbani | Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane[END_REF], suggesting a non-vesicular transport as a main path for sterol distribution. Several LTP members have a LTD able to recognize sterols [START_REF] Chiapparino | The orchestra of lipid-transfer proteins at the crossroads between metabolism and signaling[END_REF]. Some members of the START (Steroidogenic Acute Regulatory Transfer) family are LTPs for sterol [START_REF] Wilhelm | STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites[END_REF], however these proteins are not present in fungi and Archaea, where other StART-like analogous (VASt) proteins have been shown to transport Ergosterol (Marek et al., 2020).

With only an ORD domain, S. cerevisiae Osh4 is one of the simplest Osh proteins (Figure 2).

It has been studied for its implication in the regulation of sterol gradient from the ER to the PM and in the secretory pathway by modulation of PI( 4)P levels at the Golgi (de Saint-Jean et al., 2011;[START_REF] Filseck | A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes[END_REF][START_REF] Georgiev | Osh Proteins Regulate Membrane Sterol Organization but Are Not Required for Sterol Movement Between the ER and PM[END_REF][START_REF] Ling | Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature[END_REF]J. Moser von Filseck et al., 2015;[START_REF] Smindak | Lipid-dependent regulation of exocytosis in S. cerevisiae by OSBP homolog (Osh) 4[END_REF]Stefan et al., 2011). In a first place, PI(4)P concentration plays a role in vesicle maturation, since decreasing the PI(4)P levels is necessary for loading dockingrelated proteins into the vesicles [START_REF] Ling | Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature[END_REF][START_REF] Mizuno-Yamasaki | Phosphatidylinositol 4phosphate controls both membrane recruitment and a regulatory switch of the Rab GEF Sec2p[END_REF][START_REF] Schink | Phosphoinositides in Control of Membrane Dynamics[END_REF][START_REF] Smindak | Lipid-dependent regulation of exocytosis in S. cerevisiae by OSBP homolog (Osh) 4[END_REF]. In a second place, low levels of PI(4)P and high concentration of sterol both favor vesicle fusion with the PM [START_REF] Smindak | Lipid-dependent regulation of exocytosis in S. cerevisiae by OSBP homolog (Osh) 4[END_REF]. The Osh4-mediated exchange of PI(4)P/Ergosterol (Figure 3) in yeast is ensured by the interconversion of PI(4)P and PI by the phosphatase Sac1 at the ER, and the kinase Pik1 at the Golgi, respectively (de Saint-Jean et al., 2011;[START_REF] Filseck | A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes[END_REF].

Figure 3 illustrates a schematic for Osh4 function in S. cerevisiae. The PI(4)P backbone, PI, synthetized at the ER is transported to the Golgi by the LTP of the CRAL-Trio superfamily, Sec14 [START_REF] Sugiura | Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle[END_REF], where it is phosphorylated by the PI-4 kinase, Pik1, to generate PI(4)P [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF]. PI(4)P concentration being higher at the Golgi than at the ER, Osh4 transports PI(4)P to the ER along its concentration gradient and the energy generated is used to transport ergosterol in the opposite direction. PI(4)P is dephosphorylated by the phosphatase Sac1 at the ER [START_REF] Filseck | A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes[END_REF][START_REF] Moser Von Filseck | INTRACELLULAR TRANSPORT. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate[END_REF].

In vesicular transport from the Golgi to the PM, Osh4 would function in a similar fashion, participating in the maturation of secretory vesicles [START_REF] Ling | Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature[END_REF][START_REF] Mizuno-Yamasaki | Phosphatidylinositol 4phosphate controls both membrane recruitment and a regulatory switch of the Rab GEF Sec2p[END_REF][START_REF] Smindak | Lipid-dependent regulation of exocytosis in S. cerevisiae by OSBP homolog (Osh) 4[END_REF]) (Figure 4). )), respectively. Osh4 would exchange PI(4)P for ergosterol at the PM, facilitating vesicle fusion with the PM [START_REF] Smindak | Lipid-dependent regulation of exocytosis in S. cerevisiae by OSBP homolog (Osh) 4[END_REF].

S. cerevisiae has six more Osh family members with overlapping function [START_REF] Beh | Overlapping Functions of the Yeast Oxysterol-Binding Protein Homologues[END_REF], and PI(4)P is a common ligand for all these proteins (Maeda et al., 2013;[START_REF] Manik | Structure of Yeast OSBP-Related Protein Osh1 Reveals Key Determinants for Lipid Transport and Protein Targeting at the Nucleus-Vacuole Junction[END_REF]J. Moser von Filseck et al., 2015). Osh6/7 exchange PI(4)P and PS between the ER and the PM, where they localize [START_REF] D'ambrosio | Osh6 requires Ist2 for localization to ER-PM contacts and efficient phosphatidylserine transport in budding yeast[END_REF]Maeda et al., 2013;[START_REF] Schulz | Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues[END_REF]. Osh6/7 exchange PS by using the PI(4)P gradient that is sustained by Sac1 at the ER, and the PI-4 kinase Stt4 at the PM (Figure 2D) [START_REF] Lipp | Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism[END_REF]. Interestingly, Osh6/7-mediated transport is dependent on the binding capacity of PI(4)P and the PI(4)P levels, as deletion of Sac1 results in a loss of PI( 4)P gradient and Osh6 activity (J. Moser von [START_REF] Filseck | A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes[END_REF].

In filamentous fungi, the role of these transporters is poorly understood. In C. albicans, it has been suggested that Osh3 is required for invasive growth in specific nutriment-poor media [START_REF] Hur | Characterization of Osh3, an oxysterolbinding protein, in filamentous growth of Saccharomyces cerevisiae and Candida albicans[END_REF]. A. nidulans has five Osh proteins (OshA-OshE) and oshC and oshD deletion mutants show altered branching events [START_REF] Brunswik | Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans[END_REF]. In A. nidulans, OshA-E proteins are localized at different sites and, for instance, the Osh4 homologous, OshC, is in the cytoplasm and showed a subapical clustered localization in a small population of cells [START_REF] Brunswik | Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans[END_REF].

Compared to the in-depth studies of the Osh proteins in S. cerevisiae, much remains to be discovered about the role of these proteins in other fungi, for example during morphogenesis and highly polarized hyphal growth.

Membrane asymmetry:

Lipid asymmetry refers to the unequal the composition between the two leaflets of a bilayer [START_REF] Bretscher | Membrane structure: some general principles[END_REF]. Red blood cells (RBCs) are an excellent model to study PM properties, as they are a simple structure lacking endomembranes. In this model, recent lipidomics studies confirm a PC and SM enrichment in the exoplasmic membrane, and PE, PS and PI, in the inner one [START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF]. Moreover, acyl chains interaction has been speculated to participate in "Interleaflet coupling" across the PM [START_REF] Doktorova | Structural and functional consequences of reversible lipid asymmetry in living membranes[END_REF]. The aliphatic length and saturation of lipids are different between the two leaflets and mass spectroscopy analyses reveal that the level of unsaturation is higher in the inner leaflet, which means that membrane asymmetry does not rely only on the difference between the polar heads of the bilayer [START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF]. Membrane asymmetry, which is ATP-dependent, requires not only transport but also in situ modifications of the lipid acyl chain length [START_REF] Doktorova | Structural and functional consequences of reversible lipid asymmetry in living membranes[END_REF][START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF][START_REF] Makarova | Asymmetry across the membrane[END_REF].

Most eukaryotic cells have endomembranes, where lipid asymmetry is also present. In these cells, there are lipid asymmetries at the PM, the Golgi and the endosomes, while the ER has a symmetric lipid distribution, except for PS [START_REF] Doktorova | Structural and functional consequences of reversible lipid asymmetry in living membranes[END_REF][START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF][START_REF] Kobayashi | Transbilayer lipid asymmetry[END_REF][START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF][START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF]. New models have emerged to understand the extent and importance of lipid asymmetry among eukaryotes in vivo [START_REF] Ikeda | Lipid asymmetry of the eukaryotic plasma membrane: functions and related enzymes[END_REF][START_REF] Klose | Flexibility of a eukaryotic lipidome--insights from yeast lipidomics[END_REF][START_REF] Santos | Yeast as a model system for studying lipid homeostasis and function[END_REF][START_REF] Santos | Sphingolipidenriched domains in fungi[END_REF], as well as in vitro [START_REF] Florek | Lipid composition in fungal membrane models: effect of lipid fluidity[END_REF][START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF].

Yeasts share with most eukaryotic cells having phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), diacylglycerol (DAG) being the most abundant lipids at the PM, and triacylglycerols (TAG)

and sterol esters (SE) serving as storage lipids. Yeasts have Sphingolipids and PC in the outer leaflet, and PS and PE in the inner leaflet of the plasma membrane. The major yeast sterol is ergosterol (Erg) and, based partly on lipidomic approaches, it was shown that the dynamics of lipid metabolism change with environmental conditions, such as changes in growth conditions [START_REF] Klose | Flexibility of a eukaryotic lipidome--insights from yeast lipidomics[END_REF]. Mating in S. cerevisiae is dependent on the asymmetric distribution of sphingolipid-enriched lipid rafts [START_REF] Proszynski | Plasma membrane polarization during mating in yeast cells[END_REF]. Some steps in the endocytic pathway are dependent on the phospholipid asymmetry generated and maintained by P4-ATPases (presented below), as the absence of these proteins alter the FM4-64 endocytic tracer uptake (Pomorski et al., 2003).

Several mechanisms generate the asymmetric lipid distribution. As mentioned earlier, lipid exchange via LTPs, such as ORPs, is an example of how asymmetric distribution can be generated. Once a lipid is transported to a membrane, it can be trapped. For instance, at the Golgi, ceramides are transformed into sphingolipids and PI is phosphorylated into PI(4)P. The metabolic conversion of these lipids ensures that the transport is monodirectional. On the other hand, lipids, such as PS, with negatively charged polar heads, can interact with polybasic domains of membrane proteins at the PM, or via calcium-mediated interactions with luminal proteins at the ER [START_REF] Kobayashi | Transbilayer lipid asymmetry[END_REF][START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF].

Maintenance of membrane asymmetry is crucial for cell viability, as extracellular exposure of PS can result in cell death through apoptotic and non-apoptotic pathways [START_REF] Shlomovitz | Flipping the dogma -phosphatidylserine in nonapoptotic cell death[END_REF]. Membrane asymmetry is also critical for the distribution of membrane proteins [START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF][START_REF] Makarova | Asymmetry across the membrane[END_REF]. Charged lipids can be implicated in their topology, as proteins tend to position their positively charged amino acids in the inner leaflet of the PM.

Furthermore, proteins are organized in such way that the larger domains are in the inner side of the membrane, as this side is less compact and more fluid because of its lipid composition [START_REF] Doktorova | Structural and functional consequences of reversible lipid asymmetry in living membranes[END_REF][START_REF] Lorent | Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape[END_REF]. This suggests that lipid asymmetry play an important role in the asymmetric protein conformation.

In summary, lipid asymmetry is a common aspect of biomembranes, and such asymmetry must be maintained in an active and energy-dependent fashion. To this end, eukaryotes have evolved over the course of evolution a series of lipid transporters that generate and maintain membrane asymmetry.

▪ Lipid asymmetry and requirement for an active transport

Transmembrane lipid transporters differ by the substrate selectivity, transport direction and energy consumption. Flip-flop of lipid's polar head through a membrane bilayer is an energetically unfavorable movement. Scramblases transport lipids along their concentration gradient. Different scramblases are found for example in the ER, one of the most symmetrical endomembranes [START_REF] Daleke | Regulation of transbilayer plasma membrane phospholipid asymmetry[END_REF]Hankins et al., 2015b;[START_REF] Kobayashi | Transbilayer lipid asymmetry[END_REF]. These scramblases are regulated by Ca 2+ [START_REF] Daleke | Regulation of transbilayer plasma membrane phospholipid asymmetry[END_REF]. For instance, in mouse fibroblasts, PS is evenly distributed between the ER-monolayers, when Ca 2+ is present, due to the action of a Ca 2+ -dependent scramblase, TMEM16K [START_REF] Tsuji | Predominant localization of phosphatidylserine at the cytoplasmic leaflet of the ER, and its TMEM16K-dependent redistribution[END_REF]. In contrast, floppases and flippases actively transport lipids, with flippases transporting lipids towards the cytoplasmic side and floppases to the side exposed to the extracellular media or the lumen of the organelles. Flippase and floppase functions are ATP-dependent and they belong to the P4-ATPases and the ATP-Binding Cassettes (ABC) transporters, respectively (Figure 5) [START_REF] Kobayashi | Transbilayer lipid asymmetry[END_REF][START_REF] López-Marqués | Lipid flippases as key players in plant adaptation to their environment[END_REF]Lyons et al., 2020;[START_REF] Pomorski | Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping[END_REF]. Importantly, some members of the ABC superfamily have been implicated in antifungal drug resistance, for instance, the C. albicans drug resistant proteins 1 and 2 (CaCdr1/2) or the Multi-Drug-Resistance (MDR) proteins, as well as in the dynamics of Candida infections [START_REF] Banerjee | Directed Mutational Strategies Reveal Drug Binding and Transport by the MDR Transporters of Candida albicans[END_REF]. [START_REF] Kobayashi | Transbilayer lipid asymmetry[END_REF][START_REF] López-Marqués | Lipid flippases as key players in plant adaptation to their environment[END_REF]Lyons et al., 2020;[START_REF] Pomorski | Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping[END_REF])(Figure 5). Well conserved among eukaryotes, P4-ATPase function and ultrastructure have been characterized in S. cerevisiae [START_REF] Bai | Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p[END_REF]Baldridge andGraham, 2013, 2012;Pomorski et al., 2003;[START_REF] Takar | The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry[END_REF][START_REF] Timcenko | Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase[END_REF]Timcenko et al., , 2019;;[START_REF] Tsai | Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p[END_REF]. Collectively, S. cerevisiae has five members of this family, Neo1 (Neomycin resistant 1), Drs2 (Deficient for Ribosomal Subunit 2), Dnf1 (Drs2/Neo1 family), Dnf2, and Dnf3. Their localizations are different in S. cerevisiae budding cells: Neo1 localizes broadly at the Golgi and endosomes and transports PE and PS [START_REF] Takar | The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane[END_REF]; Drs2, which is specific for PS and PE, localizes with Dfn3 at the TGN and post-Golgi secretory vesicles [START_REF] Alder-Baerens | Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles[END_REF]Hua et al., 2002;Pomorski et al., 2003), and Dnf1 and Dnf2 at the PM and the endocytic membranes, where they transport PC and PE [START_REF] Alder-Baerens | Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles[END_REF]Hankins et al., 2015a;Hua et al., 2002;[START_REF] Pomorski | Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping[END_REF]. Several studies have uncovered different regulatory mechanisms for the function of flippases. For instance, Drs2 is positively regulated by PI(4)P [START_REF] Natarajan | Regulation of a Golgi flippase by phosphoinositides and an ArfGEF[END_REF]Timcenko et al., 2019), as well as by the interaction with both the Arf-like protein Arl1 and the guanine nucleotide exchange factor Gea2 [START_REF] Chantalat | The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae[END_REF][START_REF] Natarajan | Regulation of a Golgi flippase by phosphoinositides and an ArfGEF[END_REF]Timcenko et al., 2019;[START_REF] Tsai | Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p[END_REF]. Moreover, other flippases such as Dnf1/2 are activated upon kinases-mediated phosphorylation by Flippase kinase 1 (Fpk1) and Fpk2 [START_REF] Frøsig | Pseudohyphal growth in Saccharomyces cerevisiae involves protein kinase-regulated lipid flippases[END_REF][START_REF] Nakano | Protein Kinases Fpk1p and Fpk2p are Novel Regulators of Phospholipid Asymmetry[END_REF][START_REF] Roelants | A protein kinase network regulates the function of aminophospholipid flippases[END_REF]. P4-ATPases belong to the P-type ATPase superfamily (P1-5), in which P1-3 ATPases are transporters of ion and heavy metals. Na + /K + and Ca 2+ -ATPases are well characterized, and their mechanism of transport is based on a canonical binding pocket (Zhang and Zhang, 2019). P5 ATPases do not appear to have defined ligand, but P4-and P5-ATPases are only found in eukaryotic genomes [START_REF] Huang | New wine in old bottles: current progress on P5 ATPases[END_REF]. P-type ATPases have 4 functional domains, including 3 cytosolic domains, i.e., the Actuator Domain (A), the Nucleotide-binding domain (N), the phosphorylation domain (P) and a transmembrane domain (TM) [START_REF] Bai | Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p[END_REF][START_REF] Timcenko | Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase[END_REF]Timcenko et al., , 2019)). These proteins also function with a non-catalytic subunit of the ligand-effect modulator/the cell division cycle (Lem3/Cdc50) family [START_REF] Furuta | Endocytic Recycling in Yeast Is Regulated by Putative Phospholipid Translocases and the Ypt31p/32p-Rcy1p Pathway[END_REF]Lenoir et al., 2009;Saito et al., 2004). The yeast Lem3/Cdc50 family is composed by Cdc50, Lem3 and Crf1, which interact with Drs2, Dnf1/2 and Dnf3, respectively [START_REF] Furuta | Endocytic Recycling in Yeast Is Regulated by Putative Phospholipid Translocases and the Ypt31p/32p-Rcy1p Pathway[END_REF]Saito et al., 2004).

Formation of the heterodimeric complex activates and targets these flippases to their correct organelle localization, with the absence of one of the two heterodimeric members resulting in the ER-retention of the other one [START_REF] Furuta | Endocytic Recycling in Yeast Is Regulated by Putative Phospholipid Translocases and the Ypt31p/32p-Rcy1p Pathway[END_REF]Lenoir et al., 2009;Saito et al., 2004).

Consistent with this, mutation of either the catalytic or the non-catalytic part results in similar phenotypes [START_REF] Chen | Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane[END_REF][START_REF] Kato | A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma membrane in Saccharomyces cerevisiae[END_REF].

The catalytic cycle of these proteins (Post-Albers cycle), involves several intermediate stages depending on the substrate binding and the phosphorylation state, E1, E1P, E2P and E2 (Figure 6) [START_REF] Bai | Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p[END_REF]Baldridge and Graham, 2013;Lyons et al., 2020). Recently, the S. cerevisiae P4-ATPase Drs2 has been studied by cryo-electron microscopy in 3 conformations: autoinhibited, fully active and intermediate conformations (Timcenko et al., 2019). These structures reveal in particular how the Drs2 carboxy-terminus could inhibit the Drs2-Cdc50 complex activity and the auto-inhibition relief by PI(4)P [START_REF] Bai | Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p[END_REF]Baldridge and Graham, 2013;Lyons et al., 2020;Timcenko et al., 2019). This suggests that the mechanisms involved in PI(4)P regulation may directly affect the function of this flippase.

In P4-ATPases, the phospholipid binding site is different from the canonical substrate binding site in cation pumps. This was demonstrated by characterizing two S. cerevisiae P4-ATPases, Dnf1 and Drs2 (which bind PC/PE and PS/PE, respectively) and by showing that the domains implicated in substrate specificity are indeed located outside of the canonical binding region (Baldridge andGraham, 2013, 2012). The specificity of these flippases depends on a few amino acids, as in Dnf1, for instance, the mutation Y618F in TMD4 increases PS flipping activity, and, conversely, mutations in Drs2 (such as F511Y), reduce PS flipping activity (Baldridge and Graham, 2012). Based on directed mutagenesis and genetic approaches, the authors identified residues located on the entry (cluster located in and between TMD1-2 on the exofacial side) and exit gates (cluster located in TMD3-4 near the cytosolic side) and proposed that these gates act cooperatively for phospholipid selection and transport (Baldridge and Graham, 2013).

P4-ATPases are implicated in a number of processes. In neurological disorders in humans, some brain malfunction results from flippase dysfunction [START_REF] Martín-Hernández | New ATP8A2 gene mutations associated with a novel syndrome: encephalopathy, intellectual disability, severe hypotonia, chorea and optic atrophy[END_REF]. In some parasites, these ATPases can capture lipids, which cannot be synthesized by these organisms, in the extracellular medium [START_REF] Bansal | Role of cholesterol in parasitic infections[END_REF]. In plants, the ATPase ALA3 participates in secretion from the Golgi and is involved in the control of polarized growth of roots and shoots in Arabidopsis [START_REF] Poulsen | The Arabidopsis P4-ATPase ALA3 localizes to the golgi and requires a beta-subunit to function in lipid translocation and secretory vesicle formation[END_REF]. In fungi, the endomembrane organization and protein transport rely to some extent on ATPases, as deletion of these flippases are often related with defects in membrane trafficking in different organisms (Hankins et al., 2015b;Pomorski et al., 2003;[START_REF] Schultzhaus | Phospholipid flippases DnfA and DnfB exhibit differential dynamics within the A. nidulans Spitzenkörper[END_REF][START_REF] Schultzhaus | Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase[END_REF][START_REF] Takar | The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane[END_REF][START_REF] Wu | Neo1 and phosphatidylethanolamine contribute to vacuole membrane fusion in Saccharomyces cerevisiae[END_REF]. Moreover, membrane protein transport and localization of peripheral membrane proteins, such as the small Gproteins, depend on lipids. For instance, it was reported that the Rho GTPase Cdc42, a master regulator of polarized growth, depends on PS asymmetric distribution for membrane localization (Das et al., 2012;Haupt and Minc, 2017;[START_REF] Lopez-Marques | P4-ATPases: lipid flippases in cell membranes[END_REF]Meca et al., 2019;[START_REF] Saito | Transbilayer Phospholipid Flipping Regulates Cdc42p Signaling during Polarized Cell Growth via Rga GTPase-Activating Proteins[END_REF]. In filamentous fungi, P4-ATPases have been associated with sporulation [START_REF] Schultzhaus | The phospholipid flippase DnfD localizes to late Golgi and is involved in asexual differentiation in Aspergillus nidulans[END_REF][START_REF] Schultzhaus | Phospholipid flippases DnfA and DnfB exhibit differential dynamics within the A. nidulans Spitzenkörper[END_REF][START_REF] Schultzhaus | Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase[END_REF] and to some extent with the secretion of compounds related with fungal pathogenicity [START_REF] Balhadère | PDE1 Encodes a P-Type ATPase Involved in Appressorium-Mediated Plant Infection by the Rice Blast Fungus Magnaporthe grisea[END_REF][START_REF] Gilbert | A P-type ATPase required for rice blast disease and induction of host resistance[END_REF][START_REF] Hu | A P4-ATPase subunit of the Cdc50 family plays a role in iron acquisition and virulence in Cryptococcus neoformans[END_REF][START_REF] Hu | A putative P-type ATPase, Apt1, is involved in stress tolerance and virulence in Cryptococcus neoformans[END_REF]Labbaoui et al., 2017;[START_REF] Yun | Flippases play specific but distinct roles in the development, pathogenicity, and secondary metabolism of Fusarium graminearum[END_REF]. Consequently, flippases are proteins with great potential as candidates in the regulation of polarized growth and virulence of these organisms.

In the next section, I will discuss the human fungal pathogen C. albicans as a model to study polarized growth and the importance of flippases and of lipid distribution.

Chapter III: Candida albicans

Candida albicans is an opportunistic human fungal pathogen that belongs to the Candida genus.

In general Candida spp. are harmless commensal yeasts commonly populating the human mucosa, that can be pathogenic in specific conditions. Candida spp. belong to the Ascomycota phylum, Saccharomycotina sub-phylum and Saccharomycetales order [START_REF] Mcmanus | Molecular epidemiology, phylogeny and evolution of Candida albicans[END_REF]. Among the 16 families, the Saccharomycetales incertae sedis family contains the CTG clade. As part of this clade, C. albicans expresses the CTG codon as serine instead of leucine. The genus Saccharomyces, which includes Saccharomyces cerevisiae, belongs to the Saccharomycetaceae family, also called the whole genome duplication (WGD) clade, as the genomes of these species have been duplicated [START_REF] Diezmann | Phylogeny and evolution of medical species of Candida and related taxa: a multigenic analysis[END_REF][START_REF] Fitzpatrick | A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis[END_REF][START_REF] Wolfe | Molecular evidence for an ancient duplication of the entire yeast genome[END_REF]. Focusing on the diversity of these phyla, it is interesting to notice that, although C. albicans and S. cerevisiae are both yeasts that can be found in ovoid budding form, C. albicans has diverged from S. cerevisiae about 800 million years ago [START_REF] Fitzpatrick | A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis[END_REF][START_REF] Hedges | The origin and evolution of model organisms[END_REF]. Yeasts reproduce by budding, fission and/or sexual reproduction, each of these growth modes are triggered by specific environmental conditions. The genus Candida includes more than 150 species [START_REF] Calderone | Candida and candidiasis[END_REF]. The majority of Candida species belong to the CTG codon clade, such as C. luisitaniae, C. guilliermondii, C. parapsilosis, C. tropicalis and C. dubliniensis, which are the species most closely related to C. albicans [START_REF] Fitzpatrick | A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis[END_REF]; C. glabrata, on the other hand, belongs to the WGD clade [START_REF] Fitzpatrick | A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis[END_REF].

Most Candida species, about 65%, are adapted to their environmental niche and able to grow at a temperature of 37°C, which is important for commensal organisms [START_REF] Calderone | Candida and candidiasis[END_REF][START_REF] Pappas | Invasive candidiasis[END_REF]. The genome of C. albicans is completely sequenced (http://www.candidagenome.org) and consists of 8 pairs of chromosomes that code for about 6400 genes [START_REF] Jones | The diploid genome sequence of Candida albicans[END_REF]. Unlike S. cerevisiae, C. albicans is an obligate diploid and is the most prevalent among Candida spp. to cause infections [START_REF] Moyes | Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface[END_REF][START_REF] Pappas | Invasive candidiasis[END_REF], making the study of this organism particularly medically relevant.

Virulence

As introduced above, Candida spp are harmless commensal yeasts present in the oral, gastrointestinal, and urogenital mucosa in 60% of healthy individuals [START_REF] Pappas | Invasive candidiasis[END_REF].

However, these organisms can become pathogenic in response to environmental stress, such as an imbalance of the intestinal flora or an immunodeficiency of the host, resulting in superficial infections, such as vaginal infections [START_REF] Moyes | Candidalysin is a fungal peptide toxin critical for mucosal infection[END_REF]. C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei are the 5 major Candida spp that are isolated from blood or mucosal samples of immunocompromised patients, but C. albicans and C. glabrata are the two most virulent and common nosocomial diseases [START_REF] Brunke | Two unlike cousins: Candida albicans and C. glabrata infection strategies[END_REF]. When C. albicans invades the host tissues and infiltrates into the bloodstream, resulting in systemic infection [START_REF] Pappas | Invasive candidiasis[END_REF], which is particularly problematic for immunocompromised patients, such as patients with AIDS, transplant patients or those undergoing chemotherapy [START_REF] Moyes | Candidalysin is a fungal peptide toxin critical for mucosal infection[END_REF][START_REF] Pappas | Invasive candidiasis[END_REF].

C. albicans transition from a commensal to a pathogen is triggered by different environmental and biological cues. C. albicans growth is limited by the presence of bacteria that induce the secretion of Candida spp.-growth inhibitory factors by the mucosa, thus extensive or long-term use of antibiotics may indirectly favor C. albicans development [START_REF] Kullberg | Invasive Candidiasis[END_REF][START_REF] Pappas | Invasive candidiasis[END_REF]. Other factors, such as mucositis (inflammation of the mucosa), surgery and intravenous catheters can facilitate bloodstream infection in immunosuppressed patients, resulting in candidemia [START_REF] Kullberg | Invasive Candidiasis[END_REF][START_REF] Pappas | Invasive candidiasis[END_REF]. C. albicans also exhibits specific properties, such as adhesion, secretion and invasive growth to facilitate pathogenicity [START_REF] Kullberg | Invasive Candidiasis[END_REF][START_REF] Moyes | Candidalysin is a fungal peptide toxin critical for mucosal infection[END_REF][START_REF] Noble | Candida albicans cell type switches and functional plasticity in the mammalian host[END_REF][START_REF] Pappas | Invasive candidiasis[END_REF].

▪ Adhesion:

The first step in the interaction with epithelial tissues is adhesion, mediated via epithelial cell receptors and Candida adhesins [START_REF] Moyes | Candidalysin is a fungal peptide toxin critical for mucosal infection[END_REF]. The most common adhesins expressed by C. albicans belong to the Als (Agglutinin-Like Sequence) family, encoding for cell wallassociated proteins [START_REF] Hoyer | Candida albicans Agglutinin-Like Sequence (Als) Family Vignettes: A Review of Als Protein Structure and Function[END_REF] and the cell wall protein Hwp1 (Hyphal Wall Protein) [START_REF] Staab | Expression of Transglutaminase Substrate Activity on Candida albicans Germ Tubes through a Coiled, Disulfide-bonded N-terminal Domain of Hwp1 Requires C-terminal Glycosylphosphatidylinositol Modification[END_REF][START_REF] Sundstrom | Essential Role of the Candida albicans Transglutaminase Substrate, Hyphal Wall Protein 1, in Lethal Oroesophageal Candidiasis in Immunodeficient Mice[END_REF][START_REF] Zhu | Interactions of Candida albicans with epithelial cells[END_REF]. Both Als proteins and Hwp1 are Glycophosphatidylinositol (GPI)-anchored to the PM but their roles in adhesion are different. For instance, by using the loss-of-function mutants als1, als2 and als3, it was shown that Als1 has no role in adhesion, Als2 an intermediate role, and Als3 a critical role: indeed, als3 cells are not able to adhere and to infect epithelial cells in vitro [START_REF] Zhao | Analysis of the Candida albicans Als2p and Als4p adhesins suggests the potential for compensatory function within the Als family[END_REF][START_REF] Zhao | ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin; functional comparisons between Als3p and Als1p[END_REF].

▪ Secretion and invasion:

The epithelial and endothelial host cell invasion by C. albicans can be facilitated by distinct mechanisms: (i) The induction of phagocytosis by epithelial cells, with the adhesins/invasins Als3 and Ssa1 expressed on the surface of C. albicans being recognized and bound by Ecadherin and other epithelial cell receptors [START_REF] Moyes | Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface[END_REF][START_REF] Swidergall | Oropharyngeal Candidiasis: Fungal Invasion and Epithelial Cell Responses[END_REF].

(ii) Active penetration, which is thought to be facilitated by mechanical forces generated during the hyphal growth, coupled to an adhesin-mediated anchoring [START_REF] Moyes | Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface[END_REF][START_REF] Swidergall | Oropharyngeal Candidiasis: Fungal Invasion and Epithelial Cell Responses[END_REF]. (iii) The secretion of proteins can also play an important role in Candida pathogenesis. For example, the Secreted Aspartyl Proteinase (SAP) family can damage the epithelium, hence increasing the probability of penetration through epithelial cells [START_REF] Moyes | Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface[END_REF][START_REF] Swidergall | Oropharyngeal Candidiasis: Fungal Invasion and Epithelial Cell Responses[END_REF][START_REF] Zhu | Interactions of Candida albicans with epithelial cells[END_REF]. SAP1-3 contribute to superficial infections; SAP4-6 are important to escape from macrophages and for systemic infections [START_REF] Naglik | Candida albicans proteinases and host/pathogen interactions[END_REF][START_REF] Naglik | Candida albicans secreted aspartyl proteinases in virulence and pathogenesis[END_REF], and to invade epithelial cells in vitro [START_REF] Dalle | Cellular interactions of Candida albicans with human oral epithelial cells and enterocytes[END_REF][START_REF] Zhu | Interactions of Candida albicans with epithelial cells[END_REF]. Another secreted protein, candidalysin, which is a cytolytic peptide toxin, can induce epithelial cell damage [START_REF] Allert | Candida albicans-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers[END_REF][START_REF] Moyes | Candidalysin is a fungal peptide toxin critical for mucosal infection[END_REF]. The ECE1 (extent of cell elongation) gene encodes this toxin that is necessary for efficient host infection, although not essential for hyphal formation per se [START_REF] Allert | Candida albicans-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers[END_REF][START_REF] Moyes | Candidalysin is a fungal peptide toxin critical for mucosal infection[END_REF].

Candida hyphal development is essential for biofilm formation [START_REF] Cavalheiro | Candida Biofilms: Threats, Challenges, and Promising Strategies[END_REF].

Biofilms are microbial communities of bacteria, fungi, or both, that confer different properties than those of individual cells, such as drug resistance [START_REF] Cavalheiro | Candida Biofilms: Threats, Challenges, and Promising Strategies[END_REF][START_REF] Rodríguez-Cerdeira | Pathogenesis and Clinical Relevance of Candida Biofilms in Vulvovaginal Candidiasis[END_REF]. The first step of a biofilm formation is adhesion to the host tissues, therefore Candida adhesins such as Hwp1, Als1, and Als2 play a crucial role in this process [START_REF] Rodríguez-Cerdeira | Pathogenesis and Clinical Relevance of Candida Biofilms in Vulvovaginal Candidiasis[END_REF]. Consistently, mutants of transcription factors, such as Efg1 and Cph1 that control the expression of ECE1, HWP1, and ALS3, are critical for biofilm formation [START_REF] Cavalheiro | Candida Biofilms: Threats, Challenges, and Promising Strategies[END_REF][START_REF] Ramage | The filamentation pathway controlled by the Efg1 regulator protein is required for normal biofilm formation and development in Candida albicans[END_REF][START_REF] Rodríguez-Cerdeira | Pathogenesis and Clinical Relevance of Candida Biofilms in Vulvovaginal Candidiasis[END_REF].

Candida albicans morphogenesis

C. albicans can be found as budding yeasts (white cells), morphologically similar to S. cerevisiae cells, opaque cells, gray cells, and GUT (Gastrointestinally Induced Transition) cells [START_REF] Noble | Candida albicans cell type switches and functional plasticity in the mammalian host[END_REF]. Opaque cells are produced from white cells that are homozygous (a/a or α∕α) for the Mating Type like Locus (MTL) [START_REF] Perry | Unraveling How Candida albicans Forms Sexual Biofilms[END_REF]. This transition occurs in 1 in 10,000 cell divisions and passes through an intermediate stage, gray cells [START_REF] Perry | Unraveling How Candida albicans Forms Sexual Biofilms[END_REF].

Opaque cells are competent for mating and the GUT cells, which are also competent for mating, are thought to be a morphology adapted to commensalism [START_REF] Noble | Candida albicans cell type switches and functional plasticity in the mammalian host[END_REF][START_REF] Pande | Passage through the mammalian gut triggers a phenotypic switch that promotes Candida albicans commensalism[END_REF]. Despite the mating competence of opaque cells, C. albicans was considered as an asexual organism, since no meiosis or sporulation has been observed [START_REF] Noble | Candida albicans cell type switches and functional plasticity in the mammalian host[END_REF]. Indeed, mating is thought to be a very rare event due to the high frequency (~97%) of MTL heterozygous cell [START_REF] Perry | Unraveling How Candida albicans Forms Sexual Biofilms[END_REF]. However, opaque cells can undergo homothallic mating (between two opaque cells of the same matin type) in a glucose poor media or during oxidative stress [START_REF] Perry | Unraveling How Candida albicans Forms Sexual Biofilms[END_REF]. This is the case of the sexual biofilm where, in the deepest layer isolated from the surface, there are less nutrient and oxygen, and homothallic mating is favored [START_REF] Noble | Candida albicans cell type switches and functional plasticity in the mammalian host[END_REF][START_REF] Perry | Unraveling How Candida albicans Forms Sexual Biofilms[END_REF]. As in all sexual organisms, mating in C. albicans allows for the cells to adapt faster to different environmental conditions by generating genetic variability, and once adapted, the asexual budding and filamentous growth allows the cellular spreading [START_REF] Perry | Unraveling How Candida albicans Forms Sexual Biofilms[END_REF].

The yeast-to-hyphae transition is a virulence trait in C. albicans, but filamentous growth is not required for all Candida spp.'s ability to infect a host; for instance, C. glabrata does not form hyphae but is the second most common pathogen among Candida spp. [START_REF] Brunke | Two unlike cousins: Candida albicans and C. glabrata infection strategies[END_REF]. In C. albicans, mutants locked in either the filamentous or yeast form are less virulent, thus, the ability to switch between the two morphological states is essential for virulence [START_REF] Braun | NRG1, a repressor of filamentous growth in C.albicans, is down-regulated during filament induction[END_REF][START_REF] Braun | Identification and characterization of TUP1-regulated genes in Candida albicans[END_REF][START_REF] Lo | Nonfilamentous C. albicans Mutants Are Avirulent[END_REF][START_REF] Murad | NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans[END_REF][START_REF] Pappas | Invasive candidiasis[END_REF][START_REF] Schweizer | The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans[END_REF]Zheng et al., 2004). In addition, most of the infection steps detailed above, such as active penetration and induced endocytosis occur during C. albicans hyphal state [START_REF] Dalle | Cellular interactions of Candida albicans with human oral epithelial cells and enterocytes[END_REF][START_REF] Moyes | Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface[END_REF][START_REF] Naglik | Candida albicans proteinases and host/pathogen interactions[END_REF][START_REF] Naglik | Candida albicans secreted aspartyl proteinases in virulence and pathogenesis[END_REF][START_REF] Noble | Candida albicans cell type switches and functional plasticity in the mammalian host[END_REF][START_REF] Swidergall | Oropharyngeal Candidiasis: Fungal Invasion and Epithelial Cell Responses[END_REF][START_REF] Zhu | Interactions of Candida albicans with epithelial cells[END_REF] and the genes implicated are mostly hyphal specific genes. For example, ALS3, which is only expressed in hyphae, induces C. albicans endocytic uptake by epithelial cells through Als3-E-cadherin interactions. Yeast forms colonize the surface of the tissue, without apparent damage [START_REF] Moyes | Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface[END_REF][START_REF] Noble | Candida albicans cell type switches and functional plasticity in the mammalian host[END_REF][START_REF] Swidergall | Oropharyngeal Candidiasis: Fungal Invasion and Epithelial Cell Responses[END_REF]. Furthermore, recent work supports the idea that filamentous growth is dispensable for virulence in specific infection models [START_REF] Dunker | Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain[END_REF]. A C. albicans strain lacking the Epithelial Escape and Dissemination 1 (EDD1) gene is able to form germ tubes but does not undergo hyphal extension, resulting avirulent in an in vivo murine model of intraperitoneal infection [START_REF] Dunker | Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain[END_REF]. However, this mutant is fully virulent in a mouse model of systemic infection, most likely due to rapid proliferation supported by enhanced metabolic adaptation [START_REF] Dunker | Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain[END_REF]. In summary, while filamentation is most of the times a key trait for virulence, specific cases exist in which efficient host infection happens in absence of any filamentation.

▪ Hyphal gene induction

While C. albicans budding growth is triggered by internal cues, similar to S. cerevisiae, hyphal growth is triggered by external cues [START_REF] Bassilana | External signal-mediated polarized growth in fungi[END_REF]. In contrast to budding growth, which is cell cycle-dependent, hyphal growth can be initiated at any cell cycle stage (Hazan et al., 2002) and the expression of hyphal specific genes is regulated by several key transcription factors [START_REF] Arkowitz | Recent advances in understanding Candida albicans hyphal growth[END_REF][START_REF] Chen | The regulation of hyphae growth in Candida albicans[END_REF]Kornitzer, 2019;[START_REF] Sudbery | Growth of Candida albicans hyphae[END_REF] (Figure 7). For instance, in vitro, in response to the presence of serum, C. albicans changes from a budding yeast cell of about 5μm to a filamentous cell with a filament extension rate of ~ 0.3μm/min (Silva et al., 2019). The presence of serum activates signaling pathways that depend on Ras1 and trigger in particular a mitogen-activated protein kinase (MAPK) cascade, which ultimately converges on the activation of the transcription factor Cph1 [START_REF] Chen | The regulation of hyphae growth in Candida albicans[END_REF]Kornitzer, 2019;[START_REF] Sudbery | Growth of Candida albicans hyphae[END_REF]. Among the hyphal specific genes, the Hyphal G cyclin 1 (HGC1) gene, encoding for a cyclin associated with the G1 phase, is critical for hyphal growth [START_REF] Wang | Hgc1-Cdc28-how much does a single protein kinase do in the regulation of hyphal development in Candida albicans?[END_REF] and HGC1 is further regulated by UME6 (Carlisle and Kadosh, 2010). The hgc1 and ume6 deletion mutants are both defective in maintaining hyphal growth and are reduced in

▪ Germ tube initiation:

Filamentous growth consists in the initiation and maintenance of filament apical growth; thus, it is a highly polarized process. As conserved in other fungi, included S. cerevisiae polarized growth, C. albicans filamentous growth requires polarity establishment proteins, i.e., Cdc42/Cdc24/Bem1 (Bassilana et al., 2005(Bassilana et al., , 2003a;;[START_REF] Brand | Cdc42 GTPase dynamics control directional growth responses[END_REF][START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF][START_REF] Pulver | Rsr1 focuses Cdc42 activity at hyphal tips and promotes maintenance of hyphal development in Candida albicans[END_REF]. Cdc42 is a highly conserved Rho GTPase, which was initially described in S. cerevisiae [START_REF] Johnson | Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity[END_REF], and its activation depends on the formation of a polarity establishment complex [START_REF] Chiou | Cell Polarity in Yeast[END_REF]Woods and Lew, 2019) comprised of (i) the Guanine nucleotide exchange factor (GEF), Cdc24, which is the sole activator of Cdc42, (ii) p21 activated kinases (PAKs), which are Cdc42 effectors, and (iii) Bem1, which is a SH3 domain containing scaffold protein. Bem1 and the PAKs participate in a positive feedback loop, resulting in the formation of a polarity patch of Cdc42-GTP [START_REF] Bassilana | External signal-mediated polarized growth in fungi[END_REF][START_REF] Butty | A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization[END_REF][START_REF] Chiou | Cell Polarity in Yeast[END_REF][START_REF] Woods | Polarity establishment by Cdc42: Key roles for positive feedback and differential mobility[END_REF]. The recruitment of either Cdc24 or Bem1 appears to be sufficient to initiate a Cdc42 polarity patch [START_REF] Witte | Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization[END_REF]. Bem1 is also a Cdc42-GTP effector [START_REF] Grinhagens | A time-resolved interaction analysis of Bem1 reconstructs the flow of Cdc42 during polar growth[END_REF] that is implicated in the actin-independent localization of the exocyst subunit Exo70 [START_REF] Miller | Temporal regulation of cell polarity via the interaction of the Ras GTPase Rsr1 and the scaffold protein Bem1[END_REF]. Inactivation of Cdc42 is promoted by the GTPase activating proteins (GAPs) Rga1/2 and Bem3 [START_REF] Smith | GTPase-Activating Proteins for Cdc42[END_REF].

Cell polarity is a dynamic process that allows cells to adapt to their environment in response to external cues. Cdc42 is critical for filamentous growth in fungi, for instance, it controls germ tubes directional growth in N. crassa [START_REF] Lichius | CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa[END_REF]. In C. albicans, it was shown that activated Cdc42 is recruited to the incipient germ tube and remains at the filament apex during filament extension [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF]. However, the scaffold protein, Bem1, although essential, it can still form hyphae when its expression levels are downregulated (Bassilana et al., 2003a). In contrast, Cdc42, Cdc24 are essential and its downregulation results in an impaired hyphal growth (Bassilana et al., 2005(Bassilana et al., , 2003a)). Moreover, the active Cdc42-GTP was lethal when expressed in a constitutive manner [START_REF] Ushinsky | CDC42 Is Required for Polarized Growth in Human Pathogen Candida albicans[END_REF] and caused loss of polarity when conditionally expressed in hyphae [START_REF] Brand | Cdc42 GTPase dynamics control directional growth responses[END_REF][START_REF] Court | Regulation of Cdc42 GTPase activity in the formation of hyphae in Candida albicans[END_REF].

In S. cerevisiae Cdc42-GTP has been shown to interact with several proteins, such as the exocyst component Sec3, thereby promoting polarized secretion [START_REF] Zhang | Membrane association and functional regulation of Sec3 by phospholipids and Cdc42[END_REF], and the formin Bni1p, critical for actin polymerization . In C. albicans, the formin Bni1 is localized to the Spitzenkörper and necessary for hyphal growth maintenance [START_REF] Li | The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans[END_REF][START_REF] Martin | Ras1-Induced Hyphal Development in Candida albicans Requires the Formin Bni1[END_REF]. Sec3 is necessary for C. albicans hyphal growth maintenance upon septum formation [START_REF] Li | Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation[END_REF]. Interestingly, upon optogenetic disruption of active Cdc42 localization, Sec3 localization at the hyphal tip is perturbed (Silva et al., 2019).

▪ Hyphal extension:

Hyphal growth is independent of cell cycle, yet its maintenance is regulated by the Cyclindependent kinases (the Cdk Cdc28) [START_REF] Desai | Candida albicans Hyphae: From Growth Initiation to Invasion[END_REF][START_REF] Wang | Hgc1-Cdc28-how much does a single protein kinase do in the regulation of hyphal development in Candida albicans?[END_REF]. C. albicans has three G1 cyclins (Ccn1, Cln3, and Hgc1) and two B-type mitotic cyclins (Clb2, Clb4), their expression oscillates during the cell cycle and differently regulate the same target kinase, Cdk1, which is always active but finely tuned [START_REF] Desai | Candida albicans Hyphae: From Growth Initiation to Invasion[END_REF]. Decoupling hyphal growth from the cell cycle allows for cell tip continuous growth, thus Cdc28 is also finely regulated by the different cyclins. The forkhead transcription factor Fkh2 is phosphorylated by the Ccn1/Cln3-Cdc28 complex during hyphal growth [START_REF] Greig | Cell Cycle-Independent Phospho-Regulation of Fkh2 during Hyphal Growth Regulates Candida albicans Pathogenesis[END_REF]. Fkh2 activity is independent from the cell cycle and targets HGC1. HGC1 and UME6 are critical for filament extension [START_REF] Desai | Candida albicans Hyphae: From Growth Initiation to Invasion[END_REF]Zheng et al., 2004). The complex Hgc1-Cdc28 also phosphorylates Rga2, inactivating this Cdc42 GAP at the hyphal tip to maintain Cdc42 activity [START_REF] Zheng | Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth[END_REF]. Rga2 localization at the septum probably contributes to restricting Cdc42 activity at the hyphal apex.

Interestingly, deletion of this GAP does not impair filamentation [START_REF] Court | Regulation of Cdc42 GTPase activity in the formation of hyphae in Candida albicans[END_REF]. Hgc1-Cdc28 phosphorylates (i) the polarisome protein Spa2, which recruits the actin-binding proteins Bni1, Bud6, and Aip5 [START_REF] Li | The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans[END_REF][START_REF] Martin | Ras1-Induced Hyphal Development in Candida albicans Requires the Formin Bni1[END_REF][START_REF] Xie | Orchestrated actin nucleation by the Candida albicans polarisome complex enables filamentous growth[END_REF] and (ii) the exocyst subunit Exo84 and the Rab GTPase Sec4 GEF Sec2, promoting their localization at the filament apex [START_REF] Bishop | Hyphal growth in Candida albicans requires the phosphorylation of Sec2 by the Cdc28-Ccn1/Hgc1 kinase[END_REF][START_REF] Caballero-Lima | In Candida albicans, phosphorylation of Exo84 by Cdk1-Hgc1 is necessary for efficient hyphal extension[END_REF][START_REF] Wang | CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth[END_REF].

Other proteins such as septins are regulated by Hgc1-Cdc28-mediated phosphorylation [START_REF] Sinha | Cyclin-Dependent Kinases Control Septin Phosphorylation in Candida albicans Hyphal Development[END_REF]. Septins play important roles in C. albicans morphogenesis and virulence [START_REF] Douglas | Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans[END_REF][START_REF] González-Novo | Fungal septins: one ring to rule it all?[END_REF][START_REF] Warenda | Candida albicans Septin Mutants Are Defective for Invasive Growth and Virulence[END_REF][START_REF] Warenda | Septin Function in Candida albicans Morphogenesis[END_REF]. The hypha-specific phosphorylation that regulates the septin Cdc11 depends on the Cdc28-Ccn1 and Cdc28-Hgc1 during initiation and extension, respectively [START_REF] Sinha | Cyclin-Dependent Kinases Control Septin Phosphorylation in Candida albicans Hyphal Development[END_REF].

The septum formation is particularly regulated during hyphal growth since no cell separation occur after cell division and Hgc1-Cdc28 also targets the Sep7 septin, whose phosphorylation prevents cytokinesis [START_REF] González-Novo | Fungal septins: one ring to rule it all?[END_REF][START_REF] Lodish | Molecular cell biology[END_REF] and Efg1, a transcription factor that competes with Ace2, another transcription factor implicated in septum degradation postcytokinesis [START_REF] Wang | Hyphal Chain Formation in Candida albicans: Cdc28-Hgc1 Phosphorylation of Efg1 Represses Cell Separation Genes[END_REF]. At the septum, septins act as barriers to retain the machinery necessary for septum formation (e.g., the polarisome, the exocyst subunit Sec3, as well as the actomyosin ring) [START_REF] Douglas | Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans[END_REF][START_REF] González-Novo | Fungal septins: one ring to rule it all?[END_REF]. In S. cerevisiae, septins are a conserved family of GTP-binding proteins that serve as scaffolds or as diffusion barriers between mother and daughter cells, and are used as markers of cell division progression [START_REF] Cheffings | Actomyosin Ring Formation and Tension Generation in Eukaryotic Cytokinesis[END_REF][START_REF] Douglas | Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans[END_REF][START_REF] González-Novo | Fungal septins: one ring to rule it all?[END_REF]. Septins can delimit active Cdc42 region by recruiting its GAPs [START_REF] Chiou | Cell Polarity in Yeast[END_REF]. Septins also recruit proteins implicated in the actomyosin ring during cytokinesis in budding yeast [START_REF] Cheffings | Actomyosin Ring Formation and Tension Generation in Eukaryotic Cytokinesis[END_REF][START_REF] González-Novo | Fungal septins: one ring to rule it all?[END_REF]. In C. albicans filamentous cells, septins mark the germ tube site. Just before septation, the septin cap at the tip generates a ring, that remains in a fixed position as the tip continues to elongate [START_REF] Douglas | Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans[END_REF][START_REF] González-Novo | Fungal septins: one ring to rule it all?[END_REF]. When mitosis is completed, one nucleus segregates back to the mother cell. At this point, the septin ring splits to allow the septum construction (Figure 8) [START_REF] Finley | Microtubules in Candida albicans hyphae drive nuclear dynamics and connect cell cycle progression to morphogenesis[END_REF][START_REF] Thomson | High frame-rate resolution of cell division during Candida albicans filamentation[END_REF]. Thus, septins are critical to regulate C. albicans cell division and morphogenesis. [START_REF] Thomson | High frame-rate resolution of cell division during Candida albicans filamentation[END_REF].

▪ Hyphal organization:

Hyphal growth is a highly polarized process which requires a rapid extension of the of the tip of the hypha. This implies the need for an apical organization of different structures involved in the initiation and maintenance of hyphal growth. In C. albicans, Cdc42-GTP is maintained at the apex of the germ tube and filament, where it promotes the activation and/or recruitment of critical effector proteins for cytoskeleton organization and membrane traffic regulating apical tip organization [START_REF] Bassilana | External signal-mediated polarized growth in fungi[END_REF](Bassilana et al., , 2005;;[START_REF] Brand | Cdc42 GTPase dynamics control directional growth responses[END_REF][START_REF] Chen | The regulation of hyphae growth in Candida albicans[END_REF][START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF][START_REF] Court | Regulation of Cdc42 GTPase activity in the formation of hyphae in Candida albicans[END_REF][START_REF] Pulver | Rsr1 focuses Cdc42 activity at hyphal tips and promotes maintenance of hyphal development in Candida albicans[END_REF].

The exocyst:

In S. cerevisiae, the exocyst complex is comprised of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 [START_REF] Terbush | The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae[END_REF], and Cdc42 interacts with Sec3 [START_REF] Zhang | Membrane association and functional regulation of Sec3 by phospholipids and Cdc42[END_REF].

In C. albicans, exocyst subunits localize to a crescent at the hyphal apex, while secretory vesicles associated proteins, the Rab-type GTPase Sec4, its GEF Sec2 and the myosin light chain protein 1 (Mlc1) localize to a subapical cluster [START_REF] Bishop | Hyphal growth in Candida albicans requires the phosphorylation of Sec2 by the Cdc28-Ccn1/Hgc1 kinase[END_REF]Crampin et al., 2005;[START_REF] Jones | Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties[END_REF][START_REF] Li | Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation[END_REF][START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF] which is consistent with the model in Figure 9. Sec15 has been shown to interact with Rsr1, Bem1 and the myosin V, Myo2 [START_REF] Guo | Sec15 links bud site selection to polarised cell growth and exocytosis in Candida albicans[END_REF]. Several members of the exocyst have been shown to be essential for a correct hyphal growth. For example, phosphorylation of Exo84 by the Hgc1-Cdc28 is required for correct recycling to the PM (Caballero-Lima and Sudbery, 2014) and Sec3 is necessary for maintaining hyphal growth upon septum formation [START_REF] Li | Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation[END_REF].

The polarisome:

The S. cerevisiae polarisome complex includes Spa2, Pea2 [START_REF] Valtz | Pea2 protein of yeast is localized to sites of polarized growth and is required for efficient mating and bipolar budding[END_REF], the formin Bni1 and the formin-actin-binding proteins Bud6 and Aip5 [START_REF] Xie | Polarisome scaffolder Spa2-mediated macromolecular condensation of Aip5 for actin polymerization[END_REF]. These proteins are conserved in C. albicans (Crampin et al., 2005;[START_REF] Xie | Orchestrated actin nucleation by the Candida albicans polarisome complex enables filamentous growth[END_REF]. As an example, Bud6 and Spa2 form an apical crescent at the filament apex (Crampin et al., 2005). In filamentous fungi such as Ashbya gossypii and N. crassa, the crescent localization of Spa2 depends on the growth rate; during the slow growth phase, Spa2 becomes a "cap" in Ashbya gossypii. In N. crassa during fast growth, Spa2 localizes as patches [START_REF] Araujo-Palomares | The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkörper[END_REF][START_REF] Köhli | Growth-speedcorrelated localization of exocyst and polarisome components in growth zones of Ashbya gossypii hyphal tips[END_REF]. In C. albicans, the spatiotemporal localization of the polarisome is altered during hyphal growth, where Cdc28-Clb2 and Cdc28-Hgc1 cooperate to phosphorylate Spa2, allowing the polarisome to be maintained at the filament tip [START_REF] Wang | CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth[END_REF].

Additionally, deletion of SPA2 disrupts filamentous growth, resulting in formation of thick filaments in the mutant [START_REF] Wang | CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth[END_REF][START_REF] Zheng | CaSPA2 is important for polarity establishment and maintenance in Candida albicans[END_REF]. The polarisome member Bni1 is a Cdc42-GTP effector implicated in actin cable polarization in S. cerevisiae [START_REF] Chen | Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p[END_REF][START_REF] Grinhagens | A time-resolved interaction analysis of Bem1 reconstructs the flow of Cdc42 during polar growth[END_REF]. In C. albicans, it was shown that deletion of BNI1 causes cells to be able to initiate but not to maintain polarized hyphal growth [START_REF] Li | The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans[END_REF][START_REF] Martin | Ras1-Induced Hyphal Development in Candida albicans Requires the Formin Bni1[END_REF], suggesting a specific role in hyphal extension.

The Spitzenkörper:

The organization of the secretory pathway can have specific functions in distinct cell types. For example, there is an accumulation of vesicles in presynaptic cells ready to be secreted upon electrical stimulus [START_REF] Binotti | An overview of the synaptic vesicle lipid composition[END_REF]. In the yeast Schizosaccharomyces pombe, during mating, it is proposed that Cdc42-GTP zones dictate regions of pheromone secretion that are ultimately stabilized when the cell encounter pheromones from cells of opposite sex (Martin, 2019b;[START_REF] Merlini | Local Pheromone Release from Dynamic Polarity Sites Underlies Cell-Cell Pairing during Yeast Mating[END_REF].

In filamentous fungi the secretory pathway is highly organized and compartmentalized at the apex of the filaments, which is the site of growth [START_REF] Bassilana | External signal-mediated polarized growth in fungi[END_REF][START_REF] Riquelme | Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures[END_REF][START_REF] Takeshita | Coordinated process of polarized growth in filamentous fungi[END_REF] . The secretory vesicles, prior to anchoring and fusion with the plasma membrane, accumulate at the apex of the filaments and this accumulation is called the Spitzenkörper (SPK) [START_REF] Riquelme | Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures[END_REF][START_REF] Takeshita | Coordinated process of polarized growth in filamentous fungi[END_REF]. The SPK, first identified in 1924, was observed in fixed samples of Coprinus narcoticus and Coprinus sterquilinus as an iron hematoxylin stained apical body [START_REF] Brunswik | Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans[END_REF], and subsequently observed as a dense portion at the Polystictus versicolor filament tip by phase contrast microscopy (Girbardt, 1957).

Although it is a common structure among different fungi (Crampin et al., 2005;[START_REF] Jones | Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties[END_REF][START_REF] Pantazopoulou | Organization and Dynamics of the Aspergillus nidulans Golgi during Apical Extension and Mitosis[END_REF][START_REF] Riquelme | The Spitzenkörper: a choreographer of fungal growth and morphogenesis[END_REF][START_REF] Schultzhaus | Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase[END_REF][START_REF] Steinberg | On the move: endosomes in fungal growth and pathogenicity[END_REF][START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF], the SPK composition varies from fungus to fungus and is proposed to be a Vesicle Supply Center (VSC) [START_REF] Riquelme | Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures[END_REF]. For example, in Neurospora crassa, the SPK is formed by different types of secretory vesicles, a central core of microvesicles surrounded by macrovesicles [START_REF] Riquelme | The Spitzenkörper: a choreographer of fungal growth and morphogenesis[END_REF]. In other fungi, such as Candida albicans, the SPK is composed of vesicles homogeneous in size [START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF], loaded with the Rab8 homolog Sec4 and its GEF Sec2, the myosin light chain Mlc1, the formin Bni1, and the Rab11 homolog Ypt31 (Crampin et al., 2005;[START_REF] Jones | Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties[END_REF]Silva et al., 2019). Very recent work in the laboratory showed that a SPK is observed in the absence of Mlc1, yet, in such mutants the filaments are wider and extend faster (Puerner et al., 2021b). This SPK is present in C. albicans hyphal cells during all stages of the cell cycle, including septum formation (Crampin et al., 2005).

Perturbation of actin cables or secretion disrupted the Spitzenkörper (Crampin et al., 2005;[START_REF] Jones | Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties[END_REF][START_REF] Li | Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation[END_REF][START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF], its integrity and polarized localization are also dependent on the polarisome member Spa2 and Bud6 (Crampin et al., 2005). In C. albicans, as in other fungi, the absence of SPK results in reduced filamentous growth (Puerner et al., 2021b;[START_REF] Zheng | Spitzenkörper assembly mechanisms reveal conserved features of fungal and metazoan polarity scaffolds[END_REF]. Interestingly, a cluster of vesicles that has all the components present in the SPK is observed in the absence of tip growth and is highly dynamic moving by hops and jumps, suggesting that such a cluster can form in the absence of directional growth (Silva et al., 2019).

.

The actin cytoskeleton:

In filamentous fungi, actin cytoskeleton and microtubules are keys for material delivery to the growing apex [START_REF] Riquelme | Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures[END_REF][START_REF] Takeshita | Coordinated process of polarized growth in filamentous fungi[END_REF]. In C. albicans, drugs such as latrunculin A or cytochalasin A, which inhibit actin polymerization, alter filamentous growth (Hazan and Liu, 2002;[START_REF] Jones | Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties[END_REF]. In filamentous fungi, such as A. nidulans, secretory vesicles transport requires microtubules [START_REF] Riquelme | Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures[END_REF]. This long-distance transport is not critical in C. albicans, where microtubules are dispensable for hyphal formation [START_REF] Rida | Yeast-to-hyphal transition triggers formin-dependent Golgi localization to the growing tip in Candida albicans[END_REF]. Instead, in C. albicans, it is suggested that the secretory pathway organization, with the ER extending along the filament, a sub-apical Golgi and a SPK comprised of vesicles of uniform size, facilitates short-range delivery [START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF] (Figure 9).

The actin cytoskeleton is comprised of 3 principal structures.(i) The cortical actin patches form a subapical ring during hyphal development in A. nidulans and N. crassa [START_REF] Araujo-Bazán | Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans[END_REF][START_REF] Berepiki | Actin organization and dynamics in filamentous fungi[END_REF][START_REF] Delgado-Álvarez | Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa[END_REF][START_REF] Upadhyay | The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans[END_REF]. In C. albicans, a collar 2 micrometers back from the filament tip was observed (Ghugtyal et al., 2015;[START_REF] Sudbery | Growth of Candida albicans hyphae[END_REF]. The actin-binding protein Abp1, which is implicated in clathrin mediated endocytosis (Epp et al., 2013a;Ghugtyal et al., 2015), localizes to this endocytic collar, yet ABP1 is not essential and hyphal morphogenesis was not altered in abp1 deletion mutants [START_REF] Caballero-Lima | The spatial distribution of the exocyst and actin cortical patches is sufficient to organize hyphal tip growth[END_REF]Ghugtyal et al., 2015;[START_REF] Martin | Functional analysis of Candida albicans genes whose Saccharomyces cerevisiae homologues are involved in endocytosis[END_REF]. In contrast, the AP-2 endocytic adaptor complex is required for C. albicans hyphal morphology and the recycling of the chitin synthase Chs3 [START_REF] Chapa-Y-Lazo | Yeast endocytic adaptor AP-2 binds the stress sensor Mid2 and functions in polarized cell responses[END_REF][START_REF] Knafler | AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans[END_REF]. The Arp2/3 (Actin Related Protein) complex is also required for C. albicans hyphal formation but, in contrast to S. cerevisiae, not for viability, indicating that actin-driven polymerization during endocytosis can occur via Arp2/3-independent routes (Epp et al., 2013a[START_REF] Epp | Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis[END_REF]. (ii) The actin cables are also independent of Arp2/3 in C. albicans (Epp et al., 2013a[START_REF] Epp | Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis[END_REF]. It was recently shown in C. albicans that the S. cerevisiae Aip5 homolog binds G-actin, Bni1, and Bud6 to form the nucleation complex to polymerize F-actin [START_REF] Xie | Orchestrated actin nucleation by the Candida albicans polarisome complex enables filamentous growth[END_REF]. Beside Bni1, another formin is present in C. albicans, Bnr1, which functions at the division site and is partially redundant with Bni1 (Crampin et al., 2005;[START_REF] Dünkler | Candida albicans Rho-type GTPase-encoding genes required for polarized cell growth and cell separation[END_REF][START_REF] Martin | Ras1-Induced Hyphal Development in Candida albicans Requires the Formin Bni1[END_REF]. Actin cables, which serve as rails for the transport of organelles such as secretory vesicles and the Golgi, are disrupted with specific drugs, resulting in a growth inhibition and in a blocked filamentation (Crampin et al., 2005;Hazan and Liu, 2002;[START_REF] Jones | Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties[END_REF][START_REF] Pointer | Boric acid destabilizes the hyphal cytoskeleton and inhibits invasive growth of Candida albicans[END_REF].

This filament arrest was concomitant with a disruption of Cdc42 localization, which was not observed in budding growth after latrunculin A treatment (Hazan and Liu, 2002), suggesting a specific requirement of actin cytoskeleton for Cdc42 localization during hyphal growth (Hazan and Liu, 2002). (iii) The actomyosin ring, which is a contractile structure composed of actin and myosin II in S. cerevisiae [START_REF] Cheffings | Actomyosin Ring Formation and Tension Generation in Eukaryotic Cytokinesis[END_REF]. Myosin II and I are regulated by the Myosin light chain (Mlc1) protein (Stevens and Davis, 1998). In S. cerevisiae, Mlc1 is recruited at the site of cytokinesis and interacts with Myo1 [START_REF] Boyne | Yeast myosin light chain, Mlc1p, interacts with both IQGAP and class II myosin to effect cytokinesis[END_REF], where it functions in parallel with the septins (Graziano et al., 2014). In C. albicans, Mlc1 is recruited at the SPK and also at the actomyosin ring (Crampin et al., 2005). In the mlc1 mutant, unfinished cytokinesis results in the formation of chains of cells (Puerner et al., 2021b). In contrast, mlc1 mutant has a faster hyphal extension rate than a WT strains, even though the cell were wider (Puerner et al., 2021b). In C. albicans, the ER (shown in brown) and the Trans Golgi Network (TGN; depicted as green spheres) extend to the hyphal tip to constitute an "on-site secretory vesicles delivery" by which vesicles are locally budded and delivered, resulting in a short-range trafficking within the tip region [START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF]. Secretory vesicles (represented as grey spheres), loaded with the Rab-type GTPase Sec4 and the GEF Sec2, are transported on actin cables (black strings) by the class V myosin Myo2/Mlc1 complex and accumulate at the Spitzenkörper (SPK) located at the hyphal tip. Actin cable nucleation is generated by the formin Bni1, which is assisted by the polarisome composed by Spea2, Aip5 and Bud6 (molecular components of this structure are schematized in the inset magnification). From the SPK, the vesicles are delivered to the PM. The exocyst, which is composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84, participates in vesicle docking before fusion. Endocytosis takes place at the endocytosis collar, an accumulation of actin patches (red rounds in the scheme) located 1-3µm sub-apically from the apex. Based on models from [START_REF] Sudbery | Growth of Candida albicans hyphae[END_REF][START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF]

Lipids in C. albicans.

The major lipids in fungi, based on S. cerevisiae, are sphingolipids, sterols and glycerophospholipids [START_REF] Klug | Yeast lipid metabolism at a glance[END_REF]; the latter group includes the phosphatidic acid (PA) and phosphatidylcholine (PC), which are less abundant than phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and its phosphorylated derivatives (PIPs), these three latest are ~80% of phospholipids at the PM. Phosphatidylinositol-4-Phosphate (PI(4)P) and phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) at the plasma membrane result from the phosphorylation of PI by the single kinases Stt4 and Mss4 [START_REF] Cutler | STT4 is an essential phosphatidylinositol 4kinase that is a target of wortmannin in Saccharomyces cerevisiae[END_REF][START_REF] Desrivières | MSS4, a Phosphatidylinositol-4phosphate 5-Kinase Required for Organization of the Actin Cytoskeleton in Saccharomyces cerevisiae *[END_REF][START_REF] Homma | Phosphatidylinositol-4-phosphate 5-kinase localized on the plasma membrane is essential for yeast cell morphogenesis[END_REF][START_REF] Trotter | A genetic screen for aminophospholipid transport mutants identifies the phosphatidylinositol 4-kinase, STT4p, as an essential component in phosphatidylserine metabolism[END_REF][START_REF] Yoshida | A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae[END_REF], while Golgi PI(4)P is produced by another kinase, Pik1 [START_REF] Flanagan | Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability[END_REF][START_REF] Garcia-Bustos | PIK1, an essential phosphatidylinositol 4-kinase associated with the yeast nucleus[END_REF]. A single synthase, encoded by the CHO1 gene, converts CDP-DAG (CDPdiacylglycerol) to PS (K. Atkinson et al., 1980;K. D. Atkinson et al., 1980;[START_REF] Letts | Isolation of the yeast structural gene for the membrane-associated enzyme phosphatidylserine synthase[END_REF].

These enzymes are conserved among fungi, with some exceptions, as an example, A. nidulans and Cryptococcus neoformans appear to have a Stt4 PI-4-kinase that is essential for viability, however, Pik1 orthologs are absents (De Souza et al., 2013;[START_REF] Cassilly | SB-224289 Antagonizes the Antifungal Mechanism of the Marine Depsipeptide Papuamide A[END_REF].

During C. albicans yeast-to-hyphal transition, lipids, especially phospholipids and ergosterol, are redistributed and enriched at the growing apex (Ghugtyal et al., 2015;Labbaoui et al., 2017;Martin and Konopka, 2004a;Vernay et al., 2012). The enzymes abovementioned implicated in phospholipid synthesis are also present in C. albicans (Figure 10), and their downregulation has a very deleterious effect in hyphal growth (Chen et al., 2010;Ghugtyal et al., 2015;Vernay et al., 2012). Suggesting an essential role of phospholipids in C. albicans morphogenic transition (Chen et al., 2010;Ghugtyal et al., 2015;Vernay et al., 2012).

Ergosterol is the major sterol in the budding yeast and ergosterol synthesis is a target for antifungal agents (Figure 11) [START_REF] Bhattacharya | Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility in Saccharomyces cerevisiae[END_REF]. Equivalent to cholesterol in mammals, ergosterol concentration in the ER is low but increases along the secretory pathway up to the PM [START_REF] Klug | Yeast lipid metabolism at a glance[END_REF]. In S. cerevisiae, 25 known ER-enzymes encoded by the ERG genes regulate ergosterol biosynthesis [START_REF] Bhattacharya | Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility in Saccharomyces cerevisiae[END_REF]. Misregulation of these enzymes results in altered susceptibilities to antifungal drugs such as fluconazole [START_REF] Bhattacharya | Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility in Saccharomyces cerevisiae[END_REF]. Fluconazole blocks 14-α-dymethylation of lanosterol in the ergosterol biosynthetic pathway [START_REF] Bhattacharya | Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility in Saccharomyces cerevisiae[END_REF].

▪ Lipid function in C. albicans:

In C. albicans, blocking the synthesis of ergosterol and sphingolipid results in a filamentous growth defect (Martin and Konopka, 2004a). Ergosterol filament apex polarization is dependent on the AP-2 complex implicated in clathrin-dependent endocytosis [START_REF] Knafler | AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans[END_REF], which supports the idea of the "raftophilic" cargo-sorting function of lipids rafts during vesicular transport [START_REF] Alvarez | Sterol-Rich Plasma Membrane Domains in Fungi[END_REF]Martin and Konopka, 2004b). Similar to ergosterol, PI(4)P, PI(4,5)P2, and PS are dramatically enriched at the end of filament (Ghugtyal et al., 2015;Labbaoui et al., 2017;Vernay et al., 2012) and the enzymes implicated in their synthesis are essential for C. albicans morphogenesis, as the repression or deletion of these enzymes impairs filamentous growth (Chen et al., 2010;Ghugtyal et al., 2015;Vernay et al., 2012). These phospholipids are important to localize and/or stabilize key components for polarized growth in fungi:

-PI(4,5)P2. In S. cerevisiae, this lipid is important for the function and proper localization of proteins such as the MAPK scaffold Ste5 during formation of mating projections [START_REF] Garrenton | Pheromoneinduced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling[END_REF][START_REF] Garrenton | Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain[END_REF] and invasive growth (Adhikari and Cullen, 2015;[START_REF] Guillas | Phosphatidylinositol 4,5bisphosphate is required for invasive growth in Saccharomyces cerevisiae[END_REF]. In C. albicans, during hyphal growth, PI [START_REF] Haupt | Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast[END_REF][START_REF] Kay | Distribution, dynamics and functional roles of phosphatidylserine within the cell[END_REF]P2 forms a steep gradient with the highest concentration at the subapical zone of the filament. This gradient is generated and maintained by a local production at the tip, where the PI-4 kinase Mss4 is localized (Vernay et al., 2012) and by clearing of PI [START_REF] Haupt | Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast[END_REF][START_REF] Kay | Distribution, dynamics and functional roles of phosphatidylserine within the cell[END_REF]P2 at the back of the cell. As the levels of PI [START_REF] Haupt | Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast[END_REF][START_REF] Kay | Distribution, dynamics and functional roles of phosphatidylserine within the cell[END_REF]P2 decrease in the rest of the cell, this lipid gets accumulated at the germ tube tip (Vernay et al., 2012). The downregulation of Mss4 impairs yeast-to-hyphal transition. In addition, this kinase is required to stabilize active Rho1 at the growth site during budding growth [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF]. These data indicate a close functional link between phospholipids and the localization of key proteins regulating hyphal morphogenesis.

-PI(4)P. In S. cerevisiae, PI(4)P, which is found principally at the Golgi and plasma membranes, participates in vesicle budding, maturation and fusion [START_REF] Bard | The formation of TGN-to-plasma-membrane transport carriers[END_REF][START_REF] Moser Von Filseck | Running up that hill: How to create cellular lipid gradients by lipid counter-flows[END_REF][START_REF] Smindak | Lipid-dependent regulation of exocytosis in S. cerevisiae by OSBP homolog (Osh) 4[END_REF]. PI(4)P facilitates the recruitment of proteins implicated in polarized growth, such as the Cdc42 effector, Cla4 [START_REF] Wild | The p21-activated Protein Kinaserelated Kinase Cla4 Is a Coincidence Detector of Signaling by Cdc42 and Phosphatidylinositol 4-Phosphate*[END_REF]. As in S. cerevisiae, there are two functionally independent pools of PI( 4)P in C. albicans, one at the Golgi and another one at the PM [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF]Ghugtyal et al., 2015;[START_REF] Santiago-Tirado | PI4P and Rab inputs collaborate in myosin-V-dependent transport of secretory compartments in yeast[END_REF]. Golgi PI(4)P levels in C. albicans are important for vesicular traffic, as down-regulation of the Pik1 kinase alters the number of Golgi cisternae and their dynamics (Ghugtyal et al., 2015). Indeed, the pik1 repressible mutant shows reduced Golgi PI(4)P levels when PIK1 gene is downregulated, which results in perturbed secretory vesicle budding and decreased secretory vesicle targeting to the site of growth (Ghugtyal et al., 2015). PM PI(4)P was observed as a tight cap at the hyphal filament tip (Ghugtyal et al., 2015). This polarized localization has been proposed to be maintained by the action of phosphatases, such as Sac1, rather than by endocytic recycling (Ghugtyal et al., 2015). Both PI(4,5)P2 and PI(4)P shows very polarized distribution (Ghugtyal et al., 2015;Vernay et al., 2012). However, a less dramatic defect in filamentation is observed upon reduction of the PI-4-kinase Stt4, responsible for the synthesis of the PI( 4)P pool at PM (Vernay et al., 2012), raising questions about the dispensability of this pool for the yeast-to-hyphal transition. -PS: Phospholipids are important for membrane electrostatic properties (Lenoir et al., 2021).

As introduced above, PS is the most abundant among the anionic phospholipids at the PM and, together with PI(4)P, it is enriched at the cytosolic leaflet from late secretory organelles to the PM in S. cerevisiae [START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF][START_REF] Klug | Yeast lipid metabolism at a glance[END_REF]Lenoir et al., 2021). In S. cerevisiae, deletion of the unique PS synthase Cho1 impairs polarized endocytosis [START_REF] Sun | The functions of anionic phospholipids during clathrin-mediated endocytosis site initiation and vesicle formation[END_REF]. PS is critical for Cdc42 and Bem1 polarized localization in S. cerevisiae (Fairn et al., 2011;Meca et al., 2019) and for Rho1 localization in S. pombe (Haupt and Minc, 2017). However, in S. cerevisiae cell viability is not affected in the cho1 mutant, indicating that the essential function of Cdc42 is not compromised upon Cho1 loss (Fairn et al., 2011;[START_REF] Sun | The functions of anionic phospholipids during clathrin-mediated endocytosis site initiation and vesicle formation[END_REF]. Together, these results suggest that the interactions between the PM and Bem1 is based on more general lipid properties. In fact, it was shown that Bem1 localization can be partially rescued by synergic lipid-protein interactions between Bem1 and other anionic lipids such as PI(4)P and PI(4,5)P2 [START_REF] Meca | Mlc1 plays a role in regulating cell robustness necessary for invasive growth (Puerner et al[END_REF]. In C. albicans, an enriched distribution of PS is observed at the hyphal apex (Labbaoui et al., 2017) and deletion of the PS synthase Cho1 results in a reduced ability to form filaments and in an altered cell wall structure (Chen et al., 2010;[START_REF] Davis | Masking of β(1-3)-glucan in the cell wall of Candida albicans from detection by innate immune cells depends on phosphatidylserine[END_REF].

Indeed, the cho1 mutant shows an increased cell wall thickness (Chen et al., 2010) andβ(1-3)-glucan unmasking [START_REF] Davis | Masking of β(1-3)-glucan in the cell wall of Candida albicans from detection by innate immune cells depends on phosphatidylserine[END_REF]. Furthermore, mutants lacking Cho1, or the PS decarboxylases (Psd1 and Psd2) have a similar compromised virulence in murine models (Chen et al., 2010;[START_REF] Davis | Candida albicans Cannot Acquire Sufficient Ethanolamine from the Host To Support Virulence in the Absence of De Novo Phosphatidylethanolamine Synthesis[END_REF]. Both cho1 and psd1/2 mutant virulence were rescued after providing these mutants with the capacity to capture higher concentrations of ethanolamine [START_REF] Davis | Candida albicans Cannot Acquire Sufficient Ethanolamine from the Host To Support Virulence in the Absence of De Novo Phosphatidylethanolamine Synthesis[END_REF]. However, the rescue of the cho1 mutant was not due to PE itself, but to a reverse Psd1/2 enzymatic reaction that produce PS when the concentration of PE is high enough [START_REF] Davis | Candida albicans Cannot Acquire Sufficient Ethanolamine from the Host To Support Virulence in the Absence of De Novo Phosphatidylethanolamine Synthesis[END_REF]. Recent work has shown that the absence of PS in the cho1 mutant improved copper-stress resistance (Douglas and Konopka, 2019). As copper level increase is an immune response against pathogens, these results suggest a critical role for PS in C. albicans pathogenesis and virulence.

▪ The P4-ATPase Drs2:

PS asymmetric distribution with strong enrichment at the cytosolic leaflet is regulated by proteins from the P4-ATPase family (Hankins et al., 2015a;Lenoir et al., 2021;[START_REF] Lopez-Marques | P4-ATPases: lipid flippases in cell membranes[END_REF]Pomorski et al., 2003;[START_REF] Roland | Decoding P4-ATPase substrate interactions[END_REF](See section II.3. Membrane asymmetry). One of the five P4-ATPases in S. cerevisiae, Drs2, was identified, together with 6 others (Drs1-Drs7), in a deficient in ribosomal subunits mutant screening [START_REF] Ripmaster | DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae[END_REF], and was visualized for the first time at the Golgi, co-localizing with another P4-ATPase Dnf3 (Chen et al., 1999;Hua et al., 2002).

Among the five genes encoding the P4-ATPases, a certain degree of redundancy has been demonstrated for the DRS2/DNF genes, yet NEO1 is the sole required for cell viability in S. cerevisiae (Hua et al., 2002). Although Drs2 and Neo1 transport PS and PE, little to none functional overlap has been shown [START_REF] Takar | The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane[END_REF]. Nonetheless, overexpression of NEO1 can suppress a cold sensitivity defect of the cdc50 mutant (Saito et al., 2004) and Drs2 and Neo1 are redundant in the absence of the PQ-loop protein Any1 [START_REF] Takar | The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry[END_REF], suggesting a segregation of Drs2 and Neo1 function induced by the Any1-Neo1 interaction [START_REF] Takar | The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry[END_REF]. In contrast, in filamentous fungi, Neo1 homologs are not essential. The A. nidulans homologous, DnfD, which localizes to the late Golgi, is also dispensable for viability and necessary for conidia formation [START_REF] Schultzhaus | The phospholipid flippase DnfD localizes to late Golgi and is involved in asexual differentiation in Aspergillus nidulans[END_REF]. In C albicans, the neo1 mutant is also viable and has an increased sensitivity to duramycin, indicating a defect in PE asymmetry at the PM (Douglas and Konopka, 2019).

Drs2 was associated with clathrin-dependent vesicle budding (Chen et al., 1999), protein transport in clathrin-dependent vesicles from TGN to the PM, and AP3-dependent budding to the vacuole in S. cerevisiae [START_REF] Gall | Drs2p-Dependent Formation of Exocytic Clathrin-Coated Vesicles In Vivo[END_REF]Hua et al., 2002). Drs2 is necessary for Golgi organization. The drs2 mutant, similar to clathrin mutants, accumulates aberrant Golgi structures called "Berkeley bodies" (Chen et al., 1999), and has a reduced number of post-Golgi exocytic vesicles [START_REF] Gall | Drs2p-Dependent Formation of Exocytic Clathrin-Coated Vesicles In Vivo[END_REF]; Moreover, Drs2 participates in the generation of membrane curvature in the TGN/Early endosome membranes [START_REF] Xu | Phosphatidylserine flipping enhances membrane curvature and negative charge required for vesicular transport[END_REF] and in the recruitment of the golgin Imh1in S. cerevisiae [START_REF] Tsai | Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p[END_REF]. Drs2, the GEF Gea2 and its effector Arl1 form a complex regulated by PI(4)P, that is implicated in the activation of Drs2 and in budding of clathrin vesicles associated to AP1 adaptor protein [START_REF] Chantalat | The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae[END_REF][START_REF] Natarajan | Regulation of a Golgi flippase by phosphoinositides and an ArfGEF[END_REF]. PM-proteins Pma1 and Can1 sorting and localization of ergosterol are restored after deletion of the OSBP-homologous protein, Osh4, in the drs2 mutant (Hankins et al., 2015b;Muthusamy et al., 2009). Thus, lipid homeostasis seems to be more important that the presence of the lipid transporters per se (Hankins et al., 2015b;Muthusamy et al., 2009).

The role of P4-ATPases has been previously investigated in filamentous fungi. In A. nidulans, PS distribution is dependent on the flippases DnfA and DnfB, the respective homologues of Dnf1 and Drs2 [START_REF] Schultzhaus | Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase[END_REF]. Enriched at the SPK, PS shows a very polarized distribution at the hyphal site of growth, in contrast to its homogeneous distribution in subapical regions or PM of nongrowing cells. Thus, PS distribution correlates with DnfA/B localization.

Consistently, the absence of DnfA reduces PS localization to the DnfB-loaded microvesicles [START_REF] Schultzhaus | Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase[END_REF]. DnfA/B have been implicated in endocytosis in A. nidulans and F. graminearum, as they interact with the AP-2 complex [START_REF] Martzoukou | The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi[END_REF]Zhang et al., 2019), which is essential for hyphal maintenance in fungi, included C. albicans [START_REF] Knafler | AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans[END_REF].

The regulation of these P4-ATPases by their non-catalytic subunits is conserved and essential for a correct function. Indeed, the deletion of the Lem3/Cdc50 members phenocopies the flippase deleted mutants [START_REF] Chen | Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane[END_REF][START_REF] Kato | A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma membrane in Saccharomyces cerevisiae[END_REF][START_REF] Schultzhaus | Phospholipid flippases DnfA and DnfB exhibit differential dynamics within the A. nidulans Spitzenkörper[END_REF]Xu et al., 2019). In A. nidulans, Cdc50 deletion impairs viability [START_REF] Schultzhaus | Phospholipid flippases DnfA and DnfB exhibit differential dynamics within the A. nidulans Spitzenkörper[END_REF]. A similar defect was observed upon deletion of both DnfA and DnfB [START_REF] Schultzhaus | Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase[END_REF], suggesting that Cdc50 regulates both flippases [START_REF] Schultzhaus | Phospholipid flippases DnfA and DnfB exhibit differential dynamics within the A. nidulans Spitzenkörper[END_REF]. In Cryptococcus neoformans, the Drs2 homologue Apt1 can complement a drs2 mutant of Saccharomyces cerevisiae [START_REF] Hu | A putative P-type ATPase, Apt1, is involved in stress tolerance and virulence in Cryptococcus neoformans[END_REF]. Moreover, based on phenotypic similarities of the cdc50 and apt1 mutants, Cdc50 has been proposed to function together with Apt1 to contribute to flippase function [START_REF] Hu | A P4-ATPase subunit of the Cdc50 family plays a role in iron acquisition and virulence in Cryptococcus neoformans[END_REF][START_REF] Hu | A putative P-type ATPase, Apt1, is involved in stress tolerance and virulence in Cryptococcus neoformans[END_REF]. In C. albicans, deletion of DRS2 results in defect of filamentous growth, altered distribution of PS and hypersensitivity to fluconazole (Labbaoui et al., 2017). A similar phenotype has been reported for a cdc50 mutant (Xu et al., 2019). In addition, in a drs2 deletion mutant, sensitivity to copper is increased and this phenotype is more drastic than that of a dnf1 or neo1 mutant (Douglas and Konopka, 2019), indicating a role of PS in copper sensitivity.

THESIS OBJECTIVES

Candida albicans hyphal growth is associated with a filament tip enrichment of lipids, including ergosterol, the phosphorylated derivatives of phosphatidylinositol (PI(4)P, PI(4,5)P2) and phosphatidylserine (PS). Deletion of the lipid flippase Drs2 results in altered distribution of PS and defect in hyphal growth, when C. albicans is exposed to serum. In contrast, budding growth is not affected. The aim of my project was to determine the function of Drs2 in hyphal growth and the importance of PS asymmetry in this process. To this end, I localized Drs2 during hyphal growth and further characterized the drs2 deletion mutant by analyzing the distribution of different lipids and key regulators of hyphal growth, such as active Cdc42. I also analyzed mutants analogous to those in S. cerevisiae that are altered for PS flippase activity. In addition, I investigated the importance of other flippases in hyphal invasive growth, as well as that of Oxysterol-binding proteins, using loss-of-function mutants. Furthermore, I examined potential genetic interactions between these transporter encoding genes and DRS2.

Growth conditions

The media and buffer composition used for this work are illustrated in Table 2.

-Budding growth: The yeasts are grown at 30°C in a rich (YEPD: Yeast extract peptone dextrose) or selective synthetic media, in liquid or solid media containing 2% agar. The effect of different compounds is tested on YEPD agar medium in the presence of 50mM of PIPES buffer, at pH 5.5 containing:

o Cell wall perturbants: Congo red (400 μg/ml), Calcofluor white (25 μg/ml) o Antifungal agents: fluconazole (10 μg / ml), caspofungin (125 ng / ml), Amphotericin B (3μg / ml).

o Toxins: papuamide A (1μg / ml), duramycin (50 μg/ml) Serial dilutions of cells in exponential growth [OD600nm= 0.6-08] are spotted (5-7μl of culture) and incubated at 30°C for 2 to 6 days.

-Filamentous growth: Filamentation is induced in liquid or agar containing media in the presence of Serum (FCS, fetal calf serum), in general 50% (Bassilana et al., 2005) or in low carbon source containing media (Spider medium) [START_REF] Calera | Defective hyphal development and avirulence caused by a deletion of the SSK1 response regulator gene in Candida albicans[END_REF].

o For filamentous growth in liquid media, the cells in exponential growth (DO600nm=0.6) are incubated with 50% FCS at 37ºC for 45, 60, 90 or 120 minutes.

o For filamentous growth in 2% agar and 50% FCS containing media, the colonies are visualized after 4 to 7 days of incubation at 30°C. o For time-lapse experiments, cells are added on 0.5% agar and 75% FCS containing media on microscope slides (pads) (Bassilana et al., 2005).

Cells at OD600nm 0.4 are added to FCS in 1:1 ratio.

-Growth of bacteria: The bacteria are grown at 37°C in rich media (TYE, Table 2) or selective (TYE Amp, Table 2) in liquid or 2% agar containing media.

Plasmid construction

The plasmids generated are illustrated in Table 3.

Plasmids pExpARG4:

-For plasmids containing DRS2 and OSH4 under the control of their own promoter, genomic DNA fragments containing the ORFs (Open Reading Frame) and 1000 base pairs upstream and downstream were amplified by PCR, using the oligonucleotide pairs CaDrs2pup1000SalI/CaDrs2m5963NotI and CaOsh4pup1000SalI/CaOsh4m3268NotI (Table 4), which contain the unique restriction sites, SalI and NotI in 5' and 3', respectively. The amplification was done using Phusion DNA polymerase and the Thermocycler Biometra Trio (Labbaoui et al., 2017), generating pExpARG4pDRS2DRS2 and pExpARG4pOSH4OSH4.

-Single directed mutagenesis (SDM) was used to introduce mutations (Drs2 F511Y and Drs2 QQ ) in Drs2, similar to that specifically altered phosphatidylserine flipping in S. cerevisiae (Baldridge and Graham, 2012). The plasmids pExpARG4pDRS2DRS2 F511Y and pExpARG4pADH1DRS2 F511Y were generated from pExpARG4pDRS2DRS2 and pExpARG4pADH1DRS2 (Labbaoui et al., 2017), respectively. The mutation F511Y

corresponds by sequence alignment to the mutation F520Y. In this work, the mutation F520Y will be called F511Y by analogy with S. cerevisiae.

The Primers CaDrs2F511Yp/ CaDrs2F511Ym (Table 4) are used to introduce this mutation, as well as a silent mutation for a KpnI restriction site, allowing to screen the transformants.

The SDM is done using the Pfu DNA polymerase, according to the program:

1. 95°C 30 sec This step is necessary to reduce the transformation background, as the plasmid that served as a matrix, unlike PCR products, is methylated. The program used is as follows:

1. 37°C 1h 2. 68°C 20min

3. 10ºC pause 4μL of product are used to transform competent XL1-Blue E. coli cells prepared by the calcium chloride method [START_REF] Inoue | High efficiency transformation of Escherichia coli with plasmids[END_REF]) (section "PLASMID AMPLIFICATION").

To verify the presence of the mutation in DRS2, after plasmid amplification a first screening is carried out using primers CaDrs2p1336 / CaDrs2m1732 to amplify a fragment of about 400pb containing the silent restriction site KpnI. Plasmids containing KpnI digested fragments were confirmed by sequencing (Eurofins MWG Operon, Esbersberg, Germany).

A similar procedure was used to generate pExpARG4pDRS2DRS2 QQ , pExpARG4pADH1DRS2 QQ . For the SDM we used primers CaDrs2QQp691/CaDrs2QQm755

to mutate in GA→QQ (Baldridge and Graham, 2013), and to introduce a silent deletion of a restriction site ClaI. To verify the presence of the mutation in DRS2, we used primers CaDrs2p446/CaDrs2m940 to amplify a fragment that we digested with ClaI. Plasmids containing fragments that were not digested with ClaI were confirmed by sequencing.

-To visualize the distribution of ergosterol, we used the genetically encoded biosensor D4H [Marek et al., 2020]. D4H was amplified from plasmid pSM2244 (Marek et al., 2020),

using

Plasmids pDup:

To select cell transformation by plasmids pDup3 and pDup5 (Gerami-Nejad et al., 2013), the Nourseothricin resistance gene SAT1 or the auxotrophy marker URA3 were used, respectively.

-Plasmids pDup3pDRS2DRS2 and pDup3pDRS2DRS2 F511Y were generated from pExpARG4pDRS2DRS2, pExpARG4PpDRS2DRS2 F511Y, respectively, after PCR amplification of a fragment containing the ORF and 1000 bp upstream and downstream using the oligonucleotide pair CaDrs2pup1000SpeI / CaDrs2m5963NotI (Table 4). PCR products digested by SpeI and NotI were inserted into pDup3 (Gerami-Nejad et al., 2013).

-For plasmids overexpression, the ORFs DRS2, OSH4 and DNF2 were amplified using primers CaDRS2p1AscI / CaDRS24963mMluI, CaOSH4p1AscI / CaOSH4m1293MluI, CaDNF2p1AscI / CaDNF2m5151MluI, which have the unique restriction sites MluI and AscI in 5 'and 3 ', respectively. After amplification as above, the PCR products were digested by MluI / AscI and inserted into the plasmid pDup3pADH1mSCARLETctRAC1tADH (Silva et al., 2019). Similar procedure was used to generate the plasmid pDup3pADH1-GFPLactC2 after GFP-LactC2 PCR amplification with primers LactC2mMluI / yeGFP3pAscI from pExpArgpACT1GFPyeLactC2 (Labbaoui et al., 2017).

-For the construction of plasmids containing pDNF2DNF2 (6Kb), PCR amplification was done in two steps: first, the pDNF2 promoter was amplified from gDNA, using primers CaDnf2pup1000XmaI / CaDnf2mup21AscI, as in section "Plasmids pExpARG4".PCR All constructions were sequenced by Eurofins MWG Operon in Esbersberg, Germany.

Plasmid amplification

The amplification of plasmids was done by transformation of competent XL1-Blue E. coli cells prepared by the calcium chloride method [START_REF] Inoue | High efficiency transformation of Escherichia coli with plasmids[END_REF]. About 0.2 μg of plasmid DNA are mixed with 100 μl of competent bacteria and subjected to:

-A heat shock: The mixture is put on ice for 10 minutes, then switch to 42°C for 2 minutes and on the ice for 2 to 5 minutes.

-An incubation period to express beta-lactamase: An equivalent volume of TYE medium is added and the suspension is incubated at 37°C for one hour to express beta-lactamase, an enzyme capable of degrading antibiotics containing the beta-lactam cycle, as is the case of ampicillin.

-The mixture is then spread on agar containing TYE + ampicillin (100μg/ml) media and incubated at 37°C for 12 hours.

Plasmid were extracted using a NucleoSpin Plasmid Kit (Macherey-Nangel) from the colonies grown on ampicillin.

Strain construction

Transformation of C. albicans: electroporation

-A colony of C. albicans is inoculated in 5 ml of YEPD + Uri and grown at 30°C overnight. This preculture is diluted 10 times in YEPD + Uri and grown again to a DO600 of 2-4.

-

The suspension is centrifuged at 3000 rpm, 3 min at 4ºC. The pellet is resuspended in 8 ml of H2O + 1 ml of lithium acetate 1M + 1 ml of TE 10X (100mMTris-HCL, 10mM EDTA, pH7.5), incubated at 30ºC, 45min, before adding 250μl of DTT 1M and incubation at 30ºC for 15 min.

-Cells are then submitted to 3 wash steps: first with 50 ml of iced water, then 25 ml of iced water and finally 10 ml of iced sorbitol 1M. The final pellet is resuspended in 100μl of sorbitol. To 40 μl of these competent cells are added 5μg of the DNA of interest.

-After electroporation at 1800V, the cells are centrifuged (1.5 min at 3000 rpm)

and resuspended in 400 μl of LM2 before inoculation on selective medium. When an antibiotic resistance gene is used (Nourseothricin or Hygromycin), 1ml of YEPD + Uri is added instead with additional incubation at 30ºC for 4 hours, followed by. centrifugation (1.5 min at 3000 rpm) and resuspension in 400μl of LM2 as before.

-Colonies appear 2-3 days after incubation at 30ºC.

Generation of C. albicans strains

•

Loss of function Mutants

The mutants ∆dnf1/∆dnf1 (dnf1), ∆dnf2/∆dnf2 (dnf2) ∆dnf3/∆dnf3 (dnf3), ∆osh2 /∆osh2 (osh2), ∆osh3 /∆osh3 (osh3), ∆osh4 /∆osh4 (osh4), ∆osh7 /∆osh7 (osh7), and

∆drs2/∆drs2∆osh2/∆osh2 (drs2osh2), ∆drs2/∆drs2∆osh3/∆osh3 (drs2osh3)
∆drs2/∆drs2∆osh4/∆osh4 (drs2osh4), ∆drs2/∆drs2∆osh7/∆osh7 (drs2osh7), as well as intermediate heterozygous strains (Table 7) were generated by homologous recombination from the original strain BWP17 (Wilson et al., 1999) (see primers for KO mutants in Table 5).

The mutant ∆drs2/∆drs2 (drs2) was previously generated (Labbaoui et al., 2017).

o For the mutants osh2, osh3, osh4, the two copies of the genes were replaced sequentially by the auxotrophy markers HIS1 and URA3. These markers were amplified by PCR from plasmids pGEMHIS1, pGEMURA3 (Wilson et al., 1999) using the primers CaOSH2mKO/CaOSH2pKO, CaOSH3mKO/CaOSH3pKO and CaOSH4mKO/CaOSH4pKO, respectively.

o For the mutant osh7, a copy of OSH7 was replaced by SAT1 [START_REF] Basso | Transformation of Candida albicans with a synthetic hygromycin B resistance gene[END_REF]Schaub et al., 2006) and the other by HIS1, amplified from plasmids pFa-SAT1 (Schaub et al., 2006) and pGEM-HIS1, using primer pairs, CaOsh7alphKOS1/CaOsh7alphKOS2 and CaOSH7A.mKO/CaOSH7A.pKO, respectively.

o

The dnf1 mutant was generated by replacing the two alleles with SAT1 and HYGB (which allows resistance to Hygromycin) amplified from pFA-SAT1 and pHB1S (Lai et al., 2016) using primers CaDNF1.S1pKO/ CaDNF1.S2mKO.

Similar deletion of DNF1 in the mutant dnf2 resulted in the dnf1dnf2 mutant.

o

The dnf2 mutant was generated by replacing the two alleles with ARG4 and HIS1 amplified from pGEM-cdARG4 (Bijlani et al., 2019) and pFA-HIS1 using primers CaDNF2mKO-pGEM/CaDNF2pKO-pGEM and CaDNF2.S1pKO/ CaDNF2KOS2, respectively.

o

The dnf3 mutant was generated by replacing the two alleles with SAT1 and URA3 amplified from pFA-SAT1 or pFA-URA3 using primers CaDNF3.S1pKO/CaDNF3.S2mKO.

The double-mutants drs2osh2, drs2osh3, drs2osh4, drs2osh7 and drs2sac1 were generated from the mutant drs2 (Labbaoui et al., 2017), in which URA3 was previously replaced by SAT1:

SAT1 was amplified from pFa-SAT1 using primers CaURAexchS1/CaURAexchS2. Then, the two copies of OSH2, OSH3, OSH4, OSH7a/α or SAC1 were sequentially replaced by URA3, ARG4 or HYGB, using pGemURA3 and primers CaOSH4mKO/CaOSH4pKO or CaOSH7A.mKO/CaOSH7A.pKO, or pFa-ARG4 and pFa-HYGB with primers CaOsh2KOS1/CaOsh2KOS2, CaOsh3KOS1/CaOsh3KOS2, CaOsh4KOS1/CaOsh4KOS2, CaOsh7alphKOS1/CaOsh7alphKOS2 and CaSAC1.S1/CaSAC1.S2. A second clone drs2osh4 was obtained from an independent clone of drs2 (PY3361, Table 7) using pFA-SAT1 and pFa-HYGB with primers CaOsh4KOS1 / CaOsh4KOS2.

To make strains prototroph the following plasmids were used (see ref in Table 3):

-pBN (ARG-URA) (Ghugtyal et al., 2015) digested with NotI for RP10 insertion of ARG4 and URA3.

-pDup5empty (Gerami-Nejad et al., 2013) digested with NgOMIV for NEUT5L insertion of URA3.

-pExpARG4 (Bassilana et al., 2003a) digested with StuI for RP10 insertion of ARG4.

-pGEM-HIS1 digested with NruI to reintroduce HIS1 in its own locus.

•

Strains expressing fluorescent reporters:

For localization of Drs2, Dnf2 and Osh4, strains expressing Drs2-GFPγ, Dnf2-GFPγ and Osh4-GFPγ were generated by integration of GFPγ 3' of the DRS2, DNF2 and OSH4 ORFs, respectively. The fragment GFPγ-HIS1 was amplified using primer pairs CaDRS2.P1(S1AFP)/CaDRS2.P2(S2), CaDNF2.S1AFP/CaDNF2.S2 and CaOsh4S1-AFP/CaOsh4S2, respectively, and plasmids pFA-GFPγ-HIS1, pFA-GFPγ-URA3, or pFA-GFPγ-ARG4 (Zhang and Konopka, 2010) (Table 3). These cassettes were integrated by homologous recombination in the heterozygous strain for each of the genes (∆ ∕+DRS2, ∆ ∕+DNF2 and ∆ ∕ +OSH4).

For localization of Mlc1, Sec7, Cdc10, Sec3 and Abp1, the strains were generated by amplifying a DNA fragment encoding a fluorescent reporter (AFP, Any Fluorescent Protein), integrated in 3' of the ORF of interest by homologous recombination. To do so, the following primers (see Table 6) and plasmids (from refs in Table 3) were used:

-Mlc1-AFP: mScarlet-ARG4 was amplified using primers

CaMlc1pFA/CaMlc1mFA and pFA-mScarlet-ARG4 (Puerner, 2020).

-Sec7-AFP: x3mScarlet-ARG4 was amplified using primers

CaSec7xFP_S1/CaSec7xFP_S2 and pFA-x3mScarlet-ARG4 (Puerner, 2020).

-Cdc10-AFP: mScarlet-ARG4 was amplified using primers CaCdc10pFa_S1AFP/CaCdc10mFA_S2.

-Sec3-AFP: x3mScarlet-ARG4 was amplified using primers

CaSec3longxFP_S1/CaSec3longxFP_S2 and pFA-x3mScarlet-ARG4.

-Abp1-AFP: GFPγ-ARG4 was amplified using primers

CaAbp140xFP_S1/CaAbp140xFP_S2 and pFA-GFPγ-ARG4.

To generate the strains expressing GFP-Sec4, the plasmid pGFP-Sec4-utr [START_REF] Li | Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation[END_REF] linearized by HindIII, was integrated in 5 'of the SEC4 ORF by homologous recombination and transformants screened for the URA3 marker. CDR1-GFP was integrated in the CDR1 locus after linearization of the plasmid pCDR1G4 (Ghugtyal et al., 2015) with XhoI and SacI by homologous recombination.

pExpArg-pACT1GFPRID and pExpArg-pACT1CRIBGFP [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF]) pExpArg-pADH1-PHFAPP1 (E50A,H54A)-GFP, pExpARG-pACT1-GFP-PHOSH2 (H340R)-PHOSH2 (H340R)- GFP (Ghugtyal et al., 2015), pExpARGpADH1-GFP-PHPLCδ1-PHPLCδ1-GFP (Vernay et al., 2012), and pExpARG-pACT1-GFP-LactC2 (Labbaoui et al., 2017) were linearized by StuI and integrated at the RP10 locus. pEApACT1mScarletD4H was also integrated at the RP10 locus after linearization with NcoI. The pDup plasmids (Gerami-Nejad et al., 2013) including pDup3pTEF1-ss-mCherryPHR2-GPI (Labbaoui et al., 2017) and pDUP3-pADHGFPLACTC2 were integrated at the NEUTL5 locus after digestion with NgoMIV.

•

GFP-nanobody (GNB) strains:

The miR-GNB tag was amplified by PCR from pFA-CamiRFP670-CaGNB-URA3 (Puerner et al., 2021b), using primers CaMlc1pFA/CaMlc1mFA. This miR-CaGNB-URA3 cassette was introduced 3' of the MLC1 ORF in strains expressing Drs2-GFP, Dnf2-GFP and Osh4-GFP to attempt to sequestrate the GFP fusion proteins at the SPK (Puerner et al., 2021b).

Verification of C. albicans mutants

The strains are verified by PCR and / or RT-PCR.

•

Genomic DNA extraction:

→ To visualize DNA fragments shorter than 2kb: 5μl of cells grown overnight at 30°C in YEPD were added to 100μl of YEPD and centrifuged at 13000 rpm for 2min. Pellets were resuspended in 100μl of LitAct-SDS lysis solution (see solution Table 2). Prior to incubation at 70ºC for 5min for cell lysis and two successive washes (in 300μl of ethanol 96% then 500μl of ethanol 70%). After centrifugation 15000g for 3min, pellets were dried at 50ºC, 20-30min, then resuspended in 50μl of TE1X. et al., 2010;Bassilana et al., 2005).

•

RT-PCR:

The RT-PCR reaction was carried out similar to the PCR reaction described above, using the same PCR program with 29 or 32 amplification cycles. Specific primers for amplifying fragments less than ~ 100 bp were used (see Table 10. RT-PCR primers).

Microscopic analyses

•

Images of colonies:

After 3-6 days of incubation at 30°C, the edge of colonies were imaged using an ImagingSource DMK 23UX174 sCMOS camera and a Leica MZ6 binocular magnifying glass at X20 magnification (Bassilana et al., 2003a).

•

Images of cells:

→Time lapses and fluorescent images were obtained using a spinning disk confocal microscope (inverted IX81 Olympus microscope with a 100X objective and a numerical aperture 1.45) and an EMCCD camera (Andor technology, UK). Z-stacks (images of 0.4 micron sections) were acquired every 5 min, as described [Ghugtyal et al., 2015]. Maximum or sum intensity projections were generated from 21 or 16 z-sections with ImageJ software. Laser illuminations of 445 nm (Turquoise), 488 nm (GFP), 561 nm (mScarlet) and 670 nm (miRFP) were used.

CRIBGFP distribution experiments were carried out as described [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF]. For the time-lapses, cells were grown on minimum media (SC-Arg) with agar (0.5%) for budding growth or supplemented with 75% FCS for hyphal growth (Bassilana et al., 2005).

→For filipin analyses, widefield images were acquired on an inverted ZEISS Axio Observer Z1 microscope with a 100X (1.3 NA) objective.

→Huygens professional software version 18.04 (Scientific-Volume Imaging) was used to deconvolve z-stack images of cells expressing PH-FAPP1 [E50A,H54A]-GFP. Bars are 5 microns.

→Filament lengths and diameters were measured from DIC images, using ImageJ software.

Golgi cisternae were quantitated from deconvolved MAX projection images.

→Quantitation of PI( 4)P distribution was performed on SUM projections, using the Matlab program HyphalPolarity (Ghugtyal et al., 2015). Quantitation of PS plasma membrane and internal mean signals was performed on central z-sections, also using the Matlab program HyphalPolarity. Unless indicated otherwise, error bars represent the standard error media. pEA-pDRS2-DRS2 QQ This study pExpARG4empty (Bassilana et al., 2003a) pExpARG4-GFPγ-ctRAC1 (Puerner et al., 2021b) pExpARG4-pACT1-CRIB-GFP [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF] pExpARG4-pACT1-GFP-lactC2 (Labbaoui et al., 2017) pExpARG4-pACT1-GFP-PHOSH2(H340R)-PHOSH2(H340R)-GFP (Ghugtyal et al., 2015) pExpARG4-pACT1-GFP-RID [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF] pExpARG4-pACT1-mScarlet-D4H Provided by H. Labbaoui pExpARG4-pADH1-DRS2 (Labbaoui et al., 2017) pExpARG4-pADH1-DRS2 F511Y This study pExpARG4-pADH1-PHFAPP1(E50A,H54A)-GFP (Ghugtyal et al., 2015) pExpARG4-pARL3-ARL3 (Labbaoui et al., 2017) pExpARG4-pDRS2-DRS2 (Labbaoui et al., 2017) pExpARG4-pDRS2-DRS2 F511Y This study pExpARG4-pOSH4-OSH4 This study pExpARGpADH1-GFP-PHPLCδ1-PHPLCδ1-GFP (Vernay et al., 2012) pFA-CaARG4 (Schaub et al., 2006) pFA-CaHIS1 (Schaub et al., 2006) pFA-GFPγ-ARG4 [START_REF] Zhang | A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans[END_REF] pFA-GFPγ-HIS1 [START_REF] Zhang | A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans[END_REF] pFA-GFPγ-URA3 [START_REF] Zhang | A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans[END_REF] pFA-HIS1 (Schaub et al., 2006) pHB1S (Lai et al., 2016) pFA-miRFP670-GNB-URA3 (GNB-A3) (Puerner et al., 2021b) pFA-mScarlet-ARG4 (Puerner, 2020) pFA-mScarlet-URA3 (Puerner et al., 2021b) pFA-x3mScarlet-ARG4 (Puerner, 2020) pFA-SAT (Schaub et al., 2006) pFA-URA3 (Schaub et al., 2006) pGem-CdARG4 (Bijlani et al., 2019) pGEM-HIS1 (Wilson et al., 1999) pGEM-URA3 (Wilson et al., 1999) pGFP-SEC4-utr [START_REF] Li | Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation[END_REF]

Introduction

Polarized growth is an essential process that is regulated in particular by cooperative interactions between key establishment proteins and specific lipids at the plasma membrane (PM). For instance in the baker yeast Saccharomyces cerevisiae, the activity and dynamic nanoclusters of the Rho-GTPase Cdc42 is regulated by multivalent interactions between its sole activator Cdc24, the scaffold protein Bem1

and anionic lipids, including phoshatidylserine (PS) [1][START_REF] Fairn | Phosphatidylserine is polarized and required for proper Cdc42 localization and for development of cell polarity[END_REF][START_REF] Meca | Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions[END_REF]. Similarly, in the fission yeast Schizosaccaromyces pombe, the localization and function of the two essential Rho-GTPases, Cdc42 and Rho1, depend on PS polarized distribution [START_REF] Haupt | Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast[END_REF]. The importance of anionic lipids, and PS in particular, for cellular function and signaling encompasses kingdoms from fungi, to mammals [START_REF] Kay | Distribution, dynamics and functional roles of phosphatidylserine within the cell[END_REF] to plants [START_REF] Noack | Functions of Anionic Lipids in Plants[END_REF]. In addition, PS has been reported to be critical for virulence in a broad spectrum of microbial pathogens (reviewed in [START_REF] Cassilly | It's Complicated: The Roles of Phosphatidylserine and Phosphatidylethanolamine in the Pathogenesis of Candida albicans and Other Microbial Pathogens[END_REF][START_REF] Rizzo | Role of lipid transporters in fungal physiology and pathogenicity[END_REF]). In particular, in the human fungal pathogen Candida albicans, the PS synthase Cho1, which is conserved in fungi but does not have an ortholog in Human, is required for virulence in a murine candidiasis model [START_REF] Chen | Phosphatidylserine synthase and phosphatidylserine decarboxylase are essential for cell wall integrity and virulence in Candida albicans[END_REF]. A cho1 deletion mutant also exhibits increased exposure of β (1,3)-glucan via up-regulation of cell wall MAPK cascades, facilitating its detection by innate immune cells [START_REF] Chen | Exposure of Candida albicans beta (1,3)-glucan is promoted by activation of the Cek1 pathway[END_REF]. PS is synthesized in the endoplasmic reticulum (ER) and its cellular distribution is regulated both by lipid transfer proteins, which function at contact sites between the ER and the target cellular compartments, and lipid transporters, such as flippases that establish PS asymmetry between membrane leaflets (reviewed in [START_REF] Lenoir | Transport Pathways That Contribute to the Cellular Distribution of Phosphatidylserine[END_REF]).

Lipid flippases are P4-ATPases, only found in eukaryotes, where they are similar in domain structures from fungi to Human (reviewed in [START_REF] Lyons | P4-ATPases: how an old dog learnt new tricks -structure and mechanism of lipid flippases[END_REF]). These proteins actively transport phospholipids from the external/luminal to cytoplasmic membrane leaflets and play an important role in polarized growth (reviewed in [START_REF] Lopez-Marques | Lipid flippases in polarized growth[END_REF]). There are 14 P4-ATPases in Human and only 5 in the yeast S. cerevisiae (Dnf1-3, Drs2, Neo1), with most of them regulated by non-catalytic subunits from the Cdc50/Lem3 family.

For instance, in S. cerevisiae, Cdc50 forms a functional complex with Drs2 [START_REF] Lenoir | Cdc50p plays a vital role in the ATPase reaction cycle of the putative aminophospholipid transporter Drs2p[END_REF][START_REF] Saito | Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae[END_REF] and very recently, the cryo-electron microscopy structure in different conformations of this complex was resolved at 2.8 to 3.7 Å [16]. This complex, which is well characterized, both in vitro and in vivo, primary transports PS [START_REF] Natarajan | Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function[END_REF][START_REF] Zhou | Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast[END_REF]. The role of lipid flippases on membrane curvature and in trafficking has been extensively studied in S. cerevisiae, as well as in mammals (reviewed in [START_REF] Best | Phospholipid flippases in membrane remodeling and transport carrier biogenesis[END_REF]). In budding S. cerevisiae, Neo1, which is the sole essential flippase [START_REF] Hua | An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system[END_REF], localizes to the Golgi and endomembranes, Drs2 and Dnf3 to the trans-Golgi network (TGN) and Dnf1-2 to the PM [START_REF] Natarajan | Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function[END_REF][START_REF] Chen | Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function[END_REF][START_REF] Pomorski | Drs2p-related P-type ATPases Dnf1p and Dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis[END_REF]. A recent study indicates that Dnf3 can also be found at the PM in a cell-cyle dependent fashion, where it regulates, together with Dnf1-2, S. cerevisiae pseudohyphal growth [START_REF] Frosig | Pseudohyphal growth in Saccharomyces cerevisiae involves protein kinase-regulated lipid flippases[END_REF]. Furthermore, in response to pheromone, it was shown that Dnf1, Dnf2 and Dnf3 localize to the schmoo tip, while Drs2 remains at the Golgi [24].

Interestingly, in the filamentous fungi Aspergillus nidulans and Fusarium graminearum, it was shown in particular that the Drs2 homolog, DnfB, localizes primarily to a cluster of vesicles at the hyphal apex, called Spitzenkorper (SPK), which is characteristic of fungi that can grow in a filamentous form [25,[START_REF] Perry | Unraveling How Candida albicans Forms Sexual Biofilms[END_REF]. Together, these data indicate that flippases can localize to different distinct compartments in specific conditions of polarized growth.

Filamentous fungi are highly polarized organisms, in which the role of flippases has been investigated. For instance, in A. nidulans, the homologs of Dnf1 and Drs2, i.e. DnfA and DnfB, regulate growth and PS asymmetry [25], while in Magnaporthe grisea, MgAPT2, one of the 4 aminophosphlipid translocase (APT) encoding genes related to Drs2, is required for plant infection but not filamentous growth [27]. In F. graminearum, flippases play redundant as well as distinct roles in vegetative growth, where FgDnfA is critical, stress response, reproduction and virulence [START_REF] Perry | Unraveling How Candida albicans Forms Sexual Biofilms[END_REF]28,29]. In the human fungal pathogen C. albicans, Neo1 is not essential and deletion of either Dnf1 or Dnf2 results in moderate increase sensitivity to copper [START_REF] Douglas | Plasma membrane architecture protects Candida albicans from killing by copper[END_REF]. The drs2 deletion mutant is hypersensitive to copper [START_REF] Douglas | Plasma membrane architecture protects Candida albicans from killing by copper[END_REF], as well as to the antifungal drug fluconazole [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF]. Deletion of Drs2 also results in altered PS distribution and impaired filamentous growth [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF], and that of its Cdc50 subunit in altered filamentous growth and reduced virulence in a murine candidiasis model [START_REF] Xu | The lipid flippase subunit Cdc50 is required for antifungal drug resistance, endocytosis, hyphal development and virulence in Candida albicans[END_REF].

Here, we sought to determine the role of Drs2 in morphogenesis and whether membrane asymmetry is critical for this process.

Our results show that in response to serum, of the four flippases, Dnf1-3 and Drs2, only Drs2 is critical for filamentous growth, with the Dnf1 and Dnf2 together having a minor role in this process. Filamentous growth was largely recovered in the drs2 mutant upon deletion of the lipid transfer protein Osh4, but not by that of other Osh proteins (Osh2, Osh3 and Osh7). Furthermore, our results demonstrate that the distributions of PS and of the phosphatidylinositol phosphate PI(4)P, which are both altered in the drs2 mutant, were restored upon deletion of OSH4. These data indicate that the requirement for flippase activity across the lipid bilayer during filamentous growth can be specifically bypassed by lipid exchange between membrane compartments.

Results

Drs2 has a unique role in C. albicans

In A. nidulans, the Drs2 homolog DnfB, is not critical for hyphal growth [25]. In contrast, in C. albicans, Drs2 is required for filamentous growth and budding growth is largely unaffected (average doubling time of 90 min, compared to 80 min in the wild-type strain) [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF]. To determine whether Drs2's role is specific, we investigated the importance of other flippases, i.e. Dnf1, Dnf2 and Dnf3, using loss of function mutants that we generated (Supplementary Figure S1A). As illustrated in Figure 1A, a drs2 deletion mutant was not invasive on a serum-containing media, and reintroduction of DRS2 in this mutant restored invasive growth (Figure 2A). In contrast, deletion of DNF1 or DNF3 did not alter serum-induced invasive growth, while deletion of DNF2 resulted in reduced invasive growth, which was further reduced in a double dnf1dnf2 deletion mutant. Over-expression of DNF2 restored invasive growth both in the dnf2 and dnf1dnf2 mutants, but not in the drs2 mutant, suggesting that Dnf2 and Drs2 function do not overlap in this invasive growth process. Upon serum-induced hyphal growth in liquid media (Figure 1B), the dnf1, dnf2 and dnf3 deletion mutants produced hyphae similar to the wild-type cells (79±4%, 78±7%, 76±10 % and 84±5 % respectively, compared to 18±3 % for the drs2 mutant); hyphal formation in the dnf1dnf2 mutant cells was somewhat reduced to 56±12%. The average hyphal filament length was nonetheless reduced in dnf2 cells, as well as in dnf1dnf2 cells (14±3 µm and 13±3 µm) compared to the WT, dnf1 and dnf3 cells (20±4 µm, 18±4 µm and 20±4 µm, respectively). These data indicate that, in contrast to the drs2 deletion mutant, cells deleted for DNF1, DNF2 and DNF3

undergo hyphal invasive growth similar to the wild-type cells, albeit with a reduced efficiency for the dnf2 mutant. Furthermore, Figure 1E shows that the dnf1-3 mutants also grew similar to the wild-type cells on cell wall perturbants, such as calcofluor white (CFW) and congo red (CR), and on antifungal drugs, such as caspofungin (Caspo) and fluconazole (FCZ), in contrast to the drs2 mutant, which was hypersensitive to CFW and FCZ [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF]. Together, these data indicate that Drs2 has a unique role in C. albicans.

Drs2 is critical for hyphal extension after septin ring formation

In response to serum, drs2 cells appear to initiate filamentous growth, although they were unable to form hyphae [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF]. Hence, we examined this mutant in a mouse model for systemic candidiasis and Figure 2B shows that the drs2 mutant is avirulent. At the transcriptional level, hyphal growth is controlled by the hyphal specific gene HGC1 [START_REF] Zheng | Hgc1, a novel hypha-specific G1 cyclin-related protein regulates Candida albicans hyphal morphogenesis[END_REF], which itself is further regulated by UME6 [START_REF] Carlisle | Candida albicans Ume6, a filament-specific transcriptional regulator, directs hyphal growth via a pathway involving Hgc1 cyclinrelated protein[END_REF]. Both hgc1 and ume6 deletion mutants are defective in hyphal extension and attenuated for virulence in a mouse infection model [START_REF] Zheng | Hgc1, a novel hypha-specific G1 cyclin-related protein regulates Candida albicans hyphal morphogenesis[END_REF][START_REF] Banerjee | UME6, a novel filament-specific regulator of Candida albicans hyphal extension and virulence[END_REF]. RT-PCR analyses in Figure 2C shows that the drs2 mutant expressed HGC1, similar to that of the WT and complemented strains after 30 min serum-induction, but at a reduced level after 120 min, and a similar pattern was observed for UME6 transcript levels. Expression of the peptide toxin candidalysin encoding ECE1 gene, [START_REF] Naglik | Candidalysin: discovery and function in Candida albicans infections[END_REF], which correlates with cell elongation, was reduced in drs2 both at 30 and 120 min, the difference compared to control cells being greatest at 120 min. Expression of the secreted protease SAP6, which is mainly associated with hyphal growth [START_REF] Hube | Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans[END_REF], was also dramatically reduced after 120 min in drs2 cells, but expression of the agglutinin-like (Als) adhesin gene ALS3, which is dispensable for hyphal formation [START_REF] Cleary | Candida albicans adhesin Als3p is dispensable for virulence in the mouse model of disseminated candidiasis[END_REF], appeared to be unaffected compared to control cells. Together, our data are consistent with a defect in drs2 hyphal extension.

To further characterize this defect at the molecular level, we used time-lapse microscopy to follow cells expressing fluorescent reporters for different cellular compartments. Figure 3A illustrates representative time courses of hyphal growth both in WT and drs2 cells expressing the septin Cdc10 [START_REF] Gonzalez-Novo | Dynamics of CaCdc10, a septin of Candida albicans, in living cells and during infection[END_REF]. Measurement of the diameter of the filament compartments, apical and distal to the Cdc10 septin ring (Figure 3B) shows that, while their diameter was constant in WT cells (~1.8 µm), the apical compartment specifically increased in drs2 cells (to ~2.5 µm). This increase in diameter was associated with a reduced extension rate after appearance of the first septin ring (0.09 µm/min in drs2 cells, compared to 0.34 µm/min in WT cells; Figure 3C). These data are consistent with a defect in polarized growth in the drs2 mutant after the first septin ring forms. In agreement with this, we observed that, upon filament extension, the localization of active Cdc42 [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF] became depolarized in the drs2 cells and that the Spitzenkörper (visualized with the myosin light chain Mlc1, [START_REF] Crampin | Candida albicans hyphae have a Spitzenkorper that is distinct from the polarisome found in yeast and pseudohyphae[END_REF][START_REF] Puerner | A Myosin Light Chain Is Critical for Fungal Growth Robustness in Candida albicans[END_REF]), was not maintained at the tip of the filament following cell division, compared to the wild-type control cells (Figures 3D and3E). Together, these data indicate that, subsequent to septin ring formation, the drs2 mutant is unable to maintain polarized growth, which ultimately results in growth arrest and/or pseudohyphal growth.

Drs2 localizes to the hyphal tip

In S, cerevisiae, Drs2 localizes to the late Golgi during budding growth [START_REF] Hua | An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system[END_REF] and mating [24], while its homolog in A. nidulans, DnfB, localizes within the Spitzenkörper core [25]. To examine the distribution of Drs2 in C. albicans, we generated a strain that expresses a DRS2-GFP fusion, which was functional (Figure 4A). Given that the homolog of Dnf1-2 in A. nidulans, DnfA, also localizes to the Spitzenkörper, albeit to the outer layer macrovesicles compared to DnfB [25], we also generated a strain that expressed a functional DNF2-GFP fusion (Figure 4A). Figure 4B shows that Drs2 was restricted to the apical region of the hyphal tip, as well as in internal structures, likely Golgi cisternae by analogy with S. cerevisiae [START_REF] Hua | An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system[END_REF]. Dnf2 was essentially localized to the plasma membrane at the apical cortex, similar to its localization in S. cerevisiae at the tip of buds and mating projections [24]. Both Dnf2 and Drs2 co-localized with a plasma membrane marker (a prenylated AFP fusion, AFP-CtRac1, [START_REF] Vauchelles | Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans[END_REF]), although Drs2 localized to a smaller region at the hyphal tip than Dnf2 (Figure 4C) and, given the proximity of the Spitzenkörper to the plasma membrane, Drs2 also partially co-localized with the Spitzenkörper marker, the Myosin Light Chain Mlc1 (Figure 4D). Together, these data indicate that Drs2 and Dnf2 have distinct distributions at the filament apex.

The drs2 mutant is altered for PI(4)P distribution

Drs2 is a P4-ATPase that flips selectively phosphatidylserine (PS) across the lipid bilayer in vitro and in vivo in S. cerevisiae [START_REF] Natarajan | Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function[END_REF][START_REF] Zhou | Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast[END_REF]. In C. albicans, using the reporter Lactadherin C2 [START_REF] Vecchio | Investigation of the phosphatidylserine binding properties of the lipid biosensor, Lactadherin C2 (LactC2), in different membrane environments[END_REF][START_REF] Leventis | The distribution and function of phosphatidylserine in cellular membranes[END_REF][START_REF] Yeung | Membrane phosphatidylserine regulates surface charge and protein localization[END_REF], we observed that the distribution of PS was altered during hyphal growth in the drs2 mutant, as the reporter was less associated with the plasma membrane and visible as intracellular punctae [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF]. To determine the impact of the DRS2 deletion on the distribution of other lipids shown to be critical for hyphal growth, such as the phosphatidylinositol phosphates PI(4P) and PI(4,5)P 2 [START_REF] Ghugtyal | Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth[END_REF][START_REF] Vernay | A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth[END_REF], as well as ergosterol [START_REF] Martin | Lipid raft polarization contributes to hyphal growth in Candida albicans[END_REF], we used specific fluorescent reporters. The distribution of PI(4,5)P 2 appears to be similar in WT cells and drs2 filamentous cells (Figure 5A), yet the distribution of PI( 4)P was substantially less polarized in the mutant, compared to the wild-type or complemented strains (Figure 5B). This depolarization is further illustrated by the graph in Figure 5B, which shows the relative concentration of PI( 4)P, as a function of filament length. In contrast, PI(4)P at the Golgi appears similar in drs2 and WT filamentous cells (Figure 5C). Furthermore, the number of Golgi cisternae was also similar (~7 cisternae per cell in drs2 cells compared to ~9 per cell in the wild-type control), indicating that the Golgi structure was largely unaffected in this mutant (Figure 5D).

Using filipin staining, it was shown that membrane sterols are highly concentrated at the apex during C. albicans hyphal growth, with such a polarization not observed in budding and pseudohyphal cells [START_REF] Martin | Lipid raft polarization contributes to hyphal growth in Candida albicans[END_REF]. We examined the sterol distribution in drs2 cells, using both filipin staining and the genetically encoded biosensor D4H [START_REF] Kishimoto | Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane[END_REF][START_REF] Marek | Sterol biosensor reveals LAM-family Ltc1dependent sterol flow to endosomes upon Arp2/3 inhibition[END_REF]. Figures 6A and6B show that sterols were highly concentrated at the apex of WT hyphal cells, whether visualized with filipin staining or the D4H reporter. In contrast, in the drs2 mutant, while we observed a polarized distribution of sterols with filipin staining (Figure 6A), albeit less pronounced than in the WT cells, there was a striking difference with D4H, as this reporter preferentially labeled internal structures in drs2 cells (Figure 6B). We attribute the difference in distribution of these reporters to different sterol accessibility from the inner PM leaflet [START_REF] Marek | Sterol biosensor reveals LAM-family Ltc1dependent sterol flow to endosomes upon Arp2/3 inhibition[END_REF]. The internal D4H associated signals were frequently observed at the filament apex and reintroduction of a copy of DRS2 in the drs2 mutant restored the ergosterol distribution to the PM (Figure 6C). Together, these data indicate that deletion of DRS2 results not only in altered distribution of PS, but also of PI( 4)P and ergosterol.

Deletion of OSH4 recovers hyphal invasive filamentous growth in the drs2

mutant Lipid transfer proteins (LTPs), and more specifically oxysterolbinding protein (OSBP)-related proteins (ORPs), are important for membrane lipid composition, via non-vesicular traffic (reviewed in [START_REF] Wong | Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes[END_REF]), including for PS distribution (reviewed in [START_REF] Lenoir | Transport Pathways That Contribute to the Cellular Distribution of Phosphatidylserine[END_REF]). LTPs can bind specific ligands such as PI(4)P, PS and sterol. For example, in S. cerevisiae, there are 7 oxysterol-binding homology (Osh) proteins, among which Osh6/Osh7 transports PS from cortical ER (cER) and PM [START_REF] Maeda | Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins[END_REF], in counter-exchange with PI(4)P [START_REF] Moser Von Filseck | A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes[END_REF]. On the other hand, another Osh protein, Kes1/Osh4, which transports sterol in counter-exchange with PI(4)P [START_REF] De Saint-Jean | Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers[END_REF] was proposed to act antagonistically with Drs2 to regulate the sterol distribution between PM and internal membranes in S. cerevisiae [START_REF] Muthusamy | Control of protein and sterol trafficking by antagonistic activities of a type IV P-type ATPase and oxysterol binding protein homologue[END_REF]. C. albicans has 5 Osh proteins (Osh1-4 and Osh7), with Osh4 and Osh7 sharing ~ 60% identity with their S.

cerevisiae counterparts, and their importance is unknown in this organism. Note that the genes encoding OHS7 (called OBPA and OBPALPHA) are located at the matingtype-like locus (MTL), similar to those encoding the Golgi phosphatidylinositol kinase Pik1 (PIKA and PIKALPHA).

To investigate the importance of Osh proteins in C. albicans invasive hyphal growth, we first generated osh4 and osh7 deletion mutants, as well as drs2 osh4 and drs2 osh7 double deletion mutants, and examined these mutants for invasive hyphal growth in response to serum induction. As illustrated in Figure 7, deletion of OSH4 or OSH7 alone did not alter invasive growth, yet deletion of OSH4 in the drs2 mutant background recovered invasive growth to a level similar to that of the WT (Figure 7A, upper panel). Reintroduction of a copy of OSH4, under the control of its own promoter in this drs2 osh4 mutant resulted in non-filamentous colonies, similar to that of drs2. In serum-containing liquid media, we also observed a recovery of hyphal growth upon deletion of OSH4 in the drs2 mutant (Figure 7B-D), with 76±9% filamentous cells, compared to 82±8% and 80±6% in the WT and the osh4 mutant, respectively. In this drs2 osh4 mutant, hyphal length was, however, slightly reduced compared to that of the WT and osh4 cells (17±3 µm, compared to 19±1 µm for WT and osh4 cells). Reintroduction of a copy of OSH4 in the drs2osh4 mutant resulted in a reduced percentage of hyphae to 54±11%, with filament lengths of 16±0.5 µm, compared to the drs2 osh4 mutant, albeit higher than the percentage of hyphae in the drs2 mutant (18±9%). The recovery of invasive filamentous growth in the drs2 mutant was specific for the OSH4 deletion, as deletion of OSH7 did not recover invasive filamentous growth in the drs2 mutant (Figure 7A, bottom panel).

To confirm the specificity of the OSH4 deletion in recovering invasive growth in the drs2 mutant, we also generated drs2 osh2 and drs2 osh3 double deletion mutants. C. albicans OSH3 has been reported be important for invasive growth in nutrient poor media (Spider media), but not in the presence of serum [START_REF] Hur | Characterization of Osh3, an oxysterolbinding protein, in filamentous growth of Saccharomyces cerevisiae and Candida albicans[END_REF]. Similarly, we observed that mutants deleted for either OSH2 or OSH3 grew invasively in the presence of serum (Figure S2). In contrast to OSH4 deletion, deletion of either OSH2 or OSH3 did not recover the drs2 invasive growth defect.

These data indicate that while Osh proteins (Osh2-4 and Osh7) are not required for serum-induced invasive filamentous growth, deletion of OSH4 bypasses the drs2 defect in this process.

While deletion of OSH4 in the drs2 mutant did also recover the growth defect on CFW, it did not recover growth on FCZ (Figure 7E), which remained similar to that of drs2, suggesting that Drs2 has specific roles that are not linked to Osh4 activity. The growth defect of drs2 cells on FCZ could result from a mislocalization of multi-drug transporters, such as Cdr1 [START_REF] Prasad | Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals[END_REF]. This is unlikely, as Figure 7F shows that a Cdr1-GFP fusion protein localized to the plasma membrane of drs2 cells, similar to that of WT cells. Together, these data indicate that DRS2 per se is not required for hyphal invasive growth, but rather that a balance in the activities of Drs2 and Osh4 regulate this process.

Drs2 is important for plasma membrane organization

To gain further insight into the mechanisms underlying the recovery of hyphal growth in the drs2 osh4 mutant, we examined whether deletion of OSH4 also restored the wild-type phospholipid distribution in the drs2 mutant. Figure 8A shows that the distribution of PI( 4)P was polarized in drs2 osh4, similar to that in WT and osh4 cells.

Similarly, the distribution of PS, as visualized with LactC2, appears to be also largely restored in drs2 osh4, as the internal punctae visualized in drs2 cells were absent in these drs2 osh4 cells, similar to the WT, complemented and osh4 strains (Figure 8B).

Quantification of the LactC2 signal in the drs2 mutant indicates that the level of PS was both reduced at the PM and increased internally, compared to WT and osh4 deleted cells, resulting in a decreased ratio of PM to intracellular signals (Figure 8C).

Deletion of OSH4 in these drs2 cells significantly recovered the PS ratio of PM to intracellular signals, with an increase in PM and a decrease in internal signals (Figure 8C). Papuamide A (PapA) is a depsipeptide toxin that binds PS in the outer leaflet of the plasma membrane [START_REF] Parsons | Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast[END_REF]. In C. albicans, the cho1 deletion mutant lacks the PS synthase and is less sensitive to PapA than WT cells [START_REF] Cassilly | SB-224289 Antagonizes the Antifungal Mechanism of the Marine Depsipeptide Papuamide A[END_REF]. As shown in Figure 8D, the drs2 mutant was hypersensitive to PapA, compared to the WT strain, reflecting increased PS in the outer PM leaflet. Interestingly, the drs2 osh4 mutant did not grow on PapA, similar to drs2, while the osh4 mutant grew similar to the WT (Figure 8C).

These results indicate that in this double mutant, the PS bilayer asymmetry was not re-established and suggest that establishment of a PM PI(4)P gradient, rather than PS bilayer asymmetry, is critical for hyphal growth.

Deletion of both DRS2 and SAC1 results in a synthetic growth defect

To further investigate the relative importance of the PS bilayer asymmetry and the PM PI(4)P gradient in hyphal growth, we generated different mutants with the aim of altering each of these components. In an attempt to alter PS asymmetry, we generated strains expressing Drs2 mutated for the residues analogous to those specifically required for PS recognition in S. cerevisiae, Drs2 F511Y and Drs2 QQ-GA (Figure S3A; [START_REF] Baldridge | Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases[END_REF][START_REF] Baldridge | Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases[END_REF]). Reintroduction of a copy of such a mutated version of C. albicans Drs2 (Drs2 F520Y ) in drs2, restored growth on PapA, similar to that of the complemented and WT strains (Figures 8D). Furthermore, the mutants expressing either Drs2 F520Y or Drs2 QQ-GA had a PS distribution similar to that of the complemented strain and generated hyphae (Figures S3B andS3C), indicating that these residues are not critical for PS distribution and morphogenesis in C. albicans.

To alter the PM PI(4)P gradient, we generated mutants deleted for the lipid phosphatase Sac1, which dephosphorylates PI(4)P, and is responsible for regulating PM PI(4)P, both in yeast and mammalian cells [START_REF] Stefan | Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites[END_REF][START_REF] Zewe | SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes[END_REF]. In C. albicans, Sac1 is critical for the steep PM PI(4)P gradient [START_REF] Ghugtyal | Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth[END_REF] and hyphal growth maintenance [START_REF] Ghugtyal | Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth[END_REF][START_REF] Zhang | The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans[END_REF]. In S. cerevisiae, deletion of either OSH4 or SAC1 is synthetically lethal in cells largely devoid of ER-PM contact sites [START_REF] Quon | Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation[END_REF], and cold-sensitive growth of a drs2 deletion mutant is partially suppressed by deletion of either SAC1 or the OSH4 homolog, KES1 [START_REF] Muthusamy | Control of protein and sterol trafficking by antagonistic activities of a type IV P-type ATPase and oxysterol binding protein homologue[END_REF]. As deletion of OSH4 restored PI(4)P polarized distribution in drs2, as well as hyphal growth, we investigated the effect of SAC1 deletion in drs2.

Figures 9A and 9B show that budding growth was altered in such a drs2 sac1 mutant, with a doubling time that increased 3-fold, compared to the WT, drs2 and sac1 strains. Furthermore, the drs2 sac1 strain did not form invasive colonies, similar to sac1 (Figure 9C). However the sac1 mutant formed filamentous cells in response to serum, albeit shorter than WT cells (Figure 9D; [START_REF] Ghugtyal | Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth[END_REF][START_REF] Zhang | The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans[END_REF]). In contrast, the drs2 sac1 strain was unable to form filamentous cells, even after 270 min incubation (Figure 9D). RT-PCR confirmed the absence of DRS2 and/or SAC1 in the respective mutants (Figure 9E). Together, these data show that DRS2 and SAC1 genetically interact in C.

albicans, indicating that they function in parallel pathways, and suggest that a PM PI(4)P gradient is critical in the absence of Drs2.

Materials and Methods

Growth conditions

Yeast extract-peptone dextrose (YEPD) or synthetic complete (SC) medium was used and strains were grown at 30°C, unless indicated otherwise. Filamentous growth induction was carried out as described previously either with 50% serum or 75% serum [START_REF] Bassilana | Cdc24, the GDP-GTP exchange factor for Cdc42, is required for invasive hyphal growth of Candida albicans[END_REF]. For filipin staining experiments, 10% of serum was used. Growth on YEPD plates containing Congo red, calcofluor white, caspofungin or fluconazole was examined as described [START_REF] Ram | Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red[END_REF]. Congo red, calcofluor white, filipin, hygromycin and fluconazole were from Fluka, Sigma-Aldrich, Saint Quentin Fallavier, France.

Papuamide A was from University British Columbia.

Strains and plasmids

Strains and oligonucleotides used are listed in Tables S1 andS2, respectively. All strains were derived from BWP17 [START_REF] Wilson | Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions[END_REF]. The deletion mutants were generated by homologous recombination. Each copy was replaced by either HIS1, URA3, ARG4, SAT1 or HYGB, using knockout cassettes generated by amplification of pGem-HIS1, pGem-URA3, pGem-CdARG4, pFa-ARG4, pFa-SAT1 and pBH1S [START_REF] Wilson | Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions[END_REF][START_REF] Bijlani | Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in Candida albicans[END_REF][START_REF] Lai | A new rapid and efficient system with dominant selection developed to inactivate and conditionally express genes in Candida albicans[END_REF][START_REF] Schaub | New pFA-cassettes for PCRbased gene manipulation in Candida albicans[END_REF] and drs2∆/drs2∆ sac1∆/sac1∆ strains were generated from the drs2∆/drs2∆ strain (PY3310 [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF]) and the dnf1∆/dnf1∆ dnf2∆/dnf2∆ strain from the dnf2∆/dnf2∆ strain (PY5804).

Plasmid pDUP3-pADH1DNF2 was constructed by amplification from gDNA of the DNF2 ORF, using primers with a unique AscI site at the 5' end (DNF2.P5) and a

unique MluI site at the 3' end (DNF2.P6) and pDUP3-pDRS2DRS2 by amplification from pExp-pDRS2DRS2 [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF] of the DRS2 ORF together with 1 kb upstream and downstream, using primers with a unique SpeI site at the 5' end (DRS2.P3) and a

unique NotI site at the 3' end (DRS2.P4). The fragments were subsequently cloned into pDUP3-SAT1 [START_REF] Gerami-Nejad | Shuttle vectors for facile gap repair cloning and integration into a neutral locus in Candida albicans[END_REF], yielding to pDUP3-pADH1DNF2 and pDUP3-pDRS2DRS2, respectively. Plasmid pExp-pOSH4OSH4 was constructed by amplification from gDNA of the OSH4 ORF, together with 1 kb upstream and downstream, using primers with a unique SalI site at the 5' end (OSH4.P3) and a unique NotI site at the 3' end (OSH4.P4) and subsequently cloned into pExpARG4, yielding to pExp-pOSH4OSH4. Site directed mutagenesis was used to mutate Drs2 to Drs2 F520Y and Drs2 QQ-GA , using the primer pairs DRS2.P7/DRS2.P8 and DRS2.P9/DRS2.P10, respectively, together with pExp-pDRS2DRS2 [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF]. To visualize Drs2 and Dnf2, GFPγ was inserted by homologous recombination at the 3' end of DRS2 or DNF2

ORF in strains heterozygous for these genes (PY3303 and PY5677), respectively, after amplification of GFPγ from the plasmid pFA-GFPγ-HIS1 [START_REF] Li | A Candida albicans temperature-sensitive cdc12-6 mutant identifies roles for septins in selection of sites of germ tube formation and hyphal morphogenesis[END_REF], using the primers DRS2.P5/DRS2.P6 or DNF2.P7/DNF2.P8. pExp-pACT1CRIBGFP [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF],

was used to transform the WT (BWP17) and drs2/drs2 (PY3310) strains. Cdr1-GFP, PH-FAPP1 [E50A,H54A]-GFP, GFP-(PH OSH2[H340R] ) 2 -GFP and GFP-PHPLCδ1-PHPLCδ1-GFP expressing strains were generated as described [START_REF] Ghugtyal | Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth[END_REF][START_REF] Vernay | A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth[END_REF]. pDUP5-mScarlet-CtRac1 [START_REF] Puerner | Mechanical force-induced morphology changes in a human fungal pathogen[END_REF] was used to transform the drs2/DRS2GFP (PY4665) and dnf2/DNF2GFP (PY5746) strains. Mlc1-mScarlet and Cdc10-mScarlet were generated by amplification of mScarlet-ARG4 from pFA-mScarlet-ARG4 [START_REF] Silva | Secretory Vesicle Clustering in Fungal Filamentous Cells Does Not Require Directional Growth[END_REF], using the primer pairs MLC1.P1/MLC1.P2 and CDC10.P1/CDC10.P2, respectively. mTurquoise-CtRac1 (mTrq-CtRac1) was constructed by replacing mScarlet in pDUP5-pADH1-mScarlet-CtRac1 [START_REF] Puerner | Mechanical force-induced morphology changes in a human fungal pathogen[END_REF] with mTrq, and subsequently cloning mTrq-CtRac1 into pDUP3 (Puerner, Bassilana & Arkowitz, in preparation). To visualize the distribution of phosphatidylserine, a fusion of GFP with the discoidin-like C2 domain of lactadherin (GFP-LactC2) was used, as previously described [START_REF] Labbaoui | Role of Arf GTPases in fungal morphogenesis and virulence[END_REF]. To visualize the distribution of ergosterol, we used filipin staining, as described [START_REF] Martin | Lipid raft polarization contributes to hyphal growth in Candida albicans[END_REF], as well as the genetically encoded biosensor D4H [START_REF] Marek | Sterol biosensor reveals LAM-family Ltc1dependent sterol flow to endosomes upon Arp2/3 inhibition[END_REF].

D4H was amplified from plasmid pSM2244 [START_REF] Marek | Sterol biosensor reveals LAM-family Ltc1dependent sterol flow to endosomes upon Arp2/3 inhibition[END_REF], using primer pair D4H. Two independent clones of each strain were generated and confirmed by PCR.

RT-PCR was also performed, where relevant, using the primers (GENE.pTm and GENE.mTm) listed in Table S2 or previously described [START_REF] Bassilana | Regulation of the Cdc42/Cdc24 GTPase module during Candida albicans hyphal growth[END_REF] and RNA extraction was carried out using Master Pure yeast RNA extraction purification kit (Epicentre). All PCR amplified products were confirmed by sequencing (Eurofins MWG Operon, Ebersberg, Germany).

Microscopy analyses

Colony and cell morphology imaging were performed as described [START_REF] Bassilana | Cdc24, the GDP-GTP exchange factor for Cdc42, is required for invasive hyphal growth of Candida albicans[END_REF]. Briefly, plates were incubated for 3-6 days prior to imaging with a Leica MZ6 binocular (x20) and cells (budding or serum-induced) were imaged by differential interference contrast with a microscope Leica DMR, using an ImagingSource DMK 23UX174 sCMOS camera.

Time lapses and fluorescent images were obtained using a spinning disk confocal microscope (inverted IX81 Olympus microscope with a 100X objective and a numerical aperture 1.45) and an EMCCD camera (Andor technology, UK). Z-stacks (images of 0.4 µm sections) acquired every 5 min, as described [START_REF] Ghugtyal | Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth[END_REF]. Maximum or sum intensity projections were generated from 21 z-sections with ImageJ software.

Laser illuminations of 445 nm (Turquoise), 488 nm (GFP) and 561 nm (mScarlet) were used. CRIBGFP distribution experiments were carried out as described [START_REF] Corvest | Spatiotemporal regulation of Rho1 and Cdc42 activity during Candida albicans filamentous growth[END_REF]. For filipin analyses, widefield images were acquired on an inverted ZEISS Axio Observer

Virulence assays

HDC was induced in 10 Balb/C mice (Charles Rivers, Italy) per group by injecting the lateral tail vein with an inoculum of 5 x 10 5 cells [START_REF] Sanchez | Relationship between Candida albicans virulence during experimental hematogenously disseminated infection and endothelial cell damage in vitro[END_REF]. Animal body weight was transcripts were determined by RT-PCR using primer pairs described in Bassilana et al., 2005, and UME6 transcripts were determined using UME6.pTm/UME6.mTm (89 bp) primer pair. Actin (ACT1) transcript levels were used for normalization as in Bassilana et al., 2005.

1-3 μm from the tip of germ tubes and longer filaments, as previously reported (Ghugtyal et al., 2015;[START_REF] Martin | Functional analysis of Candida albicans genes whose Saccharomyces cerevisiae homologues are involved in endocytosis[END_REF]. However, in the drs2 mutant, Abp1-GFP was less polarized, as shown in Figure Ad.2.A. The distribution of secretory vesicles was followed using a GFP fusion with the small Rab GTPase Sec4 [START_REF] Li | Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation[END_REF]Puerner et al., 2021a;[START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF]. In germ tubes, both a drs2 mutant and a complemented strain exhibited a cluster of Sec4 t=15 and t=20 min). The GFP-Sec4 signal at the septum was also higher to that at the SPK (Figure Ad.3.B; t=15min and t=20 min), which was not observed in the WT. Together these data suggest that membrane traffic is altered during filament extension in the drs2 mutant, consistent with a defect in hyphal growth maintenance. critical for this process. We could speculate that Drs2 is flipping PS on membrane vesicles at the SPK, which will subsequently contribute to the PS enrichment at the PM cytosolic leaflet.

Importance of Drs2 for plasma membrane and cell wall organization:

In the past decades, pivotal roles played by lipids in cell signaling and morphogenesis in fungi have been identified. PS, for example, is enriched at sites of polarized growth, such as the shmoo projection during mating of S. cerevisiae (Fairn et al., 2011) and S. pombe (Haupt and Minc, 2017), and the absence of this lipid compromises polarized growth (Fairn et al., 2011).

Similarly, PS is enriched at the apex of C. albicans hyphae (Labbaoui et al., 2017) and the absence of PS, resulting from deletion of the PS synthase Cho1 impairs hyphal formation (Chen et al., 2010). The drs2 mutant has an aberrant PS distribution (Labbaoui et al., 2017), with decreased PM and increased internal signal (Figures 8B andC), and is hypersensitive to Papuamide A (Figure 8D) which indicates an overexposure of PS at the external PM leaflet.

This mutant is also hypersensitive to copper exposure (Douglas and Konopka, 2019), suggesting a defect in plasma membrane organization.

Loss of PS asymmetry has been associated with PM disorganization and is a common feature of strains defective for cortical actin organization and membrane trafficking (Douglas and Konopka, 2019). Consistently, the drs2 mutant failed to maintain secretory vesicles and is altered for FM4-64 endocytic uptake (Xu et al., 2019) and several lines of evidence have linked an impaired endocytosis and altered PM lipid distribution to defects in hyphal morphogenesis and cell wall integrity [START_REF] Douglas | BAR Domain Proteins Rvs161 and Rvs167 Contribute to Candida albicans Endocytosis, Morphogenesis, and Virulence[END_REF]Epp et al., 2013b;[START_REF] Knafler | AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans[END_REF]. PS absence in the cho1 mutant also results in increased cell wall thickness (Chen et al., 2010) and in exposure of β(1-3)-glucan [START_REF] Davis | Masking of β(1-3)-glucan in the cell wall of Candida albicans from detection by innate immune cells depends on phosphatidylserine[END_REF]. It is possible that altered PM PS asymmetry in drs2 results in cell wall defects, as the drs2 mutant is hypersensitive to calcofluor white (CFW), a drug that binds to chitin and prevents its assembly, negatively impacting cell wall integrity. Of note, in filamentous fungi, P4-ATPases, including the Drs2 homolog DnfB, interact with the heterotetrametric complex AP-2, necessary for clathrin-mediated endocytosis [START_REF] Martzoukou | The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi[END_REF]Zhang et al., 2019), shown to be essential for cell wall integrity and polarity in different fungi [START_REF] Chapa-Y-Lazo | Yeast endocytic adaptor AP-2 binds the stress sensor Mid2 and functions in polarized cell responses[END_REF][START_REF] De León | The AP-2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast[END_REF][START_REF] Knafler | AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans[END_REF][START_REF] Martzoukou | The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi[END_REF]Zhang et al., 2019).

Importance of Drs2 for phospholipid distribution and interaction with other lipid transporters.

In S. cerevisiae, the DRS2/DNF genes show a certain degree of redundancy for growth [START_REF] Hua | Here, we showed that during serum-induced hyphal growth, Drs2 is found essentially at the filament apex, where it appears to localize at the plasma membrane and also partly at the SPK (Figures 4C & 4D). To understand where Drs2 is critical for its function in filamentous growth, I used a synthetic physical interaction system[END_REF]. Moreover, both Drs2 and the essential flippase Neo1 transport PS and PE, but they are functionally segregated due to the PQ-loop Any1-Neo1 interaction [START_REF] Takar | The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry[END_REF][START_REF] Takar | The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane[END_REF]. In filamentous fungi such as A. nidulans, the deletion of the Drs2 homolog does not have a major defect in cell morphology or hyphal growth [START_REF] Schultzhaus | Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase[END_REF], yet the double deletion mutants dnfA/dnfB (Dnf1 and Drs2 homologs, respectively) and dnfB/dnfD (Drs2/Neo1 homologs, respectively) are non-viable [START_REF] Schultzhaus | The phospholipid flippase DnfD localizes to late Golgi and is involved in asexual differentiation in Aspergillus nidulans[END_REF][START_REF] Schultzhaus | Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase[END_REF], suggesting essential overlapping functions between these flippases. In Fusarium graminearum, despite the redundancies between FgDnfA, FgDnfB, and FgDnfD, these flippases play distinct and specific roles during toxisome biogenesis [START_REF] Yun | Flippases play specific but distinct roles in the development, pathogenicity, and secondary metabolism of Fusarium graminearum[END_REF]. In C. albicans, Drs2 appears to be particularly critical for maintaining hyphal growth, yet the importance of Neo1, which is not essential in this organism (Douglas & Konopka, 2019), should be investigated in this process.

We could speculate that, in the absence of Drs2, Dnf1-3 and/or Neo1 contribute to hyphal growth during initiation, but, once filamentation is initiated, the function of Drs2 could not be compensated, resulting in a switch to isotropic growth (Figure 3).

Alteration of PM PI(4)P (Vernay et al., 2012) in the lipid kinase mutant stt4 resulted in a defect in C. albicans hyphal growth (Vernay et al., 2012) and, similarly, loss of the PM PI(4)P gradient, as well as a defect in filamentous growth, were observed in mutants deleted for the Sac1 phosphatase (Ghugtyal et al., 2015;Zhang et al., 2015). Although there was no apparent alteration of the distribution of PI(4,5)P2 nor Golgi PI(4)P in drs2, we observed a decrease in the PM PI( 4)P gradient at the apex that was rescued after complementation with the DRS2 gene (Figure 5). Together, these data reinforced the importance of PM PI(4)P distribution in hyphal growth (Figure D2). Furthermore, given that deletion of DRS2 and SAC1 together result in a synthetic growth defect, these genes could function in parallel pathways (Figure 9). In S.

cerevisiae, there is a cross-regulation of phospholipid levels. For example, deletion of the PI(4)P phosphatase, Sac1, leads to increased level of PS in endomembranes [START_REF] Foti | Sac1 Lipid Phosphatase and Stt4 Phosphatidylinositol 4-Kinase Regulate a Pool of Phosphatidylinositol 4-Phosphate That Functions in the Control of the Actin Cytoskeleton and Vacuole Morphology[END_REF][START_REF] Tani | Involvement of Sac1 phosphoinositide phosphatase in the metabolism of phosphatidylserine in the yeast Saccharomyces cerevisiae[END_REF], probably due to a misregulation of the lipid transfer protein Osh6/7, which exchanges PS to PI( 4)P (J. Moser von [START_REF] Filseck | A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes[END_REF]. Deletion of the PM PI-4 kinase, Stt4, also increases PM PS levels [START_REF] Trotter | A genetic screen for aminophospholipid transport mutants identifies the phosphatidylinositol 4-kinase, STT4p, as an essential component in phosphatidylserine metabolism[END_REF][START_REF] Wang | Noncanonical regulation of phosphatidylserine metabolism by a Sec14-like protein and a lipid kinase[END_REF]. Given that Drs2 has been shown to transport PS but not PI(4)P, it is likely that other lipid transporters are involved in regulating PI(4)P distribution in drs2. One could speculate that Osh4 regulates Osh7 that, by analogy with S. cerevisiae, would exchange PI(4)P and PS (Maeda et al., 2013;J. Moser von Filseck et al., 2015;[START_REF] Schulz | Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues[END_REF] (Figure D3).

In drs2, we observed altered PM PI(4)P distribution, reduced PM PS and increased internal ergosterol (Figure 5B,6). Drs2 has been proposed to genetically interact with the OSBP lipid transporter, Osh4, in S. cerevisiae, as Osh4 is hyperactive in the absence of Drs2 and vice versa (Hankins et al., 2015b;Muthusamy et al., 2009). Although none of the single osh deletion S2). Strikingly, the PS distribution was also restored in this drs2osh4 (Figure 8A andB), yet the PS asymmetry was not, as this mutant was still sensitive to PapA , similar to drs2 (Figure 8C). In an attempt to address the importance of PS asymmetry, we generated Drs2 mutants analogous to those described in S. cerevisiae as critical for the PS specificity (Baldridge andGraham, 2013, 2012), yet, in such mutants, the PS distribution and growth on PapA were similar to that of the WT cells, precluding directly investigation of PS flipping activity in hyphal growth. Nonetheless, our results suggest that PS asymmetry is not critical for hyphal growth.

However, given that the drs2osh4 mutant is still hypersensitive to fluconazole, similar to drs2, one can propose that Drs2 has a role in plasma membrane organization that is independent of Osh4 and PI(4)P. Ergosterol levels are equivalent across the bilayer, however, there is a dramatic polarization at the hyphal apex. In the drs2 mutant, PS is internalized to the cytoplasm, and it is exposed to the outer leaflet of the PM. Among the PIPs, only the polarized distribution of PI( 4)P pool at the PM. Additionally, ergosterol levels at the PM are decreased and the cytoplasmic levels are augmented.

CONCLUSION

In summary, we characterized the role of flippases during invasive hyphal growth in Candida albicans, with a particular focus on Drs2. We determined that Drs2 is critical for the distribution of phospholipids, such as PS and PI(4)P, as well as of ergosterol. Our results also show a genetic interaction between Drs2 and the lipid phosphatase Sac1, which regulates the PM PI(4)P gradient. In addition, we demonstrated that deletion of OSH4 rescues the invasive hyphal growth defect of drs2, indicating that the requirement for flippase activity across the lipid bilayer can be specifically bypassed by lipid exchange between membrane compartments.

However, given that deletion of OSH4 did not rescue growth on papuamide A nor on fluconazole in drs2, this suggests that Drs2 has also specific functions in plasma membrane organization, independent of Osh4. Together, our results indicate that a critical balance between the activities of two classes of lipid transporters, as well as a lipid phosphatase, regulates hyphal growth and could be conserved during evolution.

Figure 1 .

 1 Figure 1. Physical properties of membranes based on lipid composition. The saturation state of lipids confers fluidity to membranes.Saturated lipids tend to make compacted zones in the membrane, while the unsaturated lipid chains confer fluidity by inhibiting a solid gel formation. Membrane thickness is associated with the acyl-chain length and the presence of sterol. Surface charge is based on the content in anionic lipids, such as PS or PI. The curvature is determined by lipid shape, as lipid's area ratio (polar head/ acyl chain) result in different shapes: a cone locally induces a negative curvature, while a cylinder and an inverted cone induce neutral and positive curvatures, respectively. From[START_REF] Holthuis | Lipid landscapes and pipelines in membrane homeostasis[END_REF]

Figure 2 .

 2 Figure 2. Domain composition of different LTPs. A. Comparison between a yeast OSBP-homologous (Osh4) protein and the human OSBP. Osh4p contains only an ORD containing a lid and the EQVSHHPP signature as indicated. The interaction between the domains of OSBP with PI(4)P and other Arf1 and VAP proteins are shown by black lines. PH, Pleckstrin Homology domain, FFAT, two phenylalanine in an acidic tract domain. B. In yeast, Osh4p exchanges ergosterol from the ER and PI(4)P from the trans-Golgi. C. OSBP interacts via its PH domain with PI(4)P and Arf1-GTP and, through its FFAT motif, with ER-resident VAP receptors forming ER-Golgi contact sites. D. PS is transferred by Osh6p/7p to the PM by using a PI(4)P gradient maintained by Stt4p and Sac1p. PI(4,5)P2 production by Mss4p is favored by PS-and sterol-rich domains. PI(4,5)P2 enables the recruitment of Ist2p to the PM. Ist2p-Osh6/7 interaction anchors Osh6/7 to the ER-PM contact-sites (from Lipp et al., 2020).

Figure 3 .

 3 Figure 3. Sterol and PI(4)P exchange by Osh4. (i) PI/PC exchange between ER and Golgi is performed by Sec14. The kinase Pik1 phosphorylates PI to generate PI(4)P at the Golgi. (ii) PI(4)P is transported by Osh4 to the ER. The energy generated by this PI(4)P transfer along its concentration gradient is used for the transport of ergosterol in an opposite direction. (iii) The low concentration of PI(4)P at the ER is maintained by the phosphatase Sac1, which reduces PI(4)P to PI. From de Saint-Jean et al., 2011.

Figure 4 .

 4 Figure 4. Role of Osh4 in S. cerevisiae vesicle maturation. (i) Vesicles budding off the Golgi and enriched in PI(4)P and Ypt32 recruit Sec2. (ii) The recruitment of Sec4 is then facilitated by the presence of Sec2 and a decrease in PI(4)P concentration via Osh4 (a.). Ypt32 is released from the vesicles when PI(4)P concentration is reduced. (iii) Sec2 interacts with Sec15, the effector of Sec4. Once the mature vesicle is anchored to the PM, Osh4 would continue exchanging PI(4)P for ergosterol (b.), favoring the SNARE-mediated fusion to continue. The vesicle tethering is mediated by the exocyst including Sec15 and other subunits at the PM (not represented). The exocyst subunits Exo70 and Sec3 can bind PI(4,5)P2 at the PM. Vesicle tethering is followed by vesicle fusion with the PM, mediated by the SNARE fusion machinery. Figure based on the models proposed by[START_REF] Smindak | Lipid-dependent regulation of exocytosis in S. cerevisiae by OSBP homolog (Osh) 4[END_REF][START_REF] Ling | Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature[END_REF][START_REF] Schink | Phosphoinositides in Control of Membrane Dynamics[END_REF] Mizuno-Yamakasi et al, 2010.

Figure 5 .

 5 Figure 5. Flippases, Floppases and Scramblases. Phospholipids such as PS (pink) are accumulated at the cytosolic leaflet of membranes by flippases, which belong to the P4-ATPase family. ATP-binding cassette (ABC) transporters, or floppases, facilitate membrane lipid transport in the opposite direction, from the cytosolic to the exoplasmic/luminal leaflet. Flippases and floppases function in an ATP dependent manner. Lipid scramblases translocate lipids down their concentration gradients, without using ATP.

Figure 6 .

 6 Figure 6. Model for Drs2p-Cdc50p autoinhibition and activation by PI4P from Bai et al., 2019. (a.) Auto-inhibited state (without the PI(4)P activator). Autoinhibition is done through the C-terminus wrapping around the cytosolic A, P, and N domains, occupying the ATP-binding pocket. (b.) PI(4)P activates Drs2p-Cdc50p by binding a positively charged pocket in cytosolic leaflet of the membrane. (c.) This activation consists in a 90° rotation of the helix switch uncovering cytosolic domains (A,P and N) and the interaction of the helix with Gea2. Once activated, ATP and the substrate PS bind to Drs2p and PI4P diffuses away from the flippase.

Figure 7 .

 7 Figure 7. Pathways for Hypha-specific gene induction. Signals perceived by receptor molecules are trigger the corresponding signaling cascade, which results in the activation of hypha specific transcription factors. This leads to the transition from budding to filamentous growth. High concentration of the quorum sensing molecule, farnesol, activates Tup1 which negatively regulates hyphal specific gene expression and thus, the yeast to hypha transition does not occur. From Sudbery, 2011

Figure 8 .

 8 Figure 8. Temporal and spatial sequence of cell-division events during hyphal growth in Candida albicans[START_REF] Thomson | High frame-rate resolution of cell division during Candida albicans filamentation[END_REF].

Figure 9 .

 9 Figure 9. Schematic of C. albicans hyphal tip structural organization.In C. albicans, the ER (shown in brown) and the Trans Golgi Network (TGN; depicted as green spheres) extend to the hyphal tip to constitute an "on-site secretory vesicles delivery" by which vesicles are locally budded and delivered, resulting in a short-range trafficking within the tip region[START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF]. Secretory vesicles (represented as grey spheres), loaded with the Rab-type GTPase Sec4 and the GEF Sec2, are transported on actin cables (black strings) by the class V myosin Myo2/Mlc1 complex and accumulate at the Spitzenkörper (SPK) located at the hyphal tip. Actin cable nucleation is generated by the formin Bni1, which is assisted by the polarisome composed by Spea2, Aip5 and Bud6 (molecular components of this structure are schematized in the inset magnification). From the SPK, the vesicles are delivered to the PM. The exocyst, which is composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84, participates in vesicle docking before fusion. Endocytosis takes place at the endocytosis collar, an accumulation of actin patches (red rounds in the scheme) located 1-3µm sub-apically from the apex. Based on models from[START_REF] Sudbery | Growth of Candida albicans hyphae[END_REF][START_REF] Weiner | On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans[END_REF]

Figure 10 .

 10 Figure 10. Phospholipids and enzymes implicated in their synthesis in C. albicans Schematic representation of the main phospholipids distributed in C. albicans endoplasmic reticulum (ER), mitochondrial membrane, Golgi or cellular plasma membrane (PM). Arrows show the biochemical direction of phospholipid production; enzyme regulating each biochemical step are indicated next to the arrows.

Figure 11 .

 11 Figure 11. Biochemical pathway for the synthesis of Ergosterol. Genes required for each biochemical step are indicated next to the arrows; boxed genes are nonessential ones. Antifungal drugs are showed in red. The box on the right represents an alternate pathway leading to the toxic fungistatic sterol. Reproduced from Bhattacharya et al., 2018.

  and XhoI are compatible restriction sites, PCR products digested with SalI and NotI were inserted by ligation into the plasmid pExpARG4XhoI-pARL3ARL3-NotI

  the samples are treated with DpnI, which digests methylated DNA.

  primer pair D4HpAscIBamHI / D4HmMluI and cloned into plasmid pExp-pACT1-mScarlet-CtRac1 (Silva et al., 2019), using AscI and MluI unique restriction sites, yielding pExp-pACT1-mScarlet-D4H. This plasmid was linearized with NcoI and integrated into the RP10 locus.

  products were digested by XmaI and AscI to be inserted into pDup3-(XmaI)-pTEF1-(AscI)-CRYlinker-GFPγ-CDC42-(SbfI)-tTEF1(Silva et al., 2019) generating the intermediate plasmid pDup3-(XmaI)-pDNF2-(AscI)-CRYlinker-GFPγ-CDC42-(SbfI)-tTEF1.Then, the ORF of DNF2 was similarly amplified, using primers CaDnf2p1AscI / CaDNF2m5151SbfI. PCR products were digested by AscI and SbfI to be inserted into pDup3-(XmaI)linearized with StuI for RP10 locus integration. The plasmids pDup3pDRS2DRS2, pDup3pDRS2DRS2 F511Y , pDup3pDNF2DNF2, pDup3pADHOSH4, pDup3pADHDRS2 were linearized by NgoMIV for a NEUT5L integration.

→

  To visualize DNA fragments longer than 2kb: 2 ml of cells grown overnight were centrifuged for 2 min at 13000 rpm. The cells were broken by vortexing (4 min) in the presence of acid-washed beads (0.3 g, 425-600 μm, Sigma), TENS-TX buffer (200 μl) and a solution of PCIA (200 μl), before centrifugation at room temperature for 5 min at 13000 rpm. The aqueous phase, containing DNA and RNA, was transferred to a new tube and a second extraction with PCIA (200μl) was carried out. The aqueous phase was then mixed by inversion in the presence of 1ml of ice-cold ethanol 99% and centrifuged for 10 min at 13000 rpm. Pellets were dried for 5-10 min at 50 ° C and resuspended in TE buffer (400 μl) with 3 μl of RNase (10 mM) and incubated 10 min at 37 ° C. The DNA was subsequently precipitated in ammonium acetate 4M(10 μl) and ethanol 99% (1 ml). After centrifugation (13000 rpm, 5min at ambient T °), pellets were dried at 50 ° C and resuspended in 20 μl of TE 1X buffer.•Verification of strains by PCR:The PCR reaction contains a pair of primers diluted to 1/200 (1.25μl of each primer [1 mM]), 5 μl of HF buffer (5X), 0.5μl of dNTP (10mM), 0.25μl of the phusion enzyme (1/10) and 1 μl l of genomic DNA for a final volume of 25μl. The following PCR program was used: For the RNA extraction, the protocol of the Pure Yeast RNA purification Master Kit (epicenter) of mRNA in 6 μl were added 1 μl of RNAse out (40 U / μl, Invitrogen), 1 μl of DNAse RQ1 (1U/μl, Promega) and 2 μl of MMLV-RT buffer (5X, Invitrogen). The mixture was incubated at 37 ° C 30 min, inactivated at 70 ° C 5 min, then put on ice 5 min. 2μl of random primers (random primers 100 ng / μl) were added to the mixture before incubation 10 min at 70 ° C then 5 min on ice, and addition of 4μl of MMLV-RT buffer (5X, Invitrogen), 1.25μl of dNTP (10mM), 2 μl of DTT (0.1 M), 1 μl of RNAse out and 6 μl of H2O ultrapure (Qbiogen). This mixture was incubated at 37 ° C 2 min before addition of 1 μl of MMLV-RT (Moloney Murine Leukaemia Virus Reverse Transcriptase, 200 U / μl, Invitrogen), and incubation at 37 ° C 50 min before inactivation of the reaction at 70 ° C 10 min. cDNA were kept at -20°C (Hope

  P1 and D4H.P2 and cloned into plasmid pExp-pACT1-mScarlet-CtRac1 [75], using AscI and MluI unique restriction sites, yielding pExp-pACT1-mScarlet-D4H. This plasmid was linearized with NcoI and integrated into the RP10 locus. All other pExp plasmids were linearized with StuI and integrated into the RP10 locus. pDUP3 and pDUP5 plasmids were digested with NgoMIV to release the cassette to be integrated into the NEUTL5 locus.

  Z1 microscope with a 100X(1.3 NA) objective. Huygens professional software version 18.04 (Scientific-Volume Imaging) was used to deconvolve z-stack images of cells expressing PH-FAPP1 [E50A,H54A]-GFP. Bars are 5 µm.Filament lengths and diameters were measured from DIC images, using image J software. Golgi cisternae were quantitated from deconvolved MAX projection images. Quantitation of PI4P distribution was performed on SUM projections, using the Matlab program HyphalPolarity[START_REF] Ghugtyal | Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth[END_REF]. Quantitation of PS plasma membrane and internal mean signals was performed on central z-sections, also using the Matlab program HyphalPolarity. Unless indicated otherwise, error bars represent the standard deviations.

Figure 1 :

 1 Figure 1: Drs2 has a unique role in C. albicans. A) Invasive filamentous growth

Figure 2 :

 2 Figure 2: Drs2 is critical for virulence in a mouse model of systemic candidiasis.

Figure 3 :

 3 Figure 3: Drs2 is critical for maintaining polarized filament extension. A) The

Figure 4 :

 4 Figure 4: Drs2 localizes preferentially to the filament apex. A) The Drs2-GFP and

Figure 5 :

 5 Figure 5: Plasma membrane PI4P distribution is altered in the drs2/drs2 mutant.

Figure 6 :

 6 Figure 6: Distribution of ergosterol is altered in the drs2/drs2 mutant. A) The

Figure 7 :

 7 Figure 7: Deletion of OSH4 specifically rescues hyphal invasive growth in the

Figure 8 :

 8 Figure 8: Deletion of OSH4 rescues plasma membrane PI4P gradient and

Figure 9 :

 9 Figure 9: DRS2 and SAC1 genetically interact during C. albicans development.

Figure S1 :

 S1 Figure S1: Flippase deletion mutant strains verification. A) DRS2 and DNF1-3

Figure S2 :

 S2 Figure S2: Deletion of OSH2 and OSH3 do not recover invasive growth in the

Figure S3 :

 S3 Figure S3: Drs2 residues analogous to those of S. cerevisiae required for PS

  Figure 1

  Figure 9

  at the filament apex, corresponding to the SPK (Figure Ad.2.B.), yet there appears to be an increase in Sec4 vesicles throughout the filaments in the drs2 mutant (Figure Ad.2.B.).Furthermore, Sec4 labeled vesicles were observed at the site of division in both WT and drs2 cells. However, the Sec4 signal associated with the cell division site was more intense in the drs2 mutant compared to the WT strain, and the signal at the tip was dramatically reduced in the mutant (Figure Ad.2.B).As shown inFigure 3E, the SPK is also depolarized in drs2 cells and I constructed a strain coexpressing fluorescently tagged Sec4 and Mlc1, to follow temporal dynamics of these two markers (Figure Ad.3). In WT cells, Mlc1 was found persistently at the hyphal tip during filamentous growth, however, when the maximal signal is observed, Mlc1 was also localized at the septum (Figure Ad.3.A; t=5min and t=10min). This peak at the septum was transient, as at t=25 min, Mlc1 was again observed only at the hyphal tip (Figure Ad.3.A). GFP-Sec4 was also found persistently at the tip of the hypha and the signals at the septum, besides not being observed in all WT cells, were never higher than the signals at the tip. In contrast, in the drs2 mutant, the signals associated with the SPK, both for Mlc1 and Sec4, decreased dramatically after septum recruitment; at 15-20 min little to no Mlc1 localized to the SPK (Figure Ad.3.B;

Figure Ad. 12 .

 12 Figure Ad.12. Drs2 is required for maintaining hyphal filamentous growth. The drs2 mutant filaments are altered in diameter and length. WT and drs2 mutant cells were incubated on media containing 0.5% agar and 75% FCS on microscope slides (pads) and images were acquired with a 40X objective after an overnight incubation (24h). Filamentous length and diameter were determined, as well as the distance between branches, and plotted in the bottom graphs. Each compartment corresponds to a cell (delimited by septa). Cell volume was calculated as the volume of a cylinder (V=h*π*r 2 ). n = 50 cells. Bars indicate SD. *** indicates a P value of 0.0006; **** indicates a P value < 0.0001.

Figure Ad. 2 .Figure Ad. 3 .

 23 Figure Ad.2. Polarization of exocytosis and endocytosis is impaired in the drs2 mutant. A. Drs2 is necessary for the clustered distribution of endocytosis sites. WT and drs2 cells after 60-and 90-min FCS induction, respectively, are shown. Central Z and maximum projection images (from 21 Z-sections) are shown for the indicated strains. B. Drs2 is important for the distribution of secretory vesicles. Central Z and maximum projection images of drs2 and drs2 + DRS2 cells expressing GFP-Sec4 over time. Blue arrowheads indicate the localization of the cluster of vesicles at the filament tip and at the septum. Scale bars correspond to 5μm.

Figure Ad. 4 .

 4 Figure Ad.4. Forced localization of Drs2 to the SPK does not substantially alter filamentous growth. A. Drs2-GFP localizes as a crescent at the hyphal tip, schematized on top. Images of cells expressing mScarlet-CtRac1, together with Drs2-GFP after 90 min FCS induction. Images are sum projections of 16 z-sections. B. Drs2-GFP forced localization at the SPK. Images of cells expressing Drs2-GFP as the sole copy, as well as Mlc1-miRFP-GNB. Images are sum projections of 16 z-sections. Drs2-GFP distribution is shown in brown/green and Mlc1 in orange in the schematic on top. Scale bar is 5 µm. C. Forced localization of Drs2 to the SPK does not substantially alter filamentous growth. The percentages of filamentous cells were determined for each strain. The mean percentage of hyphae +/the SD (bars) from 3 experiments (n = 100 cells each).

Figure Ad. 13 .

 13 Figure Ad.13. Forced localization of Osh4 to the SPK do not alter filamentous growth. A. Osh4-GFP localizes in the cytosol. Image of a hyphal cell from a strain expressing Osh4-GFP as the sole copy. Sum projections of 16 z-sections are shown B. Synthetic physical interactions between Mlc1-miRFP-GNB and Osh4-GFP relocalizes Osh4 to the SPK. Sum projections of 16 z-sections are shown. C. Forced localization of Osh4 to the SPK does not alter filamentous growth. The percentage of filamentous cells was counted for each strain n = 100 cells.

  endocytic patches clustering during hyphal growth, indicative of a defect in membrane traffic (Figure Ad.2.A. and Ad.3.B.). In addition, a mutant of the Drs2 non-catalytic subunit, cdc50

  mutants was affected for serum-induced hyphal invasive growth (Figure 7A-D and S2), the deletion of OSH4 rescued hyphal growth and restored the PM PI(4)P in drs2 (Figure 7A-D and

Figure D1 .

 D1 Figure D1. Drs2 is necessary for a correct localization of the polarity machinery. In WT cells, proteins implicated in polarized growth such as Cdc42, Mlc1 and Sec4 are required at the filament apex and, temporarily, at the septum site. In the drs2 mutant, the absence of Drs2 leads to a misplacement of the polarity machine to the septum, losing completely the apical localization.

Figure D3 .

 D3 Figure D3. Drs2 negatively regulates Osh4 activity as PI(4)P transporter. In WT cells, Osh4 activity would be downregulated by Drs2. In the absence of the flippase, Osh4 hyperactively transport PI(4)P all over the PM dissolving the gradient. Deletion of OSH4 abolish the hyperactivity defect. "?" could be Osh7 exchanging PI(4)P, and PS.

Table 1 .

 1 Lipid classification. Schematic representation of lipid groups. Classification is based on their structure and synthesis pathway. Condensation of thioesters give rise to fatty acids, glycerides, glycerophospholipids and sphingolipids. Sterols are synthetized by condensation of isoprene units

Table 2 . Media and buffers

 2

	Growth media
	Media	Composition for 1L
	YEPD + Uri	2.2% bacto peptone, 1.1% yeast extract, 2% Agar, 2%glucose,0.1% Uridine
	LM2	8g Yeast nitrogen base, 55mg adenine, 55mg tyrosine
	TYE	5g NaCl, 16g bactotryptone, 10g yeast extract
	TYE + Ampicillin	5g NaCl, 16g bactotryptone, 10g yeast extract, 100 μg/ml
	SPIDER	10g nutrient (1%), 10g Mannitol (1%), 2g K2HPO4 (0.2%),15g Agar 1.5%
	FCS	50% YEPD + 50% FCS
	Selective media
	Media	Composition for 1L
	-Ura	LM2, glucose 2%, 1/100 volume of -leu+leu drop out
	-His-Ura	LM2, glucose 2%, 1/100 volume -His drop out
	-His+Ura	LM2, glucose 20%, uridine(80mg), 1/100 volume -His dropout
	-His-Ura-Arg	LM2 + 20% glucose + 1/100 volume of -His-Arg drop out.
	YEPD + NAT	2.2% bacto peptone, 1.1% yeast extract, 2% Agar, 2%glucose, 0.1% Uridine, 200μg/ml NAT
	YEPD + HYG	2.2% bacto peptone, 1.1% yeast extract, 2% Hagar, 2%glucose, 0.1% Uridine, 1mg/ml hygromicin
	Buffers and solutions
	TENS-TX	10mM TrisHCl pH8.0, 1mM EDTA pH8.0, 100mM NaCl, 1%(w/v) SDS, 2% (v/v) Triton X100
	PCIA ( PhenolChloroform IsoamylAlcohol)	25ml Phenol, 24ml Chloroform, 1ml Isoamyl Alcohol
	TE 1X (Tris-EDTA)	10mM Tris HCl pH8.0, 1mM EDTA pH8.0
	Sorbitol	1M
	PBS	Phosphate Buffer Saline 1M
	LitAct-SDS	Lithium acetate 200mM, 1%SDS

Table 3 . Plasmids

 3

	Name

Table 4 . Primers for plasmid construction Name Sequence

 4

	CaDNF2m2 204	GAATAATACATCCCCACATCAG
	CaDNF2m5 151_SbfI	ATCCTGCAGGTTAATTCATTGAAGATACTCTTTTTTG
	CaDnf2m51 51MluI	ATACGCGTTTAATTCATTGAAGATACTCTTTTTTG
	CaDnf2mup 21AscI	TATAGGCGCGCCAAGAGTGTGGATAGCAATTCG
	CaDNF2N5	GACAAACAATAAAATAAAATTTAACAAGACATAATAAGACAATTC
	50Sm1968	TCGAGACATTCTTGATTGTTTAGTTGGCGTTACCCCAGC
	CaDNF2N5	GCTGGGGTAACGCCAACTAAACAATCAAGAATGTCTCGAGAATTG
	50Sp1885	TCTTATTATGTCTTGTTAAATTTTATTTTATTGTTTGTC
	CaDNF2p1 724	CCAATAGAATGAATAACTCACAATC
	CaDnf2p1A scI	GGCGCGCCATGACTAAAAATAAGCACAATTTCACGC
	CaDnf2pup 1000XmaI	TACCCGGGCTGGTGTAGGGTATAGCCTAC
	CaDrs2F51	ATATTTGATCAATTCAACCGTGACATACAATGAAATGGGTACCAAA
	1Ym	TTGGAAAATAAAATCC
	CaDrs2F51	GGATTTTATTTTCCAATTTGGTACCCATTTCATTGTATGTCACGGTT
	1Yp	GAATTGATCAAATAT
	CaDrs2m17 32	CATTTCTGGTTAATGTACCGG
	CaDrs2m49 64NotI	TATAGCGGCCGCgagttggttaataacaaaatatcag
	CaDrs2m94 0	GGACAACATCTCCCACTTG
	CaDRS2m MluI	ATACGCGTTTACTTACTTTCTGATAATTCCCC
	CaDrs2p13 36	CGTACTGATGTGGAAAGAATC
	CaDrs2p44 6	CGTCACGAGATTTTGATATAACCC
	CaDRS2pA scI	GGCGCGCCATGTCCAATTATAATCGGACAG
	CaDrs2pup 1000SalI	ATGTCGACcttttaatatagccgaattccccg
	CaDrs2pup 1000SpeI	TAACTAGTCTTTTAATATAGCCGAATTCCCCG
	CaDrs2QQ_	CTATTTGTGGGGGAAACATGAGGTACTGCTCCAATAATTGATGTAA
	m755	CAAGGAAAAATAAATTGGC
	CaDrs2QQ_	GCCAATTTATTTTTCCTTGTTACATCAATTATTGGAGCAGTACCTCA
	p691	TGTTTCCCCCACAAATAG
	CaOSH4m1 293MluI	ATACGCGTTTAAAAGACTATTTCTTTTTCATCTTCC

Table 5 . Primers for KO mutants

 5

	Name	Sequence
	CaDNF2pKO	cacctaattctactttatccaatttcgaattgctatccacactcttatgactaaaaataagcacaatttca cgcTGTGGAATTGTGAGCGGATA
	CaDNF2mKO	gtatgtgtgtttgttgtctataatgttttcttttatgttatgcaaatgaaaattgctgttaattcattgaagat actcTTTCCCAGTCACGACGTT
	CaDNF2mTETOKin	TggtgatcactagggtcgtcattgttattaaCtccatcactactattatttagcgtgaaattgtgcttat ttttagtcatCTAGTTTTCTGAGATAAAGCTG
	CaDNF2pTETOKin	Gtctagtggttgccatatttttcttatttttttattttttttttttgcacgtttgggtataattttccaacaaatc aaatacAGGAATTGATTTGGATGG
	CaOSH2mKO	caatcaacaattaaaacagtctaaaatatgtctcccgtgttcgccaaatcttgtgcctttcttctaggc caatacTTTCCCAGTCACGACGTT
	CaOSH2pKO	ccataataaattactgataaaacactaacaactttagaataaacagctaaaatcatgaatgattcacc ccaactaagtgcTGTGGAATTGTGAGCGGATA
	CaOSH3mKO	ggattctatgatctaaataatataacgtctaatagatatgaaaaagacaaagaaatagtaaaatttac cacaatttaatcTTTCCCAGTCACGACGTT
	CaOSH3pKO	cccactcaatcccattatttctttcccttgacagttgaactttcccataccaacttgaaatatggaaact ttagaagTGTGGAATTGTGAGCGGATA
	CaOSH4mKO	gatgtactactattataaacattattttacgaaattgaaaaaacctactctaaattcatttaaaagactat ttcTTTCCCAGTCACGACGTT
	CaOSH4pKO	ctatattttaaaataacacatcgtcttcaagccaacagtatatttttttgtcatgtcaaactccaaatcca gttctgaagTGTGGAATTGTGAGCGGATA

Table 6 . Primers for KI strains

 6

	CaCdc10pFa_S1AFP	caatcaaaccaagatttgaagaacacctctggtgtgccaaatgctcctatgttccaatcaactacaggtac tgctgctgctagaGGTGCTGGCGCAGGTGCTTC
		CGCGTTTTGCTTTTCAACAAACACACAAAAGAAGAGGAATAC
	CaCdc10mFA_S2	AAAAAAGTAAAATCACATTATATCAATAACAAACctgatatcatcgat
		gaattcgag
	CaMlc1pFA	gatgagttattaaaaggggtcaatgtaacttctgatggaaatgtggattatgttgaatttgtcaaatcaatttt agaccaaGGTGCTGGCGCAGGTGCTTC
		CGAACAAGACTATACAATAACTATAATTTGTAAAACTTGTAGT
	CaMlc1mFA	ATATATATTTCAATGGTTAATTGTAAATTTTCTTTTATtctgatatcat
		cgatgaattcgag
		GTGAAAAGGGTCCTTTTAAAAAGGAAAAAATTGCCACGGATA
	CaAbp140xFP_S1	GAAGATTATTAGTCAATAGAAAGAAACAATTGAAAATGTACC
		GTAATTGGCTACAGGCTGTATTTAGAGGAggtgctggcgcaggtgcttc
		GTCTCATCCTTATATTCAAGGTATTGATGTAACTTCATTAATG
	CaAbp140xFP_S2	AATCAACTGATACGAGAAATTTGGCAATTATTCAAACATTTAG
		ATAATTTTTTATATATTACTCTAAtctgatatcatcgatgaattcgag
	CaDRS2.P1(S1AFP)	caagttgaaggtcaagatcaagataaaattgttagattatatgatactactaaaaaaagaggagtttttgg ggaattatcagaaagtaagGGTGCTGGCGCAGGTGCTTC

Table 7 . KO mutant and complemented strains.

 7

	STRAIN	RELEVANT GENOTYPE	REFERENCE
	BWP17	ura3Δ::λimm434/ura3Δ::λimm434 his1Δ::hisG/his1Δ::his arg4::hisG/arg4Δ::hisG	(Wilson et al., 2000)
	PY3303-3304	Same as BWP17 with DRS2∆::URA3/DRS2	(Labbaoui et al., 2017)
	PY3310	Same as PY3303 with DRS2∆::URA3 / DRS2∆::HIS1	(Labbaoui et al., 2017)
	PY3361	Same as PY3304 with DRS2∆::URA3/ DRS2∆::HIS1	Provided by H. Labbaoui
	PY3375	Same as PY3310 with RP10::ARG4	(Labbaoui et al., 2017)
	PY3377	Same as PY3361 with RP10::ARG4	Provided by H. Labbaoui

Table 8 . Strains with KI and protein reporters:

 8

	PY2263	Same as BWP17 with RP10::ARG4-pExpArgACT1pCRIB-GFP	(Bassilana et al., 2005)
	PY3785	Same as BWP17 with ABP1/ABP1::ABP1-GFPγ-ARG4	Provided by H. Labbaoui
	PY4665-64	Same as PY3303 with DRS2∆::URA3/DRS2::DRS2-GFPγ-HIS1	This study
	PY4676-75 Same as PY4671 with SEC4/SEC4::GFP-SEC4-URA3	This study
	PY4677	Same as BWP17 with SEC4/SEC4::GFP-SEC4-URA3	This study
	PY4928-31,51,52	Same as PY4665 with MLC1/MLC1::MLC1-mScarlet-ARG4	This study
	PY4972, PY5792, 76-77, 93	Same as PY3310 with RP10::ARG4-pExpArgACT1pCRIB-GFP	This study
	PY5063-65	Same as PY4676 with RP10::ARG4-pExpARGpDRS2DRS2	This study
	PY5097-100	Same as PY3310 with ABP1/ABP1::ABP1-GFPγ-ARG4	This study
	PY5218-22	Same as PY4676 with MLC1/MLC1::MLC1mScarlet-ARG4	This study
	PY5349,47-50	Same as PY4677 with MLC1/MLC1::MLC1mScarlet-ARG4	This study
	PY5385	Same as BWP17 with MLC1/MLC1:: MLC1-miRFP670GNB-URA3	Puerner
	PY5507-06,08	Same as BWP17 with CDR1/CDR1::CDR1-GFP-SAT1 This study

Table 9 . Strains with lipid reporters:

 9

	PY1206= PY5566	Same as BWP17 with RP10::ARG4-pADHGFP-PH PLCδ1 -PH PLCδ1 -GFP	(Vernay et al., 2012)
	PY2045	Same as BWP17 with RP10::ARG4-pADH1-PH FAPP1[E50A,H54A] -GFP	Provided by Ghugtyal
	PY3239 Same as BWP17 with RP10::ARG4-pACT1GFPyeLactC2	(Labbaoui et al., 2017)
	PY3362 Same as PY3310 with RP10::ARG4-pACT1GFPyeLactC2	(Labbaoui et al., 2017)
	PY3873	Same as PY3310 with RP10::ARG4-pADH1-PH FAPP1[E50A,H54A] -GFP	This study
	PY4050	Same as PY3310 with RP10::ARG4-pADHGFP-PH PLCδ1 -PH PLCδ1 -GFP	This study (Labbaoui)
	PY5134-36	Same as PY3310 with NEUT5L::SAT1-pDup3pADHGFPyeLactC2	This study
	PY5151-53	Same as PY5134 with RP10::ARG4-pExpARGpDRS2DRS2	This study
	PY5254-55	Same as PY5246 with RP10::ARG4-pACT1GFPyeLactC2 This study
	PY5336-34,35,37	Same as PY3921 with RP10::ARG4pACT1GFPyeLactC2 This study
	PY5364-37,65	Same as PY5296 with RP10::ARG4pACT1GFPyeLactC2 This study
	PY5373 Same as PY5297 with RP10::ARG4pACT1GFPyeLactC2 This study
	PY5156,
	54,55	Same as PY5134 with RP10::ARG4-pExpARGpDRS2DRS2 F511Y	This study
	5470-73,
	PY5568, 5551	Same as PY3310 with RP10::ARG4-pACT1-GFP-(PH OSH2[H340R] )2-GFP	This study

Table 10 . RT-PCR primers

 10

	ACT1mTM	ACATTTGTGGTGAACAATGG
	ACT1pTM	ATGTTCCCAGGTATTGCTGA
	CaDRS2p3485TM	CGGCATTAGTGGTTACCATGT
	CaDRS2m3547TM	GCAGGATACCAACCTAACCATAA

Table I : Strains used in the study.

 I

	PY5003	Same as PY3375 with NEUT5L::SAT1-pADH1-GFP-(PH OSH2[H340R] ) 2 -GFP CDR1/CDR1::CDR1-GFP-SAT1	This study
	PY5674 PY6400	pADHDNF2 Same as PY5296 with RP10::ARG4-Same as PY5804 with dnf1∆::URA3/	This study This study
	PY5005	Same as PY4861 with NEUT5L::SAT1-pOSH4OSH4 dnf1∆::HYGB	This study
	STRAIN PY5677 PY6407	RELEVANT GENEOTYPE pADHDNF2 Same as BWP17 with DNF2∆::HIS1/ Same as PY5568 with NEUT5L::SAT1-	REFERENCE This study This study
	BWP17 PY5042	ura3Δ::λimm434/ura3Δ::λimm434 Same as PY3310 with RP10::ARG4-DNF2 pDRS2DRS2	[68] [31]
	PY5741 PY6408	his1Δ::hisG/his1Δ::hisG pDRS2DRS2 Same as BWP17 with dnf3∆::URA3/ Same as PY4671 with osh2∆::URA3/	This study This study
	PY5045	arg4::hisG/arg4Δ::hisG Same as PY3310 with RP10::ARG4-dnf3∆::SAT1 osh2∆::HYGB	This study
	PY1206 PY5746 PY6431	Same as BWP17 with RP10::ARG4-pDRS2DRS2F520Y Same as PY5677 with DNF2:: DNF2-Same as PY4671 with osh3∆::URA3/	[48] This study This study
	PY5134	pADH1-GFP-PHPLCδ1-PHPLCδ1-GFP Same as PY3310 with NEUT5L::SAT1-GFPγ-URA3 osh3∆::ARG4	This study
	PY2263 PY5801 PY6432	Same as BWP17 with RP10:: ARG4 pACT1GFP-yeLactC2 Same as PY5741 with RP10::ARG4-Same as PY4671 with sac1∆::ARG4/	[40] This study This study
	PY5151	pACT1-CRIBGFP Same as PY5134 with RP10::ARG4-pExp and his1Δ::HIS1 sac1∆::HYGB	This study
	PY2045 PY5804 PY6436	Same as BWP17 with RP10:: pADH1-pDRS2DRS2 Same as BWP17 with dnf2∆::HIS1/ Same as BWP17 with sac1∆::ARG4/	[47] This study This study
	PY5156	FAPP1 [E50A,H54A] GFP Same as PY5134 with RP10::ARG4-dnf2∆::ARG4 sac1∆::HYGB	This study
	PY3303 PY5814	Same as BWP17 with DRS2∆::URA3/ pDRS2DRS2F520Y Same as PY5804 with NEUT5L::URA3	[31] This study
	PY5218 PY5915	DRS2 Same as PY4676 with MLC1/MLC1-Same as PY5746 with MLC1 /MLC1-	This study This study
	PY3239	Same as BWP17 with RP10::ARG4-mScarlet-ARG4 mScarlet-ARG4	[31]
	PY5246 PY5919	pACT1GFP-yeLactC2 Same as BWP17 with osh7a∆::SAT1/ Same as PY5814 with NEUT5L::SAT1-	This study This study
	PY3310	Same as BWP17 with drs2∆::HIS1/ osh7α∆::HIS1 pADHDNF2	[31]
	PY5256 PY5922	drs2∆::URA3 Same as PY5246 with RP10::URA3 Same as PY5871 with NEUT5L:: SAT1-	This study This study
	PY3375	Same as PY3310 with RP10::ARG4 ARG4 pADHDNF2	[31]
	PY3873 PY5296 PY6037	Same as PY3310 with RP10:: pADH1-Same as PY4671 with osh4∆::URA3/ Same as BWP17 with RP10::ARG4-	[47] This study This study
		FAPP1 [E50A,H54A] GFP osh4∆::HYGB pACT1-mScarlet-D4H
	PY3921 PY5297 PY6076	Same as BWP17 with osh4∆::URA3/ Same as PY4671 with osh7α∆::HYGB/ Same as PY3921 with RP10::ARG4-	This study This study This study
		osh4∆::HIS1 OSH7a∆::URA3 pACT1-mScarlet-D4H
	PY3945 PY5336 PY6083	Same as BWP17 with osh3∆::HIS1/ Same as PY3921 with RP10::ARG4-Same as PY3310 with RP10::ARG4-	This study This study This study
		osh3∆::URA3 pACT1GFP-yeLactC2 pACT1-mScarlet-D4H
	PY3974 PY5349 PY6213	Same as PY3921 with RP10::ARG4 Same as PY4677 with MLC1/MLC1-Same as PY6083 with NEUT5L:: pACT1-	This study This study This study
	PY3977	Same as PY3945 with RP10::ARG4 mScarlet-ARG4 Turquoise-CtRAC1-SAT1	This study
	PY3995 PY5364 PY6218	Same as BWP17 with osh2∆::HIS1/ Same as PY5296 with RP10::ARG4-Same as PY6083 with NEUT5L::SAT1-	This study This study This study
		osh2∆::URA3 pACT1GFP-yeLactC2 pDRS2DRS2
	PY4002 PY5539 PY6235	Same as PY3995 with RP10::ARG4 Same as PY5296 with RP10::ARG4 Same as PY4946 with RP10::ARG4	This study This study This study
	PY4050 PY5568 PY6237	Same as PY3310 with RP10::ARG4-Same as PY3310 with RP10::ARG4-Same as PY6037 with NEUT5L::pACT1-	This study This study This study
		pADH1-GFP-PHPLCδ1-PHPLCδ1-GFP pADH1-GFP-(PH OSH2[H340R] ) 2 -GFP Turquoise-CtRAC1-SAT1
	PY4665 PY5613 PY6239	Same as PY3303 (URA3) with DRS2:: Same as BWP17 with Same as PY4665 with NEUT5L::	This study This study This study
		DRS2-GFPγ-HIS1 CDC10/CDC10::CDC10-mScarlet-ARG4 mScarlet-CtRAC1-SAT1
	PY4671 PY6241	Same as PY3310 with drs2∆::HIS1/ and NEUT5L::SAT1-Same as PY5746 with NEUT5L::	This study This study
		drs2∆::SAT1 pADH1miRFP670CtRac1 mScarlet-CtRAC1-SAT1
	PY4861 PY5615 PY6260	ura3Δ::λ imm434/ura3Δ::λ imm434 Same as 3310 with with Same as PY3310 with RP10::ARG4-	[66] This study This study
		his1::hisG/HIS1::his1::hisG CDC10/CDC10::CDC10-mScarlet-ARG4 pDRS2DRS2QQ-GA
	PY6284	arg4::hisG/URA3::ARG4::arg4::hisG and NEUT5L::SAT1-Same as PY5134 with RP10::ARG4-	This study
	PY4928	Same as PY4665 with MLC1 /MLC1-pADH1miRFP670CtRac1 pDRS2DRS2QQ-GA	This study
	PY5619 PY6286	mScarlet-ARG4 Same as BWP17 with RP10::ARG4-Same as PY6083 with osh4∆::HYGB/	[47] This study
	PY4946	Same as BWP17 with dnf1∆::SAT1/ pADH1-GFP-(PH OSH2[H340R] ) 2 -GFP osh4∆::SAT1	This study
	PY5626 PY6393	dnf1∆::HYGB Same as PY3921 with RP10::ARG4-Same as PY4861 with	This study This study
	PY4972	Same as PY3310 with RP10:: ARG4 pADH1-GFP-(PH OSH2[H340R] ) 2 -GFP CDR1/CDR1::CDR1-GFP-SAT1	This study
	PY5630 PY6395	pACT1-CRIBGFP Same as PY5296 with RP10::ARG4-Same as PY3375 with	This study This study

Table II : Primer sequences

 II

	AGAAATAGTAAAATTTACCACAATTTAATCTCTGATATCATCGATGAATTC 24. Sartorel E, Barrey E, Lau RK, Thorner J. Plasma membrane aminoglycerolipid RT.PCR
	DRS2.pTm	GAG CGGCATTAGTGGTTACCATGT
	PRIMER SAC1.P1 DRS2.mTm flippase function is required for signaling competence in the yeast mating pheromone SEQUENCE AATATAAACACTATTTTTTCCACTTTCTTTAATCTAACCTTATACGCAACTAT GCAGGATACCAACCTAACCATAA
	DNF1.P1 SAC1.P2 DNF1.pTm DNF1.mTm response pathway. Mol Biol Cell. 2015;26(1):134-50. Epub 2014/11/08. doi: CAAGTGACAACTCTCCAGTTAGCTCCTATTATTTGCATCGACCAAATCTAG AAGATCTTACTAGAACATCGAAAGTTGGAGTGACCATTTGCTGAAGCTTC ACAATTATATAACAGAAAATGGTGTTAGAAGCTTCGTACGCTGCAGGTC ATCTATTATATAAAATAAGCAGCTACAACTAAAGACAACTTTCTAAAAAAA GCACAATCAACGGGAAGTCATAC AATGCCTGCACGTTTATCCAA
	GTACGCTGCAGGTC CGAAAAATGTAAAACCGAAATCAAATGCAATGATACAATAAACTGAAACC CAATTACCACATAAAGTAGGTTAAGGATTCAAAGCTACACTATTCCTCTGA TATCATCGATGAATTCGAG AAAACAACCCCCACAGCTAATCAATTCTTTCTGATATCATCGATGAATTCG AG GGCGCGCCATGACTAAAAATAAGCACAATTTCACGC ATACGCGTTTAATTCATTGAAGATACTCTTTTTTG TGACAAGACCGGGACATTA 10.1091/mbc.E14-07-1193. PubMed PMID: 25378585; PubMed Central PMCID: DNF1.P2 DNF2.P5 DNF2.P6 DNF2.pTm DNF2.mTm TCCGTAACCCAGCCAAT DNF3.pTm AAGCATCAATGGCAGGTCATC PMCPMC4279224.
	DNF2.P1 DNF2.P2 DRS2.P3 DRS2.P4 DNF3.mTm 25. Schultzhaus Z, Yan H, Shaw BD. Aspergillus nidulans flippase DnfA is cargo of CACCTAATTCTACTTTATCCAATTTCGAATTGCTATCCACACTCTTATGACT AAAAATAAGCACAATTTCACGCGAAGCTTCGTACGCTGCAGGTC CTTGTATGTGTGTTTGTTGTCTATAATGTTTTCTTTTATGTTATGCAAATGA AAATTGCTGTTAATTCATTGAAGATACTCTCTGATATCATCGATGAATTCG AG TAACTAGTCTTTTAATATAGCCGAATTCCCCG GCTGCCGTGCTTGTTCTTG TATAGCGGCCGCGAGTTGGTTAATAACAAAATATCAG OSH4.P3 ATGTCGACAAAACAACACAAGAAAGAGCTGCTGCTG OSH4.P4 OSH4.pTm TTTGATTACTGCTGCTCCTT OSH4.mTm the endocytic collar and plays complementary roles in growth and phosphatidylserine AATGTGACCACTGGATGCTT TATAGCGGCCGCGGTTAGTAACGCTCTTAGAACGTTGG DRS2.P5 CAAGTTGAAGGTCAAGATCAAGATAAAATTGTTAGATTATATGATACTAC OSH7a.pTm TGAAGCAGCTGTCAAATCTAATGTG asymmetry with another flippase, DnfB. Mol Microbiol. 2015;97(1):18-32. Epub
	DNF2.P3 DNF2.P4 DRS2.P6 OSH7a.mTm CTGTCCCTCGTTATCCATTTCATC CACCTAATTCTACTTTATCCAATTTCGAATTGCTATCCACACTCTTATGACT AAAAATAAGCACAATTTCACGCTAAATAATAGTAGTGATGGAGTGTGGAA TTGTGAGCGGATA GTATGTGTGTTTGTTGTCTATAATGTTTTCTTTTATGTTATGCAAATGAAAA TAAAAAAAGAGGAGTTTTTGGGGAATTATCAGAAAGTAAGGGTGCTGGC GCAGGTGCTTC GGGAGTGAGATAGTTGTATATCAAACAGTTCTATAGCTTTAAATAAATAA AAAAAAACATTCATTGATTGCATTAATATACTACTCATACTCTATTTATCTG 2015/04/08. doi: 10.1111/mmi.13019. PubMed PMID: 25846564. OSH7α.pTm GTAACTCGTCTGCAGCAATCATG OSH7α.pTm CCTCGTTGTCCCATTTACCAA 26. Yun Y, Guo P, Zhang J, You H, Guo P, Deng H, et al. Flippases play specific but
	TTGCTGTTAATTCATTGAAGATACTCTTTTTTGATTTTCGTAGCTTATTCTA GATGCTCTTTCCCAGTCACGACGTT ATATCATCGATGAATTCGAG GGTAGCTACAGAAGAAATTCCATTAGAAGATTTTGATGATGAACAAAAAA AAGCCAGAAAACAGAGGGAAGAT TCACTGGATGCTTTCTCTTAACAAA distinct roles in the development, pathogenicity, and secondary metabolism of DNF2.P7 OSH2.pTm OSH2.mTm
	DNF3.P1 OSH3.pTm Fusarium graminearum. Mol Plant Pathol. 2020;21(10):1307-21. Epub 2020/09/04. CTACAGATTCAACTTTATAGAATCCTACATTGAGTCACCTGTCTCATCATTT GAGCATCTAGAATAAGCTACGAAAATCAAAAAAGAGTATCTTCAATGAAT GGGTAAACTTGCACCTACGGATT
	OSH3.mTm	GGTCATTAATTTACAACTACAGTGAATCATACAATTAGATTTGGAAGCTTC GGTGCTGGCGCAGGTGCTTC GCAGTGTCTGTATCTCCCTTTTCA
	GTACGCTGCAGGTC CCTCTCATTCTTTATATAAATATGCTTGAAATTCTTGTATGTGTGTTTGTTG TGGCTCCACTTACAAATCATAGTGA doi: 10.1111/mpp.12985. PubMed PMID: 32881238; PubMed Central PMCID: DNF2.P8 UME6.pTm
	DNF3.P2 UME6.mTm	GCTCGGGTGAATTGAGACAATTTTGATGTATCTTGTATATTTGTATAAATA TCTATAATGTTTTCTTTTATGTTATGCAAATGAAAATTGCTGTTATCTGATA GCTTTACCAATCCTAGTCCCAACTC
	TCATCGATGAATTCGAG PMCPMC7488471. CTTAGATGTACATTTCTTTTGGTTTTGGAAACTCAATAGACACCTCTGATAT MLC1.P1 GATGAGTTATTAAAAGGGGTCAATGTAACTTCTGATGGAAATGTGGATTA SAC1.pTm CCCATTCCACAGCAACAGATG CATCGATGAATTCGAG OSH4.P1 TGTTGAATTTGTCAAATCAATTTTAGACCAAGGTGCTGGCGCAGGTGCTTC SAC1.mTm TTCAACACCACCTCCCAGTGT 27. Gilbert MJ, Thornton CR, Wakley GE, Talbot NJ. A P-type ATPase required for CTATATTTTAAAATAACACATCGTCTTCAAGCCAACAGTATATTTTTTTGTC ATGTCAAACTCCAAATCCAGTTCTGAAGTGTGGAATTGTGAGCGGATA OSH4.P2 GATGTACTACTATTATAAACATTATTTTACGAAATTGAAAAAACCTACTCT MLC1.P2 ACT1.pTm ATGTTCCCAGGTATTGCTGA CGAACAAGACTATACAATAACTATAATTTGTAAAACTTGTAGTATATATAT TTCAATGGTTAATTGTAAATTTTCTTTTATTCTGATATCATCGATGAATTCG ACT1.mTm ACATTTGTGGTGAACAATGG rice blast disease and induction of host resistance. Nature. 2006;440(7083):535-9.
	AAATTCATTTAAAAGACTATTTCTTTCCCAGTCACGACGTT CCCTTCCATTAAATCTCCCTGAGATTTGTTGTTTTTTATCTGTATTCAGTAC AG CAATCAAACCAAGATTTGAAGAACACCTCTGGTGTGCCAAATGCTCCTAT Epub 2006/03/24. doi: 10.1038/nature04567. PubMed PMID: 16554820. OSH7A.P1 CDC10.P1
	GAAGTCAAGTTCACCATGGGTTTAACTGTGGAATTGTGAGCGGATA CGATTTCTAATCTAAACTAAAGATAAATATATAAAGAAGTACATTAAATTG GTTCCAATCAACTACAGGTACTGCTGCTGCTAGAGGTGCTGGCGCAGGTG CTTC 28. Li B, Dong X, Zhao R, Kou R, Zheng X, Zhang H. The t-SNARE protein OSH7A.P2
	CAATATATCAAAAACGAATTAAGTTTCTTCTTTCCCAGTCACGACGTT CGCGTTTTGCTTTTCAACAAACACACAAAAGAAGAGGAATACAAAAAAGT FgPep12, associated with FgVam7, is essential for ascospore discharge and plant CDC10.P2
	OSH7α.P1	CCAGCGAACATGCACTTTTCTTTCGTTGAAGGTTCAGGTAATCAGTATGG AAAATCACATTATATCAATAACAAACCTGATATCATCGATGAATTCGAG
	GATTAACAGCTAAATTAGATAAGTGTGGAATTGTGAGCGGATA ACGGCGCGCCGACTAGTGGATCCAAGGGAAAAATAAACTTAGATCATAG infection by trafficking Ca2+ ATPase FgNeo1 between Golgi and endosome/vacuole D4H.P1
	OSH7α.P2	TAAATTAGACAATACTACTTGATTCTAATTCAAAATCTATTAATATGACCGT TGGAGCC
	ATATTAAAAGTCCAACAATTAATTACTTTCCCAGTCACGACGTT GATACGCGTTAATTGTAAGTAATACTAGATCCAGGG in Fusarium graminearum. PLoS Pathog. 2019;15(5):e1007754. Epub 2019/05/09. D4H.P2
	OSH2.P1 DRS2.P7 doi: 10.1371/journal.ppat.1007754. PubMed PMID: 31067272; PubMed Central GGATTTTATTTTCCAATTTGGTACCCATTTCATTGTATGTCACGGTTGAATT CCATAATAAATTACTGATAAAACACTAACAACTTTAGAATAAACAGCTAAA ATCATGAATGATTCACCCCAACTAAGTGCGAAGCTTCGTACGCTGCAGGT GATCAAATAT
	C CAATCAACAATTAAAACAGTCTAAAATATGTCTCCCGTGTTCGCCAAATCT ATATTTGATCAATTCAACCGTGACATACAATGAAATGGGTACCAAATTGG AAAATAAAATCC PMCID: PMCPMC6527245. OSH2.P2 DRS2.P8
	DRS2.P9	TGTGCCTTTCTTCTAGGCCAATACTCTGATATCATCGATGAATTCGAG GCCAATTTATTTTTCCTTGTTACATCAATTATTGGAGCAGTACCTCATGTTT
	OSH3.P1	CCCACTCAATCCCATTATTTCTTTCCCTTGACAGTTGAACTTTCCCATACCA CCCCCACAAATAG
	DRS2.P10	ACTTGAAATATGGAAACTTTAGAAGGAAGCTTCGTACGCTGCAGGTC CTATTTGTGGGGGAAACATGAGGTACTGCTCCAATAATTGATGTAACAAG
	OSH3.P2	GGATTCTATGATCTAAATAATATAACGTCTAATAGATATGAAAAAGACAA GAAAAATAAATTGGC

29. Zhang J, Yun Y, Lou Y, Abubakar YS, Guo P, Wang S, et al. FgAP-2 complex is essential for pathogenicity and polarised growth and regulates the apical localisation

Acknowledgements ................................................................................................................... iv List of abbreviations ................................................................................................................. vii INTRODUCTION ...................................................................................................................... Chapter I: The fluid mosaic ...................................................................................................

Acknowledgements

I would like to sincerely thank the members of the jury for accepting this role and evaluating my PhD project.

I always thought that this section was going to be the easiest, but actually, when I think of all the people who collaborated, professionally and personally, to make this thesis a success, the task becomes more complicated...

List of abbreviations

Abbreviation Meaning

RESULTS

The following section gathers the results of this work, divided into two parts. The first part is consisting in the paper "Two distinct lipid transporters together regulate invasive filamentous growth in the human fungal pathogen Candida albicans", to be submitted shortly, while the second part shows additional results that further support the data included in the paper. The monitored daily and animals were sacrificed by cervical dislocation when they had lost more than 20% of their weight.

Ethics statement

Animal procedures were approved by the Bioethical Committee and Animal Welfare

Additional results

These results correspond to additional and/or preliminary data obtained during my experimental work. They include further characterization of the drs2 mutant, with respect to invasion and molecular analyses. Furthermore, I also investigated the importance of Drs2 localization for its function by targeting this flippase at the SPK constitutively.

Colony formation and invasion in the drs2 mutant.

Drs2 is critical for hyphal, invasive growth (Figures 2 &3). To further characterize this mutant, we investigated filamentous growth after 24h induction, on FCS containing 0.5% agar. While the WT (left panel) colonies have ~180 μm radius, with hyphae composed of ~30 μm long compartments, the drs2 mutant colonies are smaller (~90 μm radius) with filaments, composed of thicker and shorter compartments (quantified in Figure Ad1, first and second graphs). Of note, the overall compartment volume of the drs2 mutant is similar to that of the WT (Figure Ad1, third graph) and the distance between branches is shorter in the drs2 cells (Figure Ad1, fourth graph). These results further confirmed the role of Drs2 in maintaining hyphal polarized growth.

Drs2 is necessary for polarization of exocytosis and endocytosis.

In S. cerevisiae, deletion of DRS2 affects PM protein sorting at the TGN (Hankins et al., 2015b).

In addition, deletion of DRS2, DNF1 and DNF2 result in an alteration of the endocytic uptake of FM4-64 (Pomorski et al., 2003). I showed that in a drs2 mutant, active Cdc42 is depolarized (Figure 3D) and, consistently, this mutant cannot maintain hyphal growth (Figure 3A). Hence, I examined whether Drs2 is important for the organization of membrane traffic during hyphal growth, using exocytosis and endocytosis reporters.

We examined the distribution of endocytosis, by following the actin binding protein 1 (Abp1-GFP) (Ghugtyal et al., 2015;[START_REF] Martin | Functional analysis of Candida albicans genes whose Saccharomyces cerevisiae homologues are involved in endocytosis[END_REF]. In WT cells, Abp1-GFP was tightly clustered

Constitutive targeting of Drs2 to the SPK is not deleterious for C. albicans filamentous growth.

We showed that Drs2 localizes at the hyphal tip as an apical crescent, most likely at the PM, given its co-localization with a prenylated mScarlet fusion, mSc-CtRac1, (Figure 4), as well as partially co-localizes with Mlc1 (Figure 4

DISCUSSION

In this work, I characterized the role of the flippases belonging to the P4-ATPase subfamily during polarized hyphal growth in the human opportunistic fungal pathogen Candida albicans.

My results indicate that Drs2 plays a unique role in the maintenance of filamentous growth, which appears to be more critical after the first septum formation (Figure D1). Further characterization of the drs2 deletion mutant shows in particular that, in addition to PS distribution, the distribution of PI(4)P at the PM is altered (Figure D2). Deletion of the lipid transfer protein Osh4, which exchanges PI(4)P and sterol, specifically recovers both PI(4)P polarized distribution at the PM, and hyphal invasive growth in drs2 cells.

Importance of Drs2 for C. albicans filamentous growth and virulence

The deletion of the PS synthase Cho1 results in a defect in virulence in murine infection models (Chen et al., 2010;[START_REF] Davis | Candida albicans Cannot Acquire Sufficient Ethanolamine from the Host To Support Virulence in the Absence of De Novo Phosphatidylethanolamine Synthesis[END_REF]. Additionally, the cho1 mutant was trans-complemented with the Arabidopsis thaliana serine decarboxylase gene (AtSDC), providing this mutant with the capacity to capture enough ethanolamine to produce PS by a run backwards of the Psd1/2 enzymatic reaction, which rescues virulence [START_REF] Davis | Candida albicans Cannot Acquire Sufficient Ethanolamine from the Host To Support Virulence in the Absence of De Novo Phosphatidylethanolamine Synthesis[END_REF], indicating that PS is critical for virulence. Consistent with this, we show now that Drs2 is also required for invasive growth and virulence in a murine model of systemic candidiasis (Figure 2 and Ad.1). In addition, it is possible that the reduced virulence of drs2 could be due, at least in part, to the inability to maintain virulence attributes such as hyphal specific gene expression needed for tissue invasion, like Candidalysin (Ece1) or Sap6 (Figure 2C). Work on Fusarium graminearum also showed that flippases, including the Drs2 homologous, FgDnfB, are implicated in the production of virulence factors, such as the deoxynivalenol (DON) mycotoxin and in the expression of the genes implicated in DON production [START_REF] Yun | Flippases play specific but distinct roles in the development, pathogenicity, and secondary metabolism of Fusarium graminearum[END_REF].