Simon Lamy
email: simon.lamy@chu-rennes.fr

David Veillard

Helene Doyen

Anne Kerbrat

Laure Michel

Emilie Chretien

Ahmad Ousmen

Giles Edan

Emmanuelle Le

Switching from natalizumab administration at the day hospital to administration at home. A 1 year prospective study of patient experience and quality of life in 30 consecutive patients with multiple sclerosis (TYSAD-35)

Keywords: (Introduction, Method, Results and Discussion) : 3317  Multiple sclerosis  Quality of life  Home Infusion Therapy  Natalizumab

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

INTRODUCTION

Natalizumab is a disease-modifying treatment for very active forms of relapsing-remitting multiple sclerosis, which has been shown to be effective in a number of studies [START_REF] Polm N H O' Onnor Pw H Vr Ov T | l. r n omiz pl bo-controlled trial of natalizumab for relapsing multiple sclerosis[END_REF][START_REF] Rudick | Natalizumab plus interferon beta-1a for relapsing multiple sclerosis[END_REF][START_REF] Spelman | Comparative efficacy of first-line natalizumab vs IFN-β or glatiramer acetate in relapsing MS[END_REF][START_REF] Butzkueven | Long-term safety and effectiveness of natalizumab treatment in clinical practice: 10 years of real-world data from the Tysabri Observational Program (TOP)[END_REF] . It is administered intravenously, at a dose of 300 mg every four weeks, at the day hospital. Treatment via the 4 subcutaneous route has also been available in France since March 2021 (two 150 mg injections) [START_REF] Plavina | A Randomized Trial Evaluating Various Administration Routes of Natalizumab in Multiple Sclerosis[END_REF][START_REF] Trojano | A randomized study of natalizumab dosing regimens for relapsing-remitting multiple sclerosis[END_REF] .

Natalizumab is generally well tolerated, but headaches, asthenia, and mild infections (ear-nose-andthroat or urinary) have been reported in some cases. Allergic reactions, rarely severe (1% of them), are reported in 3 to 4% of patients, justifying treatment and monitoring in a hospital environment [START_REF] Polm N H O' Onnor Pw H Vr Ov T | l. r n omiz pl bo-controlled trial of natalizumab for relapsing multiple sclerosis[END_REF][START_REF] Butzkueven | Long-term safety and effectiveness of natalizumab treatment in clinical practice: 10 years of real-world data from the Tysabri Observational Program (TOP)[END_REF] . Furthermore, natalizumab can cause progressive multifocal leukoencephalopathy (PML) in patients seropositive for JC virus (cumulative risk of up to 3%). JC virus serology tests should therefore be performed rigorously, every six months, with the discontinuation of natalizumab in cases of seropositivity [START_REF] Butzkueven | Long-term safety and effectiveness of natalizumab treatment in clinical practice: 10 years of real-world data from the Tysabri Observational Program (TOP)[END_REF][START_REF] Bozic | Anti-John Cunnigham virus antibody prevalence in multiple sclerosis patients: baseline results of STRATIFY-1[END_REF][START_REF] Plavina | Anti-JC virus antibody levels in serum or plasma further define risk of natalizumab-associated progressive multifocal leukoencephalopathy[END_REF] . MS patients are generally young and active, with children at home. Regular visits to the hospital therefore impose significant personal and professional constraints. The need for such visits can also make these patients and their families feel that they are facing a serious illness [START_REF] Rath | Patient Preferences for Time and Location of Infusible Therapies in Multiple Sclerosis and Neuroimmunologic Disorders[END_REF][START_REF] Simmons | Living with multiple sclerosis: longitudinal changes in employment and the importance of symptom management[END_REF] .

The COVID-19 pandemic has recently led to radical changes in the organization of healthcare, including the administration of immunosuppressants for MS, from the points of view of both healthcare professionals and patients. At the end of April 2020, following the first lockdown of the French population, the French High Authority for Health (HAS) temporarily authorized the administration of natalizumab by the intravenous (IV) route to patients at home, via a home-hospitalization structure (HAD). The healthcare model for hospitalization at home in France was defined by Law No. 91 748 of July 31, 1991, and depends on dedicated medical and paramedical teams in a given area. Beyond the context of the pandemic and the need to ensure the continued treatment of these patients (risk of rebound when natalizumab is stopped) 11 , the decision of the HAS was based on a few previous experiences, particularly in Australia, reporting encouraging results for safety, efficacy, and patient satisfaction [START_REF] Vijayan | Establishment of the first at-home natalizumab infusion service for the treatment of relapsing remitting multiple sclerosis (rmms)[END_REF][13][START_REF] Ju Ton M Us K L Hulz | P ti nts' xp ri n s of n t lizum b tr tm nt in hom environment: a qualitative study[END_REF][15] . Indeed, it is ss nti l to t k th p ti nts' vi wpoint into ount (in addition to the clinical criteria of efficacy and safety) when implementing of new modes of organization for their

MATERIALS AND METHODS

Design: We performed a prospective, observational, descriptive study.

Population

Until 2020, patients had never been treated with natalizumab at home in France. Home-healthcare services, such as HAD-35 (one of the partners in the TYSAD-35 study) provide care for patients at home, within a defined area and with defined human resources (medical and paramedical health professionals). For this reason, the maximum number of patients who could be treated at home was set at 30.

The participants were recruited from the active list of 62 patients treated with natalizumab in the day hospital (DH) of the neurology department of Rennes University Hospital. Between June 2020 and January 2021, 49 consecutive patients visiting the DH for natalizumab infusion were asked whether they would like to be treated at home. The first 30 patients meeting the following inclusion criteria who agreed were included.

Inclusion criteria

The inclusion criteria were: Procedure: our MS team co-wrote with the local home hospitalization service (HAD-35) a standardized care protocol for the administration of natalizumab infusions at home and shared information about MS and natalizumab. HAD infusions were performed in the same way as at the DH (same dose, route of administration, monitoring, etc.), after validation by a neurologist of our department during a teleconsultation planned within 48 hours before the infusion. A report was sent to the HAD to authorize the infusion. The patients were seen every six months at the DH of Rennes University Hospital, for serological tests for JC virus infection, and for clinical and neurological evaluations. They received the corresponding monthly natalizumab infusion during this visit.

6 This questionnaire allows patients to assess the quality of their care pathway in a global manner rather than focusing specifically on infusion at home. It was completed by the included patients before the start of home infusions and then after 12 months of HAD care.

-Data on patient satisfaction and experience immediately after each infusion, collected with a numerical rating scale from 0 to 10 and a questionnaire designed for this study and based on a recognized tool (PPE-15 24 (Supplemental material 1)).

-Quality-of-life data collected with the MS-specific questionnaire, MusiQol

22

, which contains 31 items describing the following nine dimensions: daily activity, psychological well-being, relationships with friends, symptoms, relationships with family members, relationships with healthcare professionals, emotional and sex life, coping, and rejection. Each of the patients included completed this questionnaire before the start of the first infusion at home, and after each monthly infusion, over a period of 12 months.

All information was obtained prospectively from patients, who completed forms online with Sphinx IQ2 survey softwareversion 7.4.5.1.

Ethics

This study was approved by the Ethics Committee of the Rennes University Hospital (No.

35RC21_8924_TYS-HAD-35) and complies with the obligations of law number 78-17 of January 6, 1978, relating to data processing, files, and liberties. All participants signed a written informed consent form before inclusion.

STATISTICAL ANALYSES

Descriptive analyses

The sociodemographic and clinical characteristics of the patients and their disease are described with the mean and standard deviation (SD) for continuous variables and frequencies with percentages for qualitative variables.

Analysis of the questionnaires

After analyzing the shape of the distributions of the scores for each dimension, patient experience (MusiCare), and quality of life (MusiQol), were evaluated, with the results expressed as means and standard deviations for the whole population. The scores for each dimension were treated identically.

The means for each dimension (and of the overall score for MusiQol) between M0 and M12 were compared using non-parametric paired Wilcoxon tests.

For the measurement of patient satisfaction with each infusion, the responses to the questionnaire corresponding to a negative experience during an infusion at home were recorded and are presented as the percentage of patients reporting such experiences.

We used a significance threshold of 5% for all comparisons. All statistical analyses and calculations of scores were performed with SAS software version 9.4 (SAS Institute, Cary, NC, USA).

RESULTS

In total, 46 (94%) of the 49 consecutive patients answered in the affirmative when asked whether they wished to receive treatment at home, regardless of their place of residence, duration of treatment, or JC serology status. The main reasons given for wishing to switch to this mode of care or for refusing it are indicated in Table 1.

Characteristics of the 30 patients included in TYSAD-35:

The demographic and medical characteristics of the patients included in the study are presented in Table 2. The patients were young, with a median age of 38.1 years (range: 25-58 years); most were working (60%), living with a partner (73%), and with dependent children (70%). The clinical activity of the disease was well controlled before inclusion. The patients had been on natalizumab treatment for a median of 5.45 years (range: 2.7-14.0 years).

Follow-up of patients by the HAD service

Thirty patients were initially included; 27 (90%) were followed for the 12 months of the home treatment experiment and three left the study prematurely. One patient returned to the DH for treatment due to poor venous access, making it impossible to continue home infusion after the first three sessions. The second patient preferred to return to DH management after four infusions for personal reasons, being a healthcare professional at our establishment. The third patient was lost to follow-up after seven infusions, despite repeated attempts at contact by healthcare professionals from the DH and HAD service. In total, 306 infusions were performed at home during the 12 months of follow-up.

Natalizumab at home: the patients' perspective -Experienced quality of this new care pathway

 Primary endpoint: MusiCare. Twenty-six patients completed the MusiCare questionnaire at M0 and M12. The overall experience of the patients included improved between M0 and M12 for all the dimensions and it was significant for the dimension "experience of relationships with healthcare professionals" (ability to listen, empathy, relationship based on trust) (Table 3).  Mean overall satisfaction with home infusions was high, at 9.1/10, and remained constant throughout the follow-up period. However, an analysis of the 294 satisfaction questionnaires completed by the 30 patients after each infusion at home identified several areas of patient dissatisfaction. The most frequently cited points of dissatisfaction w r : " ontr i tory responses provided by the various healthcare tors" 7. % n "physi l p in during the procedure" . % . Similarly, the information shared with patients and their families was sometimes not entirely satisfactory. Finally, several patients felt that their involvement in therapeutic decisions and in the management of their disease could be improved (Table 4).

-Quality of life

MusiQol global score increased over the months, although this result was not significant (Figure 2).

Twenty-six patients completed the MusiQol questionnaire at M0 and M12. For these patients, a significant increase was observed in the scores for the "fri n ship" p = 0.047 n " oping" p = 0.0491) dimensions (Table 5).

Adverse events

No allergic reactions, not even minor, were reported. No adverse events were recorded apart from mild headaches and asthenia during treatment. On three occasions, the team of HAD nurses encountered difficulties with peripheral venous access, which led to one of these patients resuming treatment at the DH. Only one infusion had to be postponed due to reflex syncope just before initiation of the infusion.

Progression of multiple sclerosis

Multiple sclerosis activity remained low throughout the follow-up period in all 27 patients who completed follow-up. None of the patients experienced flare-ups. EDSS score remained stable at 2.0.

Annual follow-up by brain MRI showed that lesion load was stable in all patients who completed followup. No seroconversions were observed for the JC virus.

DISCUSSION

The context of the COVID-19 pandemic provided an opportunity to establish home infusions of natalizumab, which had already been requested by neurologists and patients but had not previously been approved by the French health authorities. We present here the first French study to evaluate this new procedure. This study included the largest number of infusions (306 in total) of natalizumab at home assessed to date, providing us with sufficient information to demonstrate the feasibility of this new practice. Its implementation, within the HAD care model, required the creation of a specific treatment protocol, the training of HAD healthcare professionals, the sharing of medical information between services, and close coordination between the actors of the neurology department DH at Rennes University Hospital and the HAD. As in the study by Shultz et al., the patients included in TYSAD-35 were very enthusiastic about this new practice, for organizational and logistical reasons (time savings, fewer medical visits) in particular, consistent with the characteristics of this population of young patients, little neurological impact of their disease, living with their families and continuing to work. Finally, the fear of contracting COVID-19 during visits to the DH was only rarely identified as a reason for wishing to be treated at home. All the patients had been on natalizumab treatment for more than two years (sometimes up to 14 years), accounting for their lack of concern about the risk of a serious allergic reaction. Such reactions are rare in practice, and have been reported to occur essentially at the time of the first or second infusion 1 . Consequently, a switch from DH to HAD treatment could be proposed to patients after a minimum period of six months, as in the study by Schultz et al.

The treatment protocol established specifically for this new organization of care was quickly assimilated and applied by the HAD. As a result, no infusion was postponed or not carried out due to logistical or organizational difficulties. However, three patients withdrew from the study. Two preferred to return to DH management, one because of poor venous access and the other worked at the hospital and found that this organization was more suitable. The third patient was lost to follow-up for several months and the cessation of treatment was accompanied by a resumption of disease activity.

These findings suggest that the HAD management model is a suitable option for most patients but should not be systematic, especially when more general adherence to treatment and follow-up are likely to be difficult. Access to both options should be maintained to take patient preferences into account.

This study is original because we prospectively measured, in real-life conditions, the impact of this new mode of care on the care quality from the patient's point of view, an essential perspective in the context of this innovative organization of care. W m sur r qu lity from th p ti nts' perspective with tools of the PREMs and PROMs types.

Th p ti nts' xp ri n of this mo of tr tm nt w s v lu t immediately after each monthly infusion and over the long term, at the end of the 12 months of follow-up. This approach and the tools used made it possible to highlight a generally very positive experience with the HAD management of natalizumab infusions. Indeed, the mean score of each dimension of MusiCare has increased at the end of the 12 months of follow-up. The difference was significant for the dimension concerning the p ti nts' xp ri n of th ir relationships with healthcare professionals. These observations suggest that good adherence to the proposed care model is likely, particularly as the HAD healthcare team was completely new to the patients and not specialized in MS or its care, unlike the DH team.

Similarly, the maintenance of an overall positive experience in terms of the quality of the information received and access to care suggests that the proposed model of collaboration between the HAD and the reference neurology team met the expectations of patients. These results are consistent with those reported by Juaton et al. in their recent study of home care in Australia, in which a qualitative approach based on interviews was used to study the 12 patients receiving such care 14

. They are also consistent with the findings of another Australian group that used questionnaires to ask several dozen patients with neurological diseases, including multiple sclerosis, about their preferences in terms of location for long-term infusion treatment (mostly natalizumab). Most of these patients expressed a preference for having their infusions at home 9 . In addition to the MusiCare tool, specifically validated for patients with MS and taking into account the entire care pathway, we developed a tool for assessing patient experience inspired by the French version of the PPE-15, and we used a numerical rating scale to assess the overall level of patient satisfaction. The results obtained with these two tools highlighted a very positive experience at the end of each infusion but also identified areas for the improvement for this new organization of care from th p ti nts' vi wpoint. For example, patients sometimes felt that the answers provided by healthcare professionals to questions were contradictory and the infusion procedure was sometimes painful. Information of this type is useful for guiding the necessary feedback-based discussions between the HAD and neurology teams to harmonize practices, based also on the sharing of theoretical knowledge about the disease and the technical specificities related to the frequency of infusions.

In evaluations of care results, principally with the MusiQol tool, we noted an improvement in the overall score for quality of life and a significant improvement in the dimensions relating to relationships with friends and coping. These results can be interpreted as indicating a better psychological adaptation to the disease and the way it is seen by others. Shultz et al. used other tools not specific for MS, but their results are consistent with ours.

No serious adverse effects, particularly of an allergic nature, were identified. Nevertheless, the risks had been anticipated by the creation of a dedicated protocol. According to this protocol the drug was administered in a safe environment, with twice-yearly visits to the DH to perform serological tests for JC virus and neurological evaluations, and for regular validation of the continuation of treatment according to the proposed procedure. This home procedure did not reduce our capability of detecting potential PML as teleconsultations were performed before each natalizumab home infusion and JC virus status were checked every 6 months.

This experimental study had several limitations, including, in particular, the absence of a control group and a small sample size. Furthermore, all the patients included were volunteers. Our results are therefore preliminary and require confirmation, which is the objective of the TYSATHOME study currently underway in France, with the same design but the aim of including more than 300 patients.

This study will also provide additional information, at national level, about the cost of this type of treatment relative to treatment in a hospital setting. With a view to perpetuating this practice for patients treated with natalizumab, administration by the subcutaneous route should enable HAD services to care for more patients, as less time will be required for monitoring after the injection.

care 16 , 17 . 21 .

 161721 Tools have, therefore, been available for several years for evaluating the care results from th p ti nts' point of vi w (Patient-Reported Outcome Measures -PROMs), n th p ti nts' experience of the new care organization (Patient-Reported Experience Measures -PREMs) 18,19 . MSspecific tools have been developed for measuring quality of life and experience related to the quality of the care path of these patients, and these tools are widely used in clinical studies 20,In France, the two reference tools are MusiQol (PROMs) and the more recently developed MusiCare (PREMs) 22,23 Before the COVID-19 pandemic, patients natalizumab treatment attended the day hospital for monthly infusions. In Rennes district (with almost 1 million inhabitants), this treatment was provided by the neurology department of Rennes University Hospital. Following HAS authorization of treatment at home at the end of April 2020, we developed a care protocol for the administration of natalizumab infusions at home, in collaboration with the local home hospitalization service (HAD-35). The objective of the TYSAD-35 stu y w s to ss ss prosp tiv ly th p ti nts' xp ri n of this new practice (MusiCare), together with their quality of life (MusiQol), and satisfaction, during the first 12 months of this experiment, and, finally, the safety of the procedure in terms of tolerance.

Figure 1 .

 1 Figure 1. Flow chart

Figure 2 .

 2 Figure 2. Change in mean global score for the MusiQol questionnaire

Table 1 . Desire or refusal to be treated with natalizumab at home (N = 49)

 1

	Reasons	N	%
	In favor or home infusions	46	94.0 %
	Time saved / Reduced traveling time	28	57.1 %
	Better material comfort	9	18.3 %
	Fits in better with professional activity	4	8.1 %
	Change in view of the disease (less serious)	3	6.0 %
	Lower risk of viral infection (COVID-19)	2	4.0 %
	Against home infusions	3	6.0 %
	Negative or worrying image of the infusion and the sick parent for the children	2	4.0 %
	Feeling safer in a hospital environment	1	2.0 %
	N = Number

Table 2 . Demographic and clinical characteristics at inclusion (N = 30)

 2

	Age at inclusion † (years)

Table 3 . Patient experience: MusiCare at M0 and M12 (N = 26)

 3

	MusiCare	M0		M12
	Dimensions	Mean	Standard deviation	Mean	Standard deviation	P value
	Experience of information about the disease in general	74.8	20.5	78.8	20.4	0.4577
	Experience of information about the treatment and examinations	81.4	17.9	82.8	18.4	0.9914
	Experience of relationships with healthcare professionals	81.8	19.8	91.5	13.2	0.0203 *
	Perception and experience of access to care	69.0	13.6	72.0	20.5	0.0972
	Conditions of reception at care sites	67.9	16.1	75.0	17.7	0.0995

*

P value < 0.05

Table 4 . Proportion of criteria deemed unsatisfactory by patients over the 12 months of the study, for the 294 questionnaires completed.

 4

	N	%

Table 5 . Quality of life: MusiQol at M0 and M12 (N = 26)

 5

	MusiQol	M0		M12		Significance of
						difference
	Dimensions	Mean	Standard deviation	Mean	Standard deviation	between M0 and M12
	Daily living activities	65.4	22.6	65.4	24.9	0.8566
	Psychological well-being	62.5	25.2	67.8	23.6	0.0660
	Symptoms	74.3	22.2	75.6	26.8	0.8398
	Relationships with friends	66.0	23.6	72.1	28.1	0.0478
	Relationships with family members	82.0	20.9	80.3	23.8	0.7144
	Emotional and sex life	68.5	31.3	70.0	26.8	0.6395
	Coping	67.8	24.8	75.5	26.3	0.0491
	Rejection	85.6	28.9	87.5	27.4	0.5156
	Relationship with the healthcare system	80.4	13.3	82.5	15.5	0.5031
	Global score	72.4	14.3	75.5	16.3	0.0864

E. Le Page received honoraria for consulting or lectures, invitations for national and international congresses from Biogen, Merck, Teva, Sanofi-Genzyme, Novartis, Alexion, Roche; research support from Teva and Biogen; academic research grants from PHRC and LFSEP, and travel grant from ARSEP Foundation. H. Doyen received honoraria for consulting from Biogen. L.

CONCLUSION

Our study demonstrates that home injections of natalizumab are feasible for patients with multiple sclerosis. This new practice has a positive impact on the experience of patients relative to hospital care, while remaining safe and effective for disease control. This therapeutic perspective is promising and of interest for patients who would like to be treated at home, which should help to establish its maintenance in the long term.

Declaration of interests

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


Keywords:

Mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs) form two important signaling pathways that allow plants to integrate various environmental and developmental cues into appropriate cell responses, through the phosphorylation of specific substrates on specific sites. The existence of common substrates to both MAPKs and CDPKs suggest that the biological functions of these substrates can be modulated by peculiar patterns of phosphorylation, a possibility designated under the notions of MAPK and CDPK phosphocodes. Moreover common MAPK and CDPK substrates offer an original opportunity to study the crosstalk between the two signaling pathways. The inhibitor of phosphatase ATI-2 was recently identified in the STRESS team as a new substrate common to both the MAPK 3 and 6 and the CDPK 5 and 6. In the frame of a general project investigating the MAPK and CDPK phosphocodes of ATI-2, the objective of my internship was to determine whether ATI-2 plays a role in biotic stress responses. For this I compared three different Arabidopsis thaliana backgrounds: wild-type, loss of function ati-2 and complemented lines in pathoassays as well as in growth inhibition assays in response to the immune elicitor flg22. Overall my results indicate that ATI-2 is not involved in the response to biotic stresses, although further replicates and controls are still needed to come to a definitive conclusion.

Résumé

Les mitogen-activated protein kinases (MAPKs) et les calcium-dependent protein kinases (CDPKs) forment deux voies de signalisation importantes permettant à la plante d'intégrer différents signaux environnementaux et développementaux pour y donner une réponse cellulaire appropriée, via la phosphorylation de substrats spécifiques sur des sites spécifiques. L'existence de substrats communs aux MAPKs et aux CDPKs suggère que les fonctions biologiques de ces substrats puissent être modulées selon des schémas particuliers, une possibilité désignée par la notion de phosphocodes MAPK et CDPK. De plus, ces substrats communs aux MAPK et CDPK offrent une opportunité intéressante pour l'étude des interactions entre ces deux voies de signalisation. L'inhibiteur de phosphatases ATI-2 a été récemment identifié par l'équipe STRESS comme un nouveau substrat, commun aux MAPK 3 et 6 et aux CDPK 5 et 6. S'insérant dans un projet plus global de décodage des phosphocodes MAPK et CDPK d'ATI-2, l'objectif de mon stage a été de déterminer si ATI-2 joue un rôle dans les réponses au stress biotique. Pour cela, j'ai comparé trois différents fonds génétiques : sauvage, perte-de-fonction ati-2 et lignées complémentées dans des patho-essais ainsi que dans des tests d'inhibition de croissance en présence de l'éliciteur immun flg22. Mes résultats indiquent qu'ATI-2 n'est pas impliqué dans la réponse au stress biotique même si plus de réplicats et de contrôles sont nécessaires pour parvenir à une conclusion définitive.

Introduction

Acknowledgments I would like to thank Julien LANG for taking me under his wing during the course of my internship. I would like to thank him for adapting his schedule to my sick leave despite having two other students to supervise. And I would like to thank him for teaching me new methods and then trusting me to try them on my own, for teaching me autonomy and planning which are two important skills to have, and for doing all of it with a lot of patience and attention.

I thank Jean COLCOMBET and the rest of the Stress Signaling team for welcoming me as a short-term member of the team and for helping me when I needed help.

I also thank Axelle DEROUBAIX as a member of the Stress Signaling team and as a fellow M1 student, for being my companion during those two months.

Finally, I thank Marie GARMIER, Karine ALIX, and the rest of the pedagogical team for accommodating to my sick leave and thus allowing me to enjoy the full experience of the internship as any other student.

Introduction

MAPK and CDPK pathways

Plants are sessile organisms and as such must adapt to local environmental perturbations due to biotic and abiotic stresses. When a stress is perceived, the stimulus activates intracellular signaling pathways. Plants have evolved sophisticated signaling modules which allow the integration of different extracellular cues into appropriate cellular responses.

These modules often involve kinase proteins, enzymes able to phosphorylate other proteins in order to change their properties. Due to its versatility and reversibility, phosphorylation is a very efficient and common way of transmitting information from one actor to the next, and to regulate the proteins involved in the response specificity. Plant genomes generally encode for more than a thousand kinase proteins (In Viridiplantae, about 5% of the genome encodes protein kinases) which underlines the importance of this type of signaling [START_REF] Colcombet | Arabidopsis MAPKs: a complex signaling network involved in multiple biological processes[END_REF]. Among them, CDPK and MAPK cascades form two crucial signaling modules.

The MAPK modules are composed of a Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK, also known as MKKK or MEKK), a Mitogen-Activated Protein Kinase Kinase (MAPKK, also known as MKK or MEK), and a MAPK (or MPK) that are activated one after the 1 other by a cascade of phosphorylation. In turn the activated MAPKs can phosphorylate different substrates on specific sites, thereby contributing to implement appropriate cell responses. In plants, MAPK modules are encoded by multi-genic families (the whole genome sequencing of Arabidopsis thaliana revealed, based on homology analyses, that this species could have 80 MAPKKKs, 10 MAPKKs, and 20 MAPKs) that can form multiple possible signaling connections. Some MAPK modules are known to be involved in plant development, such as the module YODA-MKK4/5-MPK3/6 that is implicated in stomata formation [START_REF] Wang | Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases in Arabidopsis[END_REF].

In the context of immune responses, two MAPK cascades MEKK1-MKK1/MKK2-MPK4 and MAPKKK5-MKK4/MKK5-MPK3/MPK6 have been well characterized [START_REF] Zhang | Mitogen-activated protein kinase cascades in plant signaling[END_REF]. The plant immune system is usually described as a two-layered system. The first level is triggered by conserved pathogen-associated molecular patterns (PAMPs) that are recognized by surface-localized pattern recognition receptors and leads to PAMP-triggered immunity (PTI). The second level of plant innate immunity, termed effector-triggered immunity (ETI) is triggered by the presence of pathogen effectors which are molecules produced by the pathogen to counter the PTI and that can be recognized by intracellular receptors belonging to the family of nucleotide-binding leucine-rich-repeat-containing receptors (NLRs). Several recent studies reported that PTI and ETI do not constitute independent processes, but are connected at different nodes, sharing similar signaling pathways and resulting in comparable outputs [START_REF] Lu | Intimate Association of PRR-and NLR-Mediated Signaling in Plant Immunity[END_REF]. For instance MPK3/6 are activated in a rapid and transitory manner during PTI, and in a delayed and sustained manner in ETI, but both types of kinetic converge towards the upregulation of NLR genes that thereby constitutes a common feature of the two types of immunity [START_REF] Lang | MPK3 and MPK6 control salicylic acid signaling by up-regulating NLR receptors during pattern-and effector-triggered immunity[END_REF].

As for CDPKs, they are capable of translating elevations in calcium levels in the cytoplasm or nuclei in phosphorylation signals. Like MAPK modules, CDPKs are encoded by multi-genic families (A. thaliana has 34 CDPK-coding genes in its genome). Several CPKs are involved in various aspects of plant development. For example, CPK2/6/20 are involved in pollen tube growth and CPK28 in shoot development, through the regulation of multiple hormone homeostasis [START_REF] Delormel | Properties and functions of calcium-dependent protein kinases and their relatives in Arabidopsis thaliana[END_REF]. Regarding plant immunity CPK5/6/11 were reported as redundant positive regulators of defense responses downstream of multiple PAMPs and damage-associated molecular patterns whose recognition induces Ca 2+ influx in the cell [START_REF] Boudsocq | CDPKs in immune and stress signaling[END_REF]. Remarkably CPK5/6 was shown to follow the same difference of activity kinetics as MPK3/6 in the contexts of PTI and ETI (Gao et al., 2013).

Potential crosstalk between the pathways

As phosphorylation of proteins can modify their stability, activity or subcellular localization, the characterization of MAPK and CDPK substrates is critical to fully understand their roles and still constitutes a lengthy quest. To date, only a few dozen of MAPK and CDPK substrates have been clearly characterized. For instance, the ACC synthase (ACS) 6 involved in ethylene production is phosphorylated by MPK6, while the respiratory burst oxidase homolog D involved in reactive oxygen species (ROS) production during immune responses is phosphorylated by CPK1/2/5/6/11 [START_REF] Dóczi | The Quest for MAP Kinase Substrates: Gaining Momentum[END_REF][START_REF] Simeunovic | Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases[END_REF][START_REF] Delormel | Properties and functions of calcium-dependent protein kinases and their relatives in Arabidopsis thaliana[END_REF].

The fact that some MAPKs and CDPKs are activated in analogous conditions, and have similar activation kinetics, especially in immune responses, raises the possibility of eventual interactions between the two pathways. In line with this hypothesis it has been shown that MAPK and CDPK signaling can have additive and synergistic effects on gene reprogramming in the context of PTI [START_REF] Boudsocq | Differential innate immune signaling via Ca2+ sensor protein kinases[END_REF]. From a mechanistic perspective, two main possibilities can be considered: either CDPKs and MAPKs are substrates of each other and thereby can modulate their reciprocal activities, or CDPKs and MAPKs share a common set of substrates.

Interestingly, it has been shown that the rice CDPK18 could phosphorylate MPK5 at a different site than the MAPKK and that this dual phosphorylation modulates the functions of MPK5 in immune responses [START_REF] Xie | Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice[END_REF]. Also it has been reported in tomato that the substrate ACS2 is phosphorylated in 2 different sites by MPK3/6 and CDPK2 and that both phosphorylated sites are required for ACS2 stabilization and ethylene production [START_REF] Kamiyoshihara | Turnover of LeACS2, a woundinducible 1-aminocyclopropane-1-carboxylic acid synthase in tomato, is regulated by phosphorylation/dephosphorylation: Turnover of LeACS2 by phosphorylation/dephosphorylation[END_REF]. More recently the transcription factor WRKY33 has also been characterized as a common substrate of MAPK and CDPK. In vitro and in vivo phosphorylation assays demonstrated that WRKY33 is phosphorylated on its Threonine-229 residue by CPK5/6 whereas MPK3/6 phosphorylate WRKY33 on several of its N-terminal serine residues. Those different phosphorylations have different effects on WRKY33 but act in cooperation during the immune response: while MPK3/6 phosphorylate WRKY33 to enhance its transactivation activity, phosphorylation by CPK5/6 enhances its binding to DNA, and both cascades function cooperatively to regulate camalexin biosynthesis controlled by WRKY33 [START_REF] Zhou | Differential Phosphorylation of the Transcription Factor WRKY33 by the Protein Kinases CPK5/CPK6 and MPK3/MPK6 Cooperatively Regulates Camalexin Biosynthesis in Arabidopsis[END_REF].

The existence of substrates common to both MAPK and CDPK like WRKY33 introduces the notion of MAPK/CDPK phosphocode which corresponds to the control of the functions of these substrates by specific patterns and kinetics of phosphorylation [START_REF] Perraki | Phosphocode-dependent functional dichotomy of a common co-receptor in plant signaling[END_REF].

Deciphering the phosphocode of new MAPK/CDPK substrates would therefore provide a better understanding of the coordination between the two important signaling modules, and should shed new light on the complex mechanisms allowing plants to cope with stresses.

The common substrate ATI-2 ATI-2 (At5g55200) was identified by [START_REF] Templeton | Identification and characterization of AtI-2, an Arabidopsis homologue of an ancient protein phosphatase 1 (PP1) regulatory subunit[END_REF], based on its affinity to human protein phosphatase-1 (PP1) gamma family and its homology with Inhibitor-2 (I-2) which was originally discovered as a potent inhibitor of PP1. However, later investigations also characterized I-2 as an activator of PP1 in animals [START_REF] Lemaire | Protein phosphatase-1: dual activity regulation by Inhibitor-2[END_REF]. In plants, ATI-2 can interact with Type One Protein Phosphatases (TOPPs) which are PP1-type phosphatases.

ATI-2 and TOPPs were demonstrated to regulate abscisic acid (ABA)-mediated responses.

Single mutants ati-2 and topp1 show a phenotype of higher sensitivity to salt stress and in response to ABA, while overexpression of ATI-2 or TOPP1 leads to increased growth on ABA [START_REF] Hou | Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling[END_REF]. Moreover the septuple topp1/4/5/6/7/8/9 mutant showed an increased resistance to wild type (WT) Pseudomonas syringae pv. tomato (Pst) DC3000, indicating the implication of some TOPPs in the immune response. A yeast two-hybrid (Y2H) assay performed to investigate the potential interaction between TOPPs and several immune regulators, including MPK3/6, and MPK4 also showed direct interactions between TOPP1, TOPP4, TOPP7, and TOPP8 on one hand, and MPK3, MPK4 and MPK6 on the other hand. This was confirmed in vivo using a bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana leaves [START_REF] Liu | Type one protein phosphatases (TOPPs) contribute to the plant defense response in Arabidopsis[END_REF]. Together these data suggest a possible role for ATI-2 in defense responses.

Using Y2H, the STRESS team found ATI-2 to be interacting with MPK3 and using phosphoproteomics, to be a putative substrate of CPK5. Interactions between ATI-2 and the kinases were also observed in planta using the BiFC in tobacco. Moreover in vitro kinase assays confirmed that ATI-2 is a substrate of the kinases MPK3/6 as well as CPK5/6. Using targeted mutagenesis experiments, the team showed that the phosphorylation of ATI-2 by the MAPK takes place on the serine 47 (S47) and that by the CDPK on the serine 140 (S140) Importantly, webtools compiling phosphoproteomic data from various experiments confirmed that these two sites can be phosphorylated in vivo (www.psb.ugent.be/webtools/ptm-viewer). Moreover, preliminary mass spectrometry results showed an increase in phosphorylation of the S47 site of ATI-2 in presence of flg22 which is a peptidic fragment of 22 amino acids from the bacterial protein flagellin, routinely used as a PAMP. This further suggests the implication of ATI-2 and its For ATI-2, the MAPK phosphocode is mediated by the activation of the MPK3/6 module and the phosphorylation of S47, while the CDPK phosphocode is mediated by the activation of CPK5/6 and the phosphorylation of S140. To understand the role of the MAPK/CDPK phosphocode of ATI-2, two working hypotheses can be considered: either the MAPK and CDPK phosphocodes are independent, meaning that phosphorylations of S47 and S140 occur in different biological contexts, or they can act concomitantly, leading to possibly opposite, additive or synergistic effects.

phosphocode in the response to biotic stress.

Based on these preliminary results, the STRESS team aims at addressing different questions: How is the MAPK/CDPK phosphocode encoded in ATI-2? What is the biological translation of the ATI-2 phosphocode, especially in response to stress? And how is the phosphocode molecularly decoded to result in appropriate biological responses? (Figure 1) To help answering these questions, an approach is to compare wild type plants, a loss-of-function ati-2 line, and plants expressing phosphodeficient or phosphomimicking versions of ATI-2 in order to determine how these modifications are likely to revert the stress phenotypes observed in the mutant line. But prior to this, clear phenotypes must be established for the mutant line.

Therefore the goal of my internship was to provide initial results by characterizