Donald Davesne
email: donald.davesne@earth.ox.ac.uk

François J Meunier

Matt Friedman

Roger B J Benson

Olga Otero

Histology of the endothermic opah (Lampris sp.) suggests a new structure-function relationship in teleost fish bone

Keywords: Bone histology, endothermy, osteocytes, Teleostei, Lampridiformes, Scombridae

Endothermy, production and retention of heat by the body, appeared convergently in mammals, birds and four spiny-rayed teleost fish lineages. Of these, endothermy over most or all of the body has only appeared in two groups: tunas and the opah (Lampris). Hitherto, tunas have been the only spiny-rayed fishes known to have bones containing embedded osteocyte cells; others have acellular bone. We examined bone histology in Lampris for the first time, demonstrating the presence of cellular bone very similar to that of tunas. This contrasts with the acellular condition of its ectothermic close relatives. The distribution of this character suggests that it co-evolved with red-muscle endothermy, hinting at a common physiological mechanism that would link bone histology to endothermy in these distantly related teleost lineages.

Introduction

Endothermy, the capacity to produce and retain metabolic heat, is a well-known trait of modern mammals and birds. In a spectacular example of convergent evolution, endothermy also evolved independently in mackerel (Lamnidae) and thresher (Alopiidae) sharks and in four separate teleost lineages [1,2], allowing them to maintain active and efficient swimming in a broad range of water temperatures [3]. Teleost endothermy is little studied relative to that in tetrapods, but may provide key insights into the physiological requirements and evolution of endothermy.

Changes in bone microstructure are thought to correlate with tetrapod endothermy. This structure-function relationship has been a valuable tool for unravelling the metabolism and thermal biology of extinct taxa, including dinosaurs [e.g., 4,5]. However, the scarcity of comparative histological and microanatomical data on teleost bone hinders our understanding of the relationship between physiology and bone microstructure in endothermic teleosts.

All known endothermic teleosts belong to the mega-diverse predominantly marine clade Acanthomorpha, or spiny-rayed fishes [6]. These groups show two distinct endothermic strategies, different from those of birds and mammals: (1) billfishes (Xiphioidei) and the butterfly kingfish Gasterochisma melampus (Scombridae: Gasterochismatinae),independently developed heater organs consisting of modified extraocular muscles that locally warm the brain and eyes [1,2]; (2) tunas (Scombridae: Thunnini) generate heat via the activity of red extraocular and swimming muscles and retain it with specialised counter-current blood vessels, warming not only the head but also the axial musculature [1,2]. The opah Lampris sp.

(Lampridiformes) has been recently shown to have both heat-generating extraocular and pectoral fin muscles, and a counter-exchange blood vessel network that heats cold oxygenated blood returning from the gills [7,8]. Thanks to this unique system, Lampris is the only known teleost to show endothermy over its entire body, allowing it to spend long periods of time hunting in the cold water between 50 and 400 m in depth [8].

Bone tissues in teleosts display a great variety of structures ranging from: (i) cellular bone in which osteogenic cells (osteoblasts) are enclosed by the bone matrix, and become trophic cells (osteocytes); to (ii) acellular bone that is entirely deprived of osteocytes, the osteoblasts withdrawing from the surface during deposition of the bone matrix [9][10][11][12].

Cellular bone is primitive to osteichthyans but is lost in the clade Neoteleostei, which includes acanthomorphs [11,13]. The only acanthomorphs with osteocytes reported thus far are tunas [9][10][11], in which bone tissues also show substantial remodelling [14]. The secondary reacquisition of osteocytes and the presence of conspicuous bone remodelling in tunas have been hypothesised to be linked with their active, endothermic metabolism [11,12,14].

Nevertheless, this assertion remains conjectural in the absence of comparative observations in lineages that independently evolved endothermy. In this context, Lampris provides an opportunity to test the hypothesised link between endothermy and cellular bone in teleosts.

However, the bone microstructure of Lampris has thus far been unknown. Here we describe bone histology of this endothermic taxon and some of its ectothermic close relatives.

Material and methods

Histological sections were cut from bones of two extant lampridiforms (electronic supplementary material, Table S1): Lampris sp. (MNHN-ZA-AC-A-7506, Muséum national d'Histoire naturelle, Paris, France) and Velifer hypselopterus (MNHN-ICOS-01117), along with six Late Cretaceous neoteleosts including the stem-lampridiform [15] †'Aipichthys' velifer (MNHN.F.HAK1991). These fossils were included to document the trait in some of the earliest representatives of the clade (Table S1). Each sample was embedded in epoxy resin. The sections were sawn and ground to obtain 50-60 µm thickness, and examined with an Axiovert microscope in transmitted natural light.

Results

Primary bone in the rib of Lampris includes numerous elongate osteocyte lacunae (Fig. 1a,b), with their main axis perpendicular to the direction of growth. The canaliculi that housed the cytoplasmic extensions of the cells are fewer than in typical teleost bone [12]; they are relatively short, few are ramified (Fig. 1e), and they all point toward the periosteum (Fig. 1d). Primary bone shows a parallel-fibered structure, with layers of aligned osteocytes alternating with lines of arrested growth (Fig. 1c,d). The rib is spongy, with numerous cavities (Fig. 1a) surrounded by secondary bone limited by cementing lines (Fig. 1g). The secondary bone incorporates fewer osteocytes than areas of primary bone (Fig. 1f,g).

The scapula of Velifer is composed of acellular bone: no osteocytes are incorporated in the bone tissue. However, it is perforated by numerous canals that are winding and bifurcated, indicating that they probably housed cytoplasmic extensions from surface osteoblasts (Fig. 1h).

The rib of every Late Cretaceous taxon sampled (including the stem-lampridiform †'Aipichthys') also consists of acellular bone, without any identifiable features.

Discussion

Prior to this study, lampridiform osteohistology was only reported from an unknown bone of Trachipterus [9] and from the dorsal-fin pterygiophores of Regalecus [16], two elongate-bodied lampridiforms that collectively are the sister-group of Lampris [15]. Both have acellular non-hyperostotic bones (hyperostotic bones occasionally contain a few embedded osteocytes in bones of species that otherwise have acellular bones [16]). Therefore, Lampris is the only known lampridiform with cellular bone in normal conditions (Table S2).

Furthermore, the red muscles and counter-exchange vascular networks indicative of endothermy are absent among other lampridiforms, suggesting the absence of endothermy in all lampridiforms other than Lampris [8] (Fig. 2).

In scombrids, cellular bone is described in Auxis, Euthynnus, Katsuwonus and Thunnus [9,11,12], i.e. "true" tunas (Thunnini), which are all red-muscle endotherms [1,2].

Conversely, ectothermic scombrids such as the mackerels Scomber and Scomberomorus [1] have acellular bone [9,10] (Fig. 2, Table S2). Amongst the acanthomorphs that show cranialonly endothermy [1,2], billfishes also have acellular bone (Table S2) with histological features indicative of extensive remodelling, such as secondary osteons [17,[START_REF] Atkins | Remodeling in bone without osteocytes: billfish challenge bone structure-function paradigms[END_REF]. The absence of osteocytes in billfishes and ecologically comparable large-bodied fast-swimming pelagic predators (e.g., Coryphaena, Seriola) [9,10] excludes the possibility of a size-related distribution of osteocytes in acanthomorphs. Among teleosts virtually all taxa show either cellular or acellular bone consistently throughout all bones of the skeleton [10][11][12]. Therefore, we do not expect the presence of osteocytes to be affected by proximity with warmer regions of the body.

The widespread distribution of acellular bone among lampridiforms and other acanthomorphs (including some of the stratigraphically oldest fossils) indicates that cellular bone evolved independently from acellular ancestors in the Lampris and tuna lineages (Fig. 2, Table S2).Our survey of endothermic acanthomorphs and their immediate ectothermic relatives then suggests that osteocytes are found only in red-muscle endotherms amidst the whole diversity of the clade, this physiology correlating perfectly with the occurrence of secondarily cellular bone in the acanthomorph clade (Fig. 2). This is reinforced by the observation that, despite their independent evolutionary origins, bone tissues of Lampris and tunas share peculiar histological features: conspicuous remodelled bone [12,14] and similar-looking osteocytes with an elongate cell body and very few cytoplasmic projections, differing from the 'star-shaped' lacunae of other teleosts that are typically more rounded with numerous cytoplasmic projections [12,[START_REF] Stéphan | Recherches histologiques sur la structure du tissu osseux des poissons[END_REF]. These similarities, and their difference from the primitive condition of cellular bone in non-acanthomorph teleosts, may reflect either functional similarity of osteocytes in Lampris and tuna, and/or constraints on their morphology resulting from evolutionary reacquisition. This remains uncertain.

Whether the correspondence documented here is due to a physiological advantage of reacquiring osteocytes in red-muscle endotherms, or to a passive consequence of elevated metabolism, it provides strong evidence for a structure-function correlation between bone microstructure and endothermy in acanthomorph teleosts. Unlike in tetrapods, histological indicators of metabolism have rarely been addressed by studies of teleost bone [START_REF] Dean | The structure-mechanics relationship and the response to load of the acellular bone of neoteleost fish: a review[END_REF]. To overcome this limitation, more comparative structural studies incorporating physiological considerations, at the inter-and intraspecific scale would be needed. Applying such findings to fossil taxa may permit breakthrough investigation into fish palaeophysiology (for instance, in potentially endothermic fossil acanthomorphs), a field that is currently poorly developed but is promising considering the advances in tetrapod palaeophysiology that were enabled by palaeohistology [e.g., 4,5]. This would allow a better understanding of bone biology and evolution in a group representing half of modern vertebrate diversity.

Data accessibility. Data are available as the electronic supplementary material.

Comment on the renewed taxonomy of Lampris species

The individual of Lampris sp. studied here (MNHN-ZA-AC-A-7506) is a historical specimen previously referred to the species Lampris guttatus. The original publications in which endothermy was initially reported were also based on individuals referred to L. guttatus [1,2]. Shortly after completion of our histological work, the once circumglobal L. guttatus was divided in five species, each having a more limited geographical range [3]. As a consequence, the specimens in which whole-body endothermy has been detected [1] would belong to the North Pacific species L. incognitus (N. Wegner, pers. comm.), and our specimen might not pertain to the same species. There is no available information on where MNHN-ZA-AC-A-7506 was collected, and since the specimen consists only in a portion of vertebral column and associated ribs, it is impossible to confirm its identity based on comparative anatomy. On the other hand, the six extant species of Lampris are all very similar in terms of ecology, external morphology and behaviour [3], and all seem to share similar pectoral muscle anatomy (N.

Wegner, pers. comm., D.D. and F.J.M. pers. obs.). It is then reasonably likely that they are all capable of endothermy, which would not change the conclusions of the present paper. S1. List of specimens used for the histological observations. Only one bone was sampled for each extant taxon, due to their rarity in osteological collections. Only non-pathological and non-hyperostotic bones were used. When possible, only ribs were sampled, with one exception (Velifer hypselopterus) that we do not expect to affect the results since cellular or acellular bone has been shown to be consistent over the whole skeleton in virtually all teleosts [4,5].

ESM table

In addition to the two extant lampridiforms, six fossil acanthomorphs and close neoteleost relatives from the Cenomanian (Late Cretaceous) of Lebanon were sampled as well.

For Lampris sp. (MNHN-ZA-AC-A-7506) the 7 th abdominal rib was sectioned transversally and longitudinally ~5 mm from the articular head. For fossil taxa, we took samples where ribs were already fragmented or broken. The thin section photographs of Fig. 1

Figures:Figure 1 .

 1 Figures:

Figure 2 .

 2 Figure 2. Phylogenetic distribution of the osteohistological and metabolic parameters

table S2 .

 S2 were modified to edit out background noise and remaining soft tissues in bone cavities. All thin sections are kept in the palaeohistological collections of the Muséum national d'Histoire naturelle (MNHN, Paris, France). Selected list of acanthomorph and neoteleost taxa for which the type of bone (cellular or acellular) is known.

	ESM Systematic position Taxon	Bone type		References
	Other Neoteleostei
	Neoteleostei incertae	†Ctenothrissa	acellular		this study
	sedis	vexillifer
	Aulopiformes	†Eurypholis sp.	acellular		this study
	Acanthomorpha
	Acanthomorpha	†Pycnosteroides	acellular		this study
	incertae sedis	levispinosus
	stem-	†'Aipichthys' velifer	acellular		this study
	Lampridiformes
	Lampridiformes,	Velifer hypselopterus acellular		this study
	Veliferidae
	Lampridiformes,	Lampris sp.	cellular		this study
	Lampridae
	Lampridiformes,	Trachipterus	acellular		[6]
	Trachipteridae	trachypterus
		Zu cristatus	acellular		[6]
	Lampridiformes,	Regalecus russelii	acellular (except in	[7]
	Regalecidae		hyperostotic
			pterygiophores)
	Polymixiiformes	†Omosoma sp.	acellular		this study
	Trachichthyiformes	†Acrogaster heckeli acellular		this study
	Xiphioidei,	Xiphias gladius	acellular		[6,8]
	Xiphiidae
	Xiphioidei,	Istiophorus albicans acellular		[4,8]
	Istiophoridae	Kajikia albida	acellular		[8]
	Taxon Lampris sp. Velifer hypselopterus †'Aipichthys' velifer Scombridae, †Omosoma sp. Scombrini †Acrogaster heckeli Scombridae, †Eurypholis sp. Scomberomorini †Ctenothrissa vexillifer †Pycnosteroides Scombridae, levispinosus Thunnini	Dry skeleton specimen Makaira nigricans acellular Tetrapturus acellular number MNHN-ZA-AC-A-7506 angustirostris MNHN-ICOS-01117 Tetrapturus belone acellular MNHN.F.HAK1991 Scomber scombrus acellular MNHN.F.SHA2563d MNHN.F.SHA38g Scomberomorus acellular MNHN.F.HAK1550 cavalla MNHN.F.HAK246d Scomberomorus acellular MNHN.F.HDJ55 maculatus Auxis rochei cellular Euthynnus affinis cellular	[4,8] Bone sampled [8] 7 th abdominal rib [6] scapula [6] 1 st and 6 th abdominal rib 7 th abdominal rib [4] posteriormost abdominal rib 3 rd abdominal rib [4,9] 6 th abdominal rib posteriormost abdominal rib [6] [10]
		Euthynnus	cellular		[11]
		alletteratus
		Euthynnus lineatus	cellular		[12]
		Katsuwonus pelamis cellular		[4,5,10,13]
		Thunnus alalunga	cellular		[6,10,13-15]
		Thunnus albacares	cellular		[10]
		Thunnus obesus	cellular (scales)	[16]
		Thunnus thynnus	cellular		[6,11,17]

Acknowledgements. We thank P. Béarez and G. Clément (MNHN) for granting us permission to sample the new material studied here, M. Hautecoeur and S. Morel (MNHN) for preparing the thin sections studied and figured in the article and M. Paig-Tran (California State University, Fullerton, USA) for providing information on the histology of Regalecus.

Comments by L. Legendre and N. Wegner substantially improved the manuscript.

Funding. This research was supported by the Leverhulme Trust (grant RPG-2016-168) and by a Junior Research Fellowship at Wolfson College, University of Oxford (D.D.).


Incident stroke

is associated with acute decline in cognitive function and accelerated long-term cognitive decline.

Poststroke cognitive impairment is common, but the trajectory and magnitude of cognitive change after stroke are unclear. AIM: to measure changes in the trajectory of cognitive function following a first stroke, controlling for pre-stroke cognitive performance using diverse population-based studies from the Cohort Studies of Memory in an International Consortium (COSMIC).

• 14 longitudinal cohorts from 11 countries from Asia, Africa, North America, South America, Europe, and Australia contributed data. • Sample mean age (at baseline) = 73 years (SD=8.0); 57% were female.

• Mean follow-up time for all followed-up participants was 7.5 years (SD=4.1) and an incident stroke occurred after 4.6 years on average in those who had a stroke.

• Stroke was associated with acute decline (at time of stroke) and accelerated rate of decline after covariate adjustments in global cognition and in the 4 domains.

• The rate of cognitive decline before stroke was not significantly different from the rate of decline in those who did not experience a stroke over follow up.

• Predicted values (z-scores) of global cognition and language domain for a 73-year-old White woman with common values of covariates* and an incident stroke occurring at 4.6 years into the study were used to plot the following:

  Note: Model with global cognition included 7 studies and 10,396 participants; model with language included 9 studies and 10,571 participants. *Common values of covariates at baseline: 12 years of education, no diabetes, hypertension present, non-smoker, <1 alcoholic drink per week, not depressed, not an APOE ε4 carrier, no cerebrovascular disease, BMI=27, and baseline cognition score= 0SD). **Slope = rate of cognitive change; TIS = time in study. ***Compared with the rate of change before stroke. Lo 1* , John D. Crawford 1 , Darren M. Lipnicki 1 , Richard B. Lipton 2 , Mindy J. Katz 2 , Pierre-Marie Preux 3 , Maëlenn Guerchet 3 , Eleonora d'Orsi 4 , Karen Ritchie 5 , Ingmar Skoog 6 , Elena Rolandi 7,8 , Steffi G. Riedel-Heller 9 , Mary Ganguli 10 , Kaarin J. Anstey 11 , Allison E. Aiello 12 , Henry Brodaty 1 , Yen-Ching Chen 13 , Jen-Hau Chen 13 , Pascual Sanchez-Juan 15 , Antonio Lobo 16 , Perminder S. Sachdev 1 Created with BioRender.com and MapChart **Slope for those with no stroke: (-0.02 + 0.002 x TIS) SD/year (


Yifang Wang

Houssem Rezgui

Mukherjee Chhandak

Marina Deng

Abhishek Kumar

Jonas Müller

Guilhem Larrieu

Cristell Maneux

C Mukherjee

Evidence of Trapping and Electrothermal Effects in Vertical Junctionless Nanowire Transistors

Keywords: vertical junctionless nanowire transistors, electrothermal effects, trapping, multiphysics simulations

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Faced with the limitations of continuous scaling for the classical planar MOSFETs, novel emerging and in particular 3D technologies, such as vertical junctionless nanowire transistors (VNWFET), are being explored as beyond-CMOS alternatives [1]. One of the key advantages of VNWFETs is their inherent 3D structures which can be stacked to increase the number of integrated transistors per unit area for designing more compact, faster and power-efficient hardware targeting neuromorphic computing [START_REF] Maneux | IEEE International Electron Devices Meeting (IEDM)[END_REF]. In addition, junctionless transistors have a better electrostatic integrity and immunity to short channel effects (SCE) along with a simpler fabrication process [START_REF] Guerfi | [END_REF]. Due to low thermal stability and reduced carrier mobility, electrothermal optimizations are a key design aspect for the VNWFET technology. With the formation of temperature hot-spots within the small device dimensions along with an ultra-thin gate oxide, investigation of selfheating and charge trapping effects in VNWFETs has thus become an important prerequisite. In order to investigate these transport mechanisms, in this work, we perform pulsed I-V measurements with varying pulse widths on a p-type VNWFET (Fig. 1). The pulsed measurement results are then compared with multiphysics simulation of the VNWFETs to understand the behavior of different associated physical parameters. In the following sections, we will discuss the experimental details, the simulation framework followed by a comprehensive analysis of electrothermal and trapping effects.

Pulsed I-V measurements

The principle of pulsed I-V measurements relies on applying very short gate pulses on the device so as not to allow sufficient time for its internal temperature to change significantly or activate any charge trapping. For our measurements, a pulse width varying between 1µs and 10ms was used with a duty cycle of 10%. A staircase stepping voltage was applied at the drain to minimize the charging effect [5]. The I D -V D measurement data for all available geometries (nanowire diameters of 22 and 34 nm with 16 and 25 NWs in parallel) over the entire bias range showed a constant increase in the drain current with pulse width, a trend which is diametrically opposite to that of a classical MOS transistor. For further analyses, the VNWFET with 25 NWs having a diameter of 22 nm is selected as a representative device with the highest reproducibility and measurement stability. Fig. 2 shows its pulsed I D -V D under a V GS of -2.2V, for different pulse widths depicting this trend. It has been hypothesized that this phenomenon is a combined effect of both charge trapping and self-heating.

Results and discussion

To understand this behavior, we performed finite element method (FEM) based COMSOL Multiphysics simulation of the VNWFETs under the same pulsed conditions as the measurements. The simulation method relies on the partial differential equation (PDE) package in COMSOL for transport equations coupling phonon and electron transport by solving the non-Fourier heat equation and a drift-diffusion model with interface trapping effects [6]. In addition, simulations with the VNWFET compact model [7] have also been performed and the two simulations are compared with the experimental results in Fig. 2 demonstrating good agreement. a. Trapping effects Variation of the threshold voltage (V TH ) is principally attributed to the temperature induced increase of drain current in junctionless transistors [8]. Charge trapping is known to occur at the Si-SiO 2 interface and within the oxide layer that can also cause a shift in V TH [START_REF] Schmitz | Int. Conf. on Microelectronic Test Structures[END_REF]. However, due to drain induced barrier lowering, an increase in V D also causes a variation in V TH . To avoid this effect, we compared the V TH for all devices under test at V D = -1V, extracted using the transconductance method, in Fig. 3 that shows a continuous increase with the pulse width eventually saturating at the value extracted under DC conditions when pulse width is sufficiently large. To analyze the relationship between trap density (N T ) and the observed V TH drift (∆𝑉 𝑇𝐻 ), generally described by ∆𝑉 𝑇𝐻 = 𝛼𝑞𝑁 𝑇 /𝐶 𝑜𝑥 , where 𝛼 is a constant, 𝑞 is the electronic charge and 𝐶 𝑜𝑥 is the gate oxide capacitance, pulsed I-V curves are simulated using COMSOL. In addition, ∆𝑉 𝑇𝐻 for each geometry are extracted from the measured I-V and implemented in the analytical equation of ∆𝑉 𝑇𝐻 within the compact model. Both simulations take the ∆𝑉 𝑇𝐻 drift into account and thereby show good agreement with measurements results in Fig. 2. Fig. 4, plots the trap density N T extracted from COMSOL and ∆𝑉 𝑇𝐻 from the compact model for different pulse widths. It can be seen that the extracted parameters can be described by an analytical equation of the form 𝑁 𝑇 = 𝑁 𝑆𝑆 (1 -exp (-𝑡 𝑊 /𝜏)); here N SS is the maximum number of available traps, t w is the pulse width, τ is a time constant associated with the trap activation. This indicates that the increase in the drain current with pulse width is principally governed by the trap activation that results in an equivalent shift of the device threshold voltage.

b. Self-heating effects

To investigate the dynamics of electrothermal effects associated with the pulsed I-V, COMSOL simulation was used to extract the thermal conductivity and the rise of internal device temperature between the source and drain for different pulse widths (Fig. 5). It can be observed that the device temperature also increases due to phonon accumulation near the drain region at high bias conditions and larger pulse widths indicating the onset of self-heating. Consequently, temperature hot-spots are formed due to a reduction of thermal conductivity caused by phonon confinement in the 22nm nanowires. Phonon confinement can also lead to carrier trapping at the siliconoxide interface [1] coupled with the electrothermal phenomena. Moreover, under higher biases, scattering mechanisms increase which have significant impact on trapping density.

Conclusion

In this work, we present, for the first time, a comprehensive analysis of pulsed I-V characteristics of VNWFETs. Experimental results are analyzed with physical simulations indicting the activation of trapping and self-heating with increasing pulse widths. The increase of drain current with larger pulse width has been attributed to an increasing trap density which results in a shift of the threshold voltage. As suggested by the evidences presented in this work, dissociating the trapping and electrothermal phenomena in junctionless transistors remains a challenging task and will be the subject of our future analysis.

Fig. 1 .

 1 Fig. 1. Schematic representation of (a) Vertical array of GAA nanowires, (b) a single nanowire. (c) SEM image of a fabricated nanowire [4].

Fig. 2 .

 2 Fig. 2. Pulsed ID-VD measurements compared with simulations.

Fig. 3 .

 3 Fig. 3. VTH-tw of different VNWFET geometries.

Fig. 4 .

 4 Fig. 4. VTH and NT for different pulse widths compared with analytical solutions.

Fig. 5 .

 5 Fig. 5. Thermal conductivity and internal device temperature of the VNWFET.

ACKNOWLEDGMENT This work was supported by the project FVLLMONTI funded by European Union's Horizon 2020 research and innovation program under grant agreement N•101016776.


Nils Ferrand

Nils Ferrand1

The World Saving Gamathon

Keywords: Challenge, World Changer, Transformative Science, Game Design

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Evan John
email: evan.john@curtin.edu.au

B Karam

Richard P Singh

Jessica L Oliver

Jordi Soyer

Daniel Muria-Gonzalez

Silke Soo

Kar-Chun Jacques

Tan

Karam B Singh

Richard P Oliver

Jessica L Soyer

Jordi Muria- Gonzalez

Daniel Soo

Silke Jacques

Kar-Chun Tan

Chromatin-immunoprecipitation reveals the PnPf2 transcriptional network controlling effector-mediated virulence in a fungal pathogen of wheat

Keywords: Transcription factor, virulence, regulation, gene expression, orthologue

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Background

The Parastagonospora nodorum-wheat interaction has become a model fungal-plant pathosystem to study the molecular virulence factors underpinning infection. The fungus produces small secreted effector proteins that selectively interact with host-receptors encoded by dominant susceptibility-genes [START_REF] Mcdonald | Just the surface: advances in the discovery and characterization of necrotrophic wheat effectors[END_REF][START_REF] Kanyuka | The rise of necrotrophic effectors[END_REF]. These interactions occur in a gene-for-gene manner that causes 'effector-triggered susceptibility' in the host plant, quantitatively affecting the disease which manifests as septoria nodorum blotch. Several effectors acting in this manner have now been identified and characterised for their role in virulence [START_REF] Friesen | Emergence of a new disease as a result of interspecific virulence gene transfer. 8[END_REF][START_REF] Liu | SnTox3 acts in effector triggered susceptibility to induce disease on wheat carrying the Snn3 gene[END_REF][START_REF] Liu | The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1[END_REF][START_REF] Kariyawasam | The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll[END_REF][START_REF] Richards | A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat[END_REF]. These studies have also described a consistent pattern: the expression of these genes is maximal two to four days after infection and then declines. Furthermore, expression levels can vary by the presence or absence of their matching wheat receptors, as well as by epistasis, whereby one effector gene causes suppression of another [START_REF] Phan | Differential effector gene expression underpins epistasis in a plant fungal disease[END_REF][START_REF] Peters-Haugrud | Genetics of variable disease expression conferred by inverse gene-for-gene interactions in the wheat-Parastagonospora nodorum pathosystem[END_REF][START_REF] John | Variability in an effector gene promoter of a necrotrophic fungal pathogen dictates epistasis and effector-triggered susceptibility in wheat[END_REF]. Yet relatively little is known concerning the mechanisms governing the effector gene regulation. In particular, are there common or distinct regulatory pathways involved? Do these components specifically control effector gene expression, or co-regulate other metabolic and developmental pathways? New knowledge in this area could present suitable targets to suppress for disease control.

Many fungi possess a Zn2Cys6 transcription factor (TF) Pf2 that has been associated with the regulation of effector gene expression. One example is the AbPf2 orthologue in Alternaria brassicicola that is critical for virulence on Brassica spp. [START_REF] Cho | Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola[END_REF].

Gene deletion of AbPf2 resulted in the down-regulation of a number of effector like genes, as well as putative cell-wall degrading enzymes. In P. nodorum, at least two key effector genes, ToxA and Tox3, require PnPf2 t be expressed [START_REF] Rybak | A functionally conserved Zn2Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat[END_REF]. An RNA-seq analysis also revealed PnPf2 regulates many more putative effectors, CAZymes, hydrolases/peptidases and nutrient transporters [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. The PtrPf2 orthologue in Pyrenophora tritici-repentis controls ToxA expression and virulence on wheat, much like for the homologous ToxA gene in P. nodorum [START_REF] Rybak | A functionally conserved Zn2Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat[END_REF]. In Leptosphaeria maculans, the causal agent of blackleg disease on Brassica spp., the LmPf2 orthologue also regulates several effector genes, including AvrLm4-7, AvrLm6, AvrLm10A and AvrLm11, as well as CAZyme expression [START_REF] Clairet | Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-players game[END_REF].

The Pf2 orthologues can be traced across several Ascomycota fungal lineages that include the Dothideomycetes, Leotiomycetes and Soradiomycetes [START_REF] John | Transcription factor lineages in plantpathogenic fungi, connecting diversity with fungal virulence[END_REF]. Genedeletion in the plant pathogens Botrytis cinerea, Fusarium spp., Magnaporthe oryzae

and Zymoseptoria tritici all suppressed fungal virulence as well as their capacity to utilise alternative carbon sources [START_REF] Chung | Two conidiation-related Zn(II)2Cys6 transcription factor genes in the rice blast fungus[END_REF][START_REF] Oh | Transcription factor ART1 mediates starch hydrolysis and mycotoxin production in Fusarium graminearum and F. verticillioides[END_REF][START_REF] Habig | The transcription factor Zt107320 affects the dimorphic switch, growth and virulence of the fungal wheat pathogen Zymoseptoria tritici[END_REF][START_REF] Han | Transcription factor PdeR is involved in fungal development, metabolic change, and pathogenesis of gray mold Botrytis cinerea[END_REF]. Analogous carbohydrate regulatory roles have been attributed in the saprophytic fungi Neurospora crassa and Trichoderma reesei [START_REF] Nitta | A new Zn(II)2Cys6-type transcription factor BglR regulates β-glucosidase expression in Trichoderma reesei[END_REF][START_REF] Xiong | A fungal transcription factor essential for starch degradation affects integration of carbon and nitrogen metabolism[END_REF]. In N. crassa, the putative orthologue Col-26 is a critical component within a signalling-network that responds to glucose availability and promotes the expression of CAZymes for plant cell-wall degradation [START_REF] Wu | The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus[END_REF][START_REF] Gabriel | The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi[END_REF][START_REF] Li | RCO-3 and COL-26 form an external-to-internal module that regulates the dual-affinity glucose transport system in Neurospora crassa[END_REF]. There is a strong association between CAZyme gene content and plant-pathogenic lifestyles [START_REF] Hane | CATAStrophy," a genome-informed trophic classification of filamentous plant pathogens -how many different types of filamentous plant pathogens are there?[END_REF].

There are some key factors yet to be established among Pf2 orthologues. Which DNA-regulatory elements are bound? Are Pf2-regulated genes directly targeted or is their expression modulated by indirect factors? Are there other TFs intimately connected with Pf2 regulation of virulence? The research presented here provides such critical insight for PnPf2 in P. nodorum, and establishes its direct role in effector expression and CAZyme regulation. A subsequent functional investigation of several connected and directly targeted TFs provides further evidence for the central role held by PnPf2 in the transcriptional network that orchestrates virulence in P. nodorum. ). These features are frequently observed in Zn2Cys6 TFs and have been linked to the modulation of TF activity [START_REF] Macpherson | A fungal family of transcriptional regulators: the zinc cluster proteins[END_REF][START_REF] Schubert | Functional dissection of a Candida albicans zinc cluster transcription factor, the multidrug resistance regulator Mrr1[END_REF][START_REF] John | Transcription factor lineages in plantpathogenic fungi, connecting diversity with fungal virulence[END_REF]. A structurally disordered domain, typically associated with post-translational modifications and intermolecular interactions [START_REF] Erdős | Analyzing Protein Disorder with IUPred2A[END_REF], was also identified at the Cterminus of PnPf2. Together, these features suggested that PnPf2 possesses the typical functional domains underpinning DNA-binding Zn2Cys6 TF activity [START_REF] Macpherson | A fungal family of transcriptional regulators: the zinc cluster proteins[END_REF]. Nuclear localisation of the C-terminally tagged PnPf2-GFP fusion protein was also observed (Fig. 1B). Along with the functional domains identified, this supported DNA-binding regulatory activity by PnPf2. A ChIP-seq analysis was therefore undertaken using the Pf2-HA and Pf2-HA_OE strains to identify 'summits' within enriched 'peak' regions. Summits corresponded to the best estimate of PnPf2-DNA binding loci within the peaks [START_REF] Zhang | Model-based Analysis of ChIP-Seq (MACS)[END_REF]. The Pf2-HA and Pf2-HA_OE samples provided complementary datasets; the overexpression mutant to compensate for lower PnPf2 expression under culture conditions [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF][START_REF] Ipcho | Transcriptome analysis of Stagonospora nodorum: gene models, effectors, metabolism and pantothenate dispensability[END_REF]

Results

PnPf2 possesses typical

Text S1 HERE

File S1 HERE

Previous RNA-seq differential-expression analyses had identified an enriched consensus motif (5'-WMGGVCCGAA-3') in the promoter regions of both AbPf2 and PnPf2-regulated genes [START_REF] Cho | Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola[END_REF][START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. Despite harbouring the typical 'CGG' Zn2Cys6 binding triplet [START_REF] Macpherson | A fungal family of transcriptional regulators: the zinc cluster proteins[END_REF], an interaction with PnPf2 was not observed in a heterologous system, indicating regulatory cofactors may be required [START_REF] Siggers | Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex[END_REF][START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. A search for DNA-regulatory elements that interact with PnPf2 from the ChIP-seq dataset identified two enriched motifs in the merged Pf2-HA and Pf2-HA_OE peak regions (Fig. 2A). The first motif designated as M1 (5'-RWMGGVCCGA-3') closely matches the consensus motif from AbPf2 and PnPf2-regulated gene promoters [START_REF] Cho | Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola[END_REF][START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. The second motif designated as M2 (5'-CGGCSBBWYYKCGGC-3') is novel for PnPf2, encompassing two copies of the canonical 'CGG' Zn2Cys6 binding triplets [START_REF] Macpherson | A fungal family of transcriptional regulators: the zinc cluster proteins[END_REF], separated by eight nucleotides.

Interestingly, M2 matches the AmyR regulatory response element that was modelled in A. nidulans [START_REF] Kojima | A robust analytical pipeline for genome-wide identification of the genes regulated by a transcription factor: combinatorial analysis performed using gSELEX-Seq and RNA-Seq[END_REF]. Both M1 and M2 are close to the ChIP-seq summits for the Pf2-HA and Pf2-HA_OE datasets (Fig. 2B), suggesting they accurately reflected DNAbinding loci.

The previous RNA-seq analysis had defined genes positively or negativelyregulated by PnPf2 from their expression changes in the PnPf2-deletion mutant pf2ko relative to wildtype SN15 [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. These gene sets include the in vitro culture conditions that were replicated here for ChIP to maximise TF-DNA yields, as well as an early fungal infection stage in planta (72 hrs). Instances of M1 and M2 were then identified across the promoters of the positive (i.e. pf2ko-down) and negative (pf2ko-up) PnPf2regulated gene sets (File S2). Both M1 and M2 were significantly enriched in the positively-regulated gene promoter set only (Fig. 2A). This indicates both motifs correspond to cis-regulatory elements that induce, rather than repress, gene expression.

A novel approach here sought to detect specifically the PnPf2-motif interactions. This utilised a dTomato reporter gene fused to the promoter of SNOG_15417. The promoter was chosen to encompass a ChIP-seq peak region with both the M1 and M2 motifs for a gene that is positively-regulated by PnPf2 (Fig. 2C; File S2). Integration of the construct at a predefined genomic locus in the SN15 background permitted evaluation of the reporter-gene expression in the resultant strain (p15417_M1M2) in comparison with strains where the CGG triplets in M1 and/or M2 had been substituted (p15417_m1M2, p15417_M1m2 and p15417_m1m2).

Significantly reduced expression was observed in the strains where M1 had been mutated, indicating that it is a functionally important and direct PnPf2 target in the SNOG_15417 promoter (Fig. 2C). No significant expression change was detected where only the M2 motif was mutated.

Fig. 2 HERE

PnPf2 directly targets genes associated with the pathogenic lifestyle of P. nodorum

Genes with a ChIP-seq summit in their promoter region, considered a putative PnPf2 target, were cross-referenced with the pf2ko RNA-seq data analysis (File S2).

There were 1286 targets identified from either ChIP-seq dataset, 484 of which were considered 'high-confidence' with a promoter summit in both Pf2-HA and Pf2-HA_OE (Fig. 3A). Of the 484 direct targets, 72 genes were positively-regulated in contrast to 6 negatively-regulated genes under the same in vitro conditions used for ChIP-seq.

This indicates PnPf2 functions mainly as a positive regulator of gene expression.

When expanded to also encompass PnPf2-regulated genes in planta, 93 were positively while 27 were negatively-regulated genes (Fig. 3B). Differential expression was not detected in the pf2ko mutant for 364 genes. This suggests other regulatory factors play a considerable role in their expression.

File S2 HERE

The characterised effector genes present in P. nodorum SN15, ToxA, Tox1, Tox3 and Tox267 [START_REF] Richards | A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat[END_REF], plus 29 other effector-like genes whose expression was altered in the pf2ko mutant, were assessed for evidence of direct regulation by PnPf2. Two distinct ChIP-seq summits were identified in the bi-directional Tox3 promoter (Fig. S1).

Both the upstream gene (i.e. SNOG_08982, encoding a protein disulphide-isomerase) and downstream gene (Tox3) are positively regulated by PnPf2. A ChIP-seq summit was also identified in the Tox1 promoter, but only from the Pf2-HA_OE dataset (Fig.

S1

). Unlike Tox3, Tox1 necrosis-inducing activity is still detected in the pf2ko background [START_REF] Rybak | A functionally conserved Zn2Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat[END_REF], indicating the summit may represent a weak enhancer element. The ToxA gene is only expressed during infection but in a PnPf2-dependent manner. A weak promoter summit was observed despite multiple instances of the M1 motif, suggesting another factor(s) is required to facilitate PnPf2-DNA binding that was absent under the ChIP-seq experimental conditions. No distinct PnPf2 summit was observed in the promoter of Tox267, whose expression is not significantly altered in the pf2ko mutant, although two instances of M1 were identified >1000 bp upstream (Fig. S1). In total, 11 of the 29 PnPf2-regulated effector-like genes showed evidence of direct PnPf2-promoter binding through ChIP-seq summits (Table S1-A).

Fig. S1 HERE

A gene-ontology (GO) enrichment and network analysis was then carried out to identify major functional gene classes that are directly regulated by PnPf2. Five distinct groups representing TFs, redox molecules, CAZymes, cell-signalling molecules and nutrient transporters were significantly enriched among the GO networks (Fig. 3C-D). The enrichment of CAZymes, redox molecules and nutrient transporters is consistent with enriched functional GO classes that were observed for the pf2ko differentially expressed genes [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. In contrast, the TFs and cell-signalling molecules were not enriched, indicating they could be tightly controlled by redundant pathways in addition to PnPf2. Nevertheless, it was striking that TF genes were particularly enriched in the high-confidence set of 484 targets (Fig. 3C-D). They made up 9.1% of these genes in contrast to 3.5% of the total genes annotated for SN15.

Five TFs were directly targeted and positively regulated, providing a direct connection with PnPf2 in the regulation of virulence (Table S1-B).

Fig. 3 HERE

Table S1 HERE

PnPf2 is the central transcriptional regulator of host-specific virulence

The identification of TFs as major PnPf2 targets prompted a functional exploration of other TFs with a putative intermediate role in regulating virulence. Three directly-regulated TF genes were therefore targeted for deletion where orthologues had virulence-associated roles (Table 1). These included SNOG_03490 (PnPro1), SNOG_04486 (PnAda1) and SNOG_08237. Two additional TF-encoding genes were simultaneously investigated. This included SNOG_08565, identified in the PnPf2 lineage of fungal TFs (15) suggesting a possible common evolutionary origin. It also included SNOG_03067 (PnEbr1), which is co-expressed with PnPf2, ToxA, Tox1 and Tox3 high during early infection (Fig. S2), suggesting a similarly important role in disease.

Fig. S2 HERE

Gene deletion strains for the five TFs were phenotypically characterised in comparison to wildtype SN15 and pf2ko. The pro1_KO, ada1_KO and ebr1_KO deletion mutants presented distinct phenotypic abnormalities (Fig. 4). The pro1_KO mutants were abolished in their ability to form pycnidia and sporulate both during infection and on nutrient-rich agar. However, vegetative growth was expansive in both conditions (Fig. 4A-B), suggesting PnPro1 acts to suppress hyphal development.

Although PnPro1 is positively-regulated by PnPf2, there was no distinct phenotypic overlap with the pf2ko mutant. The ada1_KO mutant was significantly reduced in virulence on detached leaves (Fig. 4A-B). Dark brown discolouration at the site of infection suggested a hypersensitive response had contained the infection. We also observed an increased susceptibility to oxidative (H2O2) stress for ada1_KO mutants similar to pf2ko. Furthermore, sporulation was reduced in ada1_KO relative to SN15 (Fig. 4C-E). The ebr1_KO mutants exhibited vegetative growth defects with an uneven growth perimeter around the colony edges coinciding with perturbed virulence (Fig. 4A-B). Similar hyphal-branching defects were described following deletion of PnEbr1 orthologues in Fusarium spp. [START_REF] Zhao | EBR1, a novel Zn(2)Cys(6) transcription factor, affects virulence and apical dominance of the hyphal tip in Fusarium graminearum[END_REF][START_REF] Jonkers | EBR1 genomic expansion and its role in virulence of Fusarium species[END_REF]. Interestingly, the ebr1_KO mutants were also susceptible to H2O2 stress at a level comparable to pf2ko and ada1_KO. Furthermore, pycnidia were abnormally developed, although still viable for the production of conidia, but were not detected on infected leaves (Fig. 4C-D). We did not observe morphological or virulence defects for the 08237_KO or 08565_KO mutants (Text S2)

which were not investigated in further detail.

Fig. 4 HERE

During the course of this study the carbon-catabolite repressor (CCR) element was modelled as the binding site for the Cre-1 TF that suppresses CAZyme expression in N. crassa [START_REF] Wu | The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus[END_REF]. We noted this was near identical to a motif (5'-RTSYGGGGWA-3') that is also enriched in PnPf2-regulated gene promoters [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF] but not identified from the ChIP-seq peaks. Since Cre-1 orthologues are conserved CCR regulators in filamentous fungi [START_REF] Adnan | Carbon catabolite repression in filamentous fungi[END_REF][START_REF] John | Transcription factor control of virulence in phytopathogenic fungi[END_REF], and since the CCR element is also enriched in PnPf2 regulated gene promoters, a putative Cre-1 orthologue (PnCreA) was investigated in P. nodorum to identify common regulatory pathways with PnPf2. Both PnCreA overexpression and gene-deletion mutants (CreA_OE and creA_KO) were created and then investigated alongside pf2ko and a PnPf2 overexpression mutant (Pf2_OE).

Despite clear phenotypic-growth abnormalities (Fig. 5), neither the CreA_OE nor creA_KO mutants exhibited virulence defects on wheat leaves (Text S2). The creA_KO strain was enhanced in starch utilisation (Fig. 5), an indicator substrate for CCR activity [START_REF] Gomi | Regulatory mechanisms for amylolytic gene expression in the koji mold Aspergillus oryzae[END_REF]. In contrast, there was a moderate reduction of pf2ko to utilise starch, similar to observations in other fungal PnPf2-orthologue mutants [START_REF] Oh | Transcription factor ART1 mediates starch hydrolysis and mycotoxin production in Fusarium graminearum and F. verticillioides[END_REF][START_REF] Han | Transcription factor PdeR is involved in fungal development, metabolic change, and pathogenesis of gray mold Botrytis cinerea[END_REF][START_REF] Xiong | A fungal transcription factor essential for starch degradation affects integration of carbon and nitrogen metabolism[END_REF]. These results support contrasting roles between PnCreA and PnPf2 for the regulation of some CAZyme-related genes.

Discussion

Prior to this research, PnPf2 had been identified as an important regulator of P.

nodorum virulence on wheat [START_REF] Rybak | A functionally conserved Zn2Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat[END_REF][START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF], but important details and mechanistic insights were missing. We sought to take further steps and establish the DNA-binding elements targeted by PnPf2 and identify genes that were directly under its direct regulation. Two distinct regulatory motifs M1 and M2 were identified and linked to positive generegulation by PnPf2. M1 was strikingly similar to an enriched sequence in AbPf2 positively-regulated gene promoters [START_REF] Cho | Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola[END_REF], possibly representing a conserved Pf2 binding mechanism. It will be pertinent to explore this motif as a regulatory target for other fungal Pf2 orthologues [START_REF] Cho | Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola[END_REF][START_REF] Rybak | A functionally conserved Zn2Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat[END_REF][START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF][START_REF] Clairet | Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-players game[END_REF][START_REF] Chung | Two conidiation-related Zn(II)2Cys6 transcription factor genes in the rice blast fungus[END_REF][START_REF] Oh | Transcription factor ART1 mediates starch hydrolysis and mycotoxin production in Fusarium graminearum and F. verticillioides[END_REF][START_REF] Habig | The transcription factor Zt107320 affects the dimorphic switch, growth and virulence of the fungal wheat pathogen Zymoseptoria tritici[END_REF][START_REF] Han | Transcription factor PdeR is involved in fungal development, metabolic change, and pathogenesis of gray mold Botrytis cinerea[END_REF][START_REF] Nitta | A new Zn(II)2Cys6-type transcription factor BglR regulates β-glucosidase expression in Trichoderma reesei[END_REF][START_REF] Xiong | A fungal transcription factor essential for starch degradation affects integration of carbon and nitrogen metabolism[END_REF]. Interestingly, the M2 motif matches the extensively characterised AmyR regulatory-response element in A. nidulans [START_REF] Wang | Comprehensive analysis of the DNA-binding specificity of an Aspergillus nidulans transcription factor, AmyR, using a bead display system[END_REF][START_REF] Kojima | A robust analytical pipeline for genome-wide identification of the genes regulated by a transcription factor: combinatorial analysis performed using gSELEX-Seq and RNA-Seq[END_REF].

Polysaccharide metabolism has long been established as a regulatory function for

AmyR [START_REF] Tani | Characterization of the amyR gene encoding a transcriptional activator for the amylase genes in Aspergillus nidulans[END_REF][START_REF] Nakamura | Expression profile of amylolytic genes in Aspergillus nidulans[END_REF]. Therefore, some shared regulatory pathways likely exist with Pf2 orthologues given the evidence for at least one conserved binding mechanism.

However, there are major a.a polymorphisms between AmyR and Pf2 orthologues at the Zn2Cys6 DNA-binding domain (15) and M1 has not been reported as an AmyR target despite extensive motif investigation [START_REF] Wang | Comprehensive analysis of the DNA-binding specificity of an Aspergillus nidulans transcription factor, AmyR, using a bead display system[END_REF][START_REF] Kojima | A robust analytical pipeline for genome-wide identification of the genes regulated by a transcription factor: combinatorial analysis performed using gSELEX-Seq and RNA-Seq[END_REF]. It is therefore conceivable that M1 is a regulatory binding element unique to Pf2 orthologues and therefore useful to identify putative direct targets such as ToxA in P. nodorum.

The ChIP-seq PnPf2-DNA binding dataset also facilitated the identification of P. nodorum genes under direct PnPf2 regulation. Among these genes is the Tox3 effector and the adjacent gene, SNOG_08982, encoding a protein disulphide isomerase. This class of protein catalyses cysteine-cysteine bond formation which has been connected to fungal effector protein production [START_REF] Marín-Menguiano | N-glycosylation of the protein disulfide isomerase Pdi1 ensures full Ustilago maydis virulence[END_REF]. Therefore, it would be worth exploring any involvement of SNOG_08982 in the post-translational modification of Tox3 and other effectors. PnPf2 binding was also detected in the Tox1 promoter. A partial reduction in Tox1 expression was reported in the pf2ko mutant (13), indicating PnPf2 is not essential but enhances expression under favourable conditions. ToxA is only expressed in planta, but is PnPf2 dependent [START_REF] Rybak | A functionally conserved Zn2Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat[END_REF]. Despite multiple instances matching the M1 motif, there was little evidence for PnPf2-ToxA promoter binding, suggesting chromatin inaccessibility or the absence of essential binding-cofactors under the ChIP culture conditions. Direct PnPf2 regulation of Tox267 was not evident.

The other recently-cloned effector gene Tox5 is not present in the SN15 isolate used in this study, but is homologous to Tox3 may be under PnPf2 control [START_REF] Kariyawasam | The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll[END_REF]. Nevertheless, several other effector-like genes were identified as direct PnPf2 targets (Table S1-A).

Importantly this analysis provided strong evidence that PnPf2 is a key direct-regulator of effectors, the major P. nodorum virulence factors in the lifestyle of this pathogen.

Evidence for regulation of effector expression has been reported for another P.

nodorum TF PnCon7 ( 42), yet its apparent requirement for fungal viability renders it difficult to investigate a precise functional role. Here, several novel TFs were functionally investigated based on their connection to PnPf2 (Table 1). We did not observe any change in the necrosis-inducing activity on wheat of fungal culture filtrates derived from the respective mutants. However, developmental virulence roles, including oxidative stress tolerance and hyphal development, were identified for P.

nodorum PnAda1 and PnEbr1. It is possible that the direct regulation of PnAda1 by PnPf2 contributes to the susceptibility to oxidative stress also identified in the pf2ko mutant. The PnCreA orthologue of N. crassa Cre-1 was also investigated, following the striking observation that the N. crassa Cre-1 CCR element (5′-TSYGGGG-3') was enriched in PnPf2-regulated gene promoters [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. Furthermore, Cre-1 and the PnPf2 orthologue Col-26 are both key components of a transcriptional network controlling CAZyme production in N. crassa [START_REF] Wu | The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus[END_REF][START_REF] Gabriel | The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi[END_REF][START_REF] Li | RCO-3 and COL-26 form an external-to-internal module that regulates the dual-affinity glucose transport system in Neurospora crassa[END_REF][START_REF] Craig | Direct target network of the Neurospora crassa plant cell wall deconstruction regulators CLR-1, CLR-2, and XLR-1[END_REF]. Here, the creA_KO strain displayed an enhanced capacity to utilise starch, which was moderately impaired in the pf2ko mutant (Fig. 5). This indicates PnCreA and PnPf2 shared a similar function to the respective N. crassa orthologues [START_REF] Xiong | A fungal transcription factor essential for starch degradation affects integration of carbon and nitrogen metabolism[END_REF]. Surprisingly however, despite vegetative growth abnormalities on agar, there was no distinct change in the virulence profile of either the creA_KO or CreA_OE mutants (Text S2). We also failed to detect the CCR element in the promoters of ToxA, Tox1, Tox3 or Tox267 (File S2). This suggests that the regulation of host-specific virulence factors critical for P. nodorum infection are not subject to CCR by PnCreA.

This investigation, along with previous TF studies in P. nodorum [START_REF] Ipcho | The transcription factor StuA regulates central carbon metabolism, mycotoxin production, and effector gene expression in the wheat pathogen Stagonospora nodorum[END_REF][START_REF] Chooi | Functional genomics-guided discovery of a light-activated phytotoxin in the wheat pathogen Parastagonospora nodorum via pathway activation[END_REF][START_REF] Rybak | A functionally conserved Zn2Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat[END_REF][START_REF] Lin | The global regulator of pathogenesis PnCon7 positively regulates Tox3 effector gene expression through direct interaction in the wheat pathogen Parastagonospora nodorum[END_REF], indicate PnPf2 is central to the transcriptional-regulatory network controlling virulence, for which a tentative model is proposed (Fig. 6). Having expanded our understanding, it also raised some key questions. For many genes directly targeted by PnPf2, differential expression in pf2ko has not been observed (364 of 484 highconfidence targets). Such discrepancies are also reported in ChIP-seq experiments on filamentous fungi (Table S2). One aspect to consider is that functional TF binding requires specific cofactors/coregulators before gene expression is eventually modulated [START_REF] Slattery | Absence of a simple code: how transcription factors read the genome[END_REF][START_REF] Reiter | Combinatorial function of transcription factors and cofactors[END_REF]. Furthermore, TF-DNA interactions can be redundant or nonfunctional [START_REF] Carey | Confirming the Functional Importance of a Protein-DNA Interaction[END_REF][START_REF] Spivakov | Spurious transcription factor binding: non-functional or genetically redundant?[END_REF][START_REF] Osterwalder | Enhancer redundancy allows for phenotypic robustness in mammalian development[END_REF] To conclude, this study presents direct evidence of DNA binding in a Pf2 orthologue, where virulence-regulatory functions are consistently observed in phytopathogenic fungi. In P. nodorum, PnPf2 is central to the transcriptional regulation of virulence and directly controls effector expression. The current research on PnPf2 now provides a platform to further investigate its signalling pathways and molecular interactions that could be inhibited for targeted disease control.

Fig. 6 HERE

Table S2 HERE

Materials and methods

Annotations and PnPf2 domain analysis

The P. nodorum annotated genome for the reference isolate SN15 (54) was used consistent with annotations in the previous RNA-seq analysis [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. The PnPf2 polypeptide sequence was submitted to Interproscan (Release 82.0) for Interpro and Conserved-Domain-Database domain identification [START_REF] Blum | The InterPro protein families and domains database: 20 years on[END_REF]. NLStradamus was used to predict the nuclear localisation signal [START_REF] Nguyen Ba | NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction[END_REF]. The disordered region was predicted using IUPRED2A (28).

Generation and assessment of fungal mutants

The molecular cloning stages, the constructs generated and diagrammatic overview of the final transformed P. nodorum mutants generated in this study are detailed in Text S3. The respective primers designed for fragment amplification and/or screening are outlined in File S3. Fungal mutants used in this study are summarised in Table 2. Their phenotypic and gene-expression analysis procedures are also described in Text S3.

Table 2. HERE Text S3 HERE

File S3 HERE

Chromatin immunoprecipitation sample preparation

The Pf2-HA, Pf2-HA_OE and pf2-HA_KO strains were prepared following 3 days standardised growth in 100 mL For ChIP-qPCR, DNA (for both the Pf2-HA, Pf2-HA_OE and pf2-HA_KO ChIP and input control samples) was recovered from Proteinase K treated samples using the GenElute PCR purification kit (Sigma-Aldrich).

For ChIP-seq analysis, DNA (for both Pf2-HA and Pf2-HA_OE ChIP and input control samples) was purified from the Proteinase K treated samples by mixing in 1 volume (400 μL) of phenol:chloroform. This was centrifuged for 5 min at 16000 g and the aqueous phase retrieved. To this, 400μL chloroform was added, mixed and spun (16000 g 5 min) before 350 μL of the aqueous phase was transferred to a fresh tube.

35 μL sodium acetate (3 M, pH 5.2) was added with 1 μL of glycogen (20mg/mL).

Samples were mixed by inversion and 1 mL 100% ethanol added before precipitation at -80 o C for 1-2 hrs. Pellets were retrieved by spinning 16000 g for 10 min at 4 o C, then washed in 1 ml of ice-cold 70% ethanol before drying and resuspension in 30 μL

Tris-Cl (10 mM).

Two independent DNA preparations for each sample (i.e. the ChIP and input samples for both Pf2-HA and Pf2-HA_OE), beginning with the fungal growth stage in Fries3 broth, were pooled to ensure sufficient DNA was obtained for generating ChIP-seq libraries. The pooled DNA was measured using a Tapestation system (Agilent, Santa Clara, California). 10 ng of each sample was processed using the TruSeq ChIP Library Preparation Kit (Illumina, San Diego, California). Libraries were size-selected (100-300 bp) and split across four separate lanes for sequencing in a NextSeq 500 sequencer (Illumina) to obtain 2 x 75 bp paired-end reads (Australian Genome

Research Facility, Melbourne, Australia).

ChIP-seq analysis

An overview of the following data analysis pipeline from QC of raw reads through to genome mapping, ChIP-seq peak/summit calling, target gene prediction, ChIP-qPCR validation, GO enrichment analysis and motif position-weight-matrix (PWM) modelling Text S1.

Raw read filtering, mapping and peak/summit calling

Raw reads were checked using FASTQC (Version 0.11.9) (57) and the adapter sequences were trimmed using Cutadapt (Version 1.15) along with nucleotides where the Illumina quality scores were below 30 [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads. 1[END_REF]. Optical duplicates were then removed using the 'dedupe' option in Clumpify (version 1.15) from the BBTools package [START_REF] Bushnell | BBMap[END_REF].

Reads were subsequently mapped to the SN15 genome (54) using BWA-MEM [START_REF] Li | Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM[END_REF].

Reads mapping to a single locus as the best match (primary alignments) were retained for downstream analysis and the datasets from sample libraries originally split across the NextSeq lanes were merged using SAMtools (Version 1.10) to produce the final mapped-read datasets [START_REF] Li | The sequence alignment/map format and SAMtools[END_REF]. MACS (Version 2.2.7.1) was used for calling enriched regions (i.e. peaks) and summits (highest nucleotide point or points within peak regions) from ChIP sample reads relative to the input samples (for Pf2-HA and Pf2-HA_OE). A Q-value peak enrichment threshold of 0.01 was used and the BAMPE option utilised to assess read depth from cognate pairs [START_REF] Feng | Identifying ChIP-seq enrichment using MACS. 9[END_REF][START_REF] Gaspar | Improved peak-calling with MACS2[END_REF]. Paired read lengths from the cognate pairs were assessed using Deeptools 'bamPEFragmentSize' (Version 3.3.0) to verify they corresponded to 100-300 bp size selected fragments [START_REF] Ramírez | deepTools2: a next generation web server for deep-sequencing data analysis[END_REF].

Modelling binding-site motifs

The overlapping peak regions identified from the Pf2-HA and Pf2-HA_OE samples were merged using MAnorm (65) to create a consensus set of enriched peak regions containing the putative PnPf2 binding sites. From this set, overrepresented PWMs up to 20 bp long were modelled with MEME (version 5.1.1) [START_REF] Bailey | MEME SUITE: tools for motif discovery and searching[END_REF][START_REF] Bailey | Fitting a mixture model by expectation maximization to discover motifs in biopolymers[END_REF]. For the resulting PWMs, 500 bp genomic regions centred at ChIP-seq summits were extracted and analysed using CentriMo (Version 5.1.1) to verify that the motif instances were also centred at the respective summits for both the Pf2-HA and Pf2-HA_OE samples [START_REF] Bailey | Inferring direct DNA binding from ChIP-seq[END_REF]. Gene promoters (spanning annotated transcription start sites to the nearest upstream gene feature or 1500 bp) with ≥ 1 occurrence of each motif were determined using FIMO [START_REF] Grant | FIMO: scanning for occurrences of a given motif[END_REF]. These were cross-referenced with the differentially expressed genes (i.e. expressed significantly up or down in pf2ko relative to SN15) defined in a previous RNA-seq analysis (13). Fisher's exact test with Bonferroni corrected P-values (70) was used to identify pf2ko differentially expressed gene-promoter sets significantly enriched (Padj < 0.01) for the respective motifs vs the background rate in SN15.

PnPf2 target gene-promoter analysis

Genes targeted by PnPf2 were determined based on the proximity of summits to annotated genes, which were identified using ChIPseeker (Version 1.24.0) [START_REF] Yu | ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization[END_REF].

Genes with ≥1 summit falling within their promoter region from the Pf2-HA or Pf2-HA_OE datasets were considered PnPf2 targets. High-confidence PnPf2 targets corresponded to genes with a promoter summit in Pf2-HA and Pf2-HA_OE. ChIP-qPCR was then undertaken to verify that the ChIP-seq peak regions in Pf2-HA and The PnPf2 target genes were cross-referenced with the pf2ko expression patterns (expressed significantly up or down in pf2ko) defined previously [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF] to link direct binding with the modulation of gene expression. The SN15 effector-like genes annotated previously [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF] were compiled among the PnPf2 targets. Annotated homologues were identified from the corresponding records in UniProt (release 2020_05) [START_REF] Bursteinas | Minimizing proteome redundancy in the UniProt Knowledgebase[END_REF]. Both the high-confidence and total PnPf2 target-gene sets were then used for GO enrichment/network analysis using the SN15 GO annotations defined previously [START_REF] Jones | A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat[END_REF]. The 'enricher' function in the Clusterprofiler package (Version 3.16.0) [START_REF] Yu | clusterProfiler: an R package for comparing biological themes among gene clusters[END_REF] was invoked to identify the overrepresented GO classes (P < 0.01) in PnPf2 targets.

Data availability statement

The ChIP-seq reads are available under BioProject ID: PRJNA824526, corresponding to BioSamples SAMN27406642 (Pf2-HA strain) and SAMN27406643 (Pf2-HA_OE strain).

Acknowledgements

This carbohydrate-active enzymes [START_REF] Friesen | Emergence of a new disease as a result of interspecific virulence gene transfer. 8[END_REF], cell-signalling molecules [START_REF] Liu | SnTox3 acts in effector triggered susceptibility to induce disease on wheat carrying the Snn3 gene[END_REF], and trans-membrane transporters [START_REF] Liu | The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1[END_REF].

  Zn2Cys6 TF domains and localises to the nucleus. Conserved domains or distinguishing features were analysed in the 652 amino acid (a.a) PnPf2 protein. The Zn2Cys6 DNA binding domain was located N-terminally at a.a 9 to 54 with an overlapping nuclear localisation signal (NLS) (KKGPKGSR; a.a 51 to 58) (Fig. 1A). A 'fungal TF domain' was identified from a.a 223 to 294 within a conserved 'middle homology region' (a.a 104 to 320

Fig. 1 HERE 2 . 2 .

 122 Fig.1 HERE

  and broaden the DNA interactions captured. A total of 997 summits were obtained from the Pf2-HA dataset and 2196 from Pf2-HA_OE, corresponding to 740 and 1588 peak regions respectively. There were 588 shared peaks identified between the samples (File S1), indicating strong overlap between the predicted PnPf2-targeted regions. The Pf2-HA_OE dataset broadened the scope of putative binding sites. A quantitative PCR (qPCR) analysis was then undertaken on independently prepared ChIP DNA, comparing the Pf2-HA and Pf2-HA_OE samples to the pf2-HA_KO control. Foldenrichment values relative to pf2-HA_KO strongly correlated with ChIP-seq summit -Log10(Q-values), a proxy measure for PnPf2-DNA binding affinity, in both the Pf2-HA (P < 0.01 with Pearson's r = 0.77) and Pf2-HA_OE (P < 0.01 with Pearson's r = 0.74) datasets (Text S1). The high reproducibility across separate methodologies provided confidence in the robustness of ChIP-seq summit calls.

Fig. 5 HERE

 5 Fig. 5 HERE

  Fries3 liquid medium (Text S3). Prior to harvesting, a 5 mL crosslinking solution (10% w/v formaldehyde, 20 mM EDTA and 2 mM PMSF dissolved in 50 mM NaOH) was added with continuous shaking at 100 rpm for 10 min. To this, 5 mL quenching solution (1.25 M glycine) was added before another 10 min shaking. Whole protein extracts were then obtained as described (Text S3) with modifications for ChIP. The 50 mM Tris was replaced with 50 mM HEPES in the lysis buffer while gentle rotation of the resuspended fungal material was replaced by eight rounds of sonication using a Bandelin (Berlin, Germany) UW3100+SH70+MS73 tip sonicator to fragment the fungal DNA (set at 15 sec on/off with 60% amp and 0.8 duty cycle). Samples were held in an ice block during sonication. The supernatant was then retrieved from two rounds of centrifugation (5000 g, 4 o C for 5 min). A 100 μL aliquot of the supernatant was reserved as an 'input control' against which ChIP samples were to be normalised. A 1000 μL aliquot was then precleared for immunoprecipitation by gently rotating with 20 μL Protein A dynabeads (10001D -Thermofisher, Waltham, Massachusetts) for 1 hr at 4 o C. The supernatant was then retrieved and incubated with 2.5 μg anti-HA polyclonal antibody (71-5500 -Thermofisher) for 16 hrs at 4 o C. Another 20 μL Protein A dynabeads were then added and gently rotated for 2 hrs at 4 o C. The dynabeads were then retrieved and washed twice with 1mL ice-cold lysis buffer, once with high-salt buffer (lysis buffer + 500 mM NaCl), once with LiCl buffer (250 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 0.5% NP40 and 0.5% NaDOC) and once with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). Samples were then incubated in a shaking incubator for 10 min (300 rpm, 65 o C) with 200 μL elution buffer (0.1 M NaHCO3, 10 mM EDTA and 1% SDS) before transferring the supernatant to a fresh tube. The input control was also supplemented with 100μL elution buffer at this stage and 8 μL NaCl solution (5 M) was added to both samples before de-crosslinking for 16 hrs at 65 o C. To these samples, 200 μL of H2O and 100 μg RNAse A (QIAGEN, Hilden, Germany) were added before incubating for 1 hr at 65 o C. Ten μg Proteinase K (Sigma-Aldrich, St. Louis, Missouri) was then added before incubating a further 1 hr at 50 o C.

Pf2-

  HA_OE would also correlate with quantitative enrichment against the pf2-HA_KO control strain. Quantitative PCR primer pairs (File S3) were designed to flank ChIPseq summits in a selection of gene promoters (ToxA, Tox1, Tox3, SNOG_03901, SNOG_04486, SNOG_12958, SNOG_15417, SNOG_15429, SNOG_16438, SNOG_20100 and SNOG_30077) and a selection of non-summit control regions (Act1 and SNOG_15429 coding sequences and the TrpC terminator). The 'input %' values were calculated for each sample using the method described previously (72) and used to calculate fold-differences (normalised to Act1) for Pf2-HA and Pf2-HA_OE relative to the pf2-HA_KO control for comparison with the Pf2-HA and Pf2-HA_OE -Log10(Qvalues) at the respective ChIP-seq summit loci. Pearson's correlation coefficient was calculated for the ChIP-qPCR fold-difference and ChIP-seq -Log10(Q-values) at the respective loci and used as the test statistic to assess whether the association was significant (SPSS version 27.0).

Fig. 1

 1 Fig. 1 PnPf2 domain analysis and cellular localisation. A) Predicted domains and

  Fig. 1 PnPf2 domain analysis and cellular localisation. A) Predicted domains and features identified in the 652 amino acid PnPf2 protein sequence typical of a Zn2Cys6

Fig. 2

 2 Fig. 2 Identification of PnPf2 regulatory element motifs. A) The M1 motif (5'-RWMGGVCCGA-3') and M2 motif (5'-CGGCSBBWYYKCGGC-3') were modelled from the merged set of Pf2-HA and Pf2-HA_OE sample ChIP-seq peak regions. Their

Fig. 3

 3 Fig. 3 Gene expression and gene-ontology (GO) analysis of PnPf2 direct targets. A) Overview of the genes targeted by PnPf2 in their promoter region based on the respective Pf2-HA and Pf2-HA_OE ChIP-seq datasets. There were 484 genes

Fig. 4

 4 Fig. 4 Phenotypic assessment of transcription factor (TF) gene deletion mutants. A)Representative images after 12 days of growth on nutrient-rich agar (V8PDA) and

Fig. 5

 5 Fig. 5 Assessment of PnPf2 and PnCreA mutant growth on different substrates. Images following 12 days of growth on nutrient-rich V8PDA and minimal-medium agar

Fig. 6

 6 Fig. 6 The proposed model of the PnPf2-centred regulatory network in the virulence of P. nodorum. The Pf2 taxonomic orthologues and AmyR regulators in the

Table 1 HERE

 1

  study was supported by the Centre for Crop and Disease Management, a joint initiative of Curtin University (https://www.curtin.edu.au/) and the Grains Research and

	Development Corporation (https://grdc.com.au/) under the research grant CUR00023
	Project F3 awarded to KCT). EJ was supported by an Australian Government
	Research Training Program Scholarship (https://www.dese.gov.au/) administered
	through Curtin University (https:// www.curtin.edu.au/). The funders had no role in
	study design, data collection and interpretation, or the decision to submit the work for
	publication.
	We would like to acknowledge Dr. Carl Mousley for helpful suggestions relevant to
	ChIP-seq motif validation, Dr. Darcy Jones for bioinformatics advice and support, as
	well as Johannes Debbler for assistance with molecular cloning.

Tables

 Table 1.Rationale for the investigation of novel transcription factors (TFs) PnEbr1 ectopically-integrated construct A Strains are listed in corresponding order to the detailed description of their generation in Text S3. B Abbreviations: GFP -Green fluorescent protein, HA -Haemagglutinin, M1 -Motif1, M2 -Motif 2.

Supplemental item legends

Text S1 Chromatin immunoprecipitation (ChIP) strain assessment and overview of ChIP-seq/ChIP-qPCR.

Text S2 Supplemental transcription factor mutant phenotype assessment.

Text S3 Supplemental materials and methods.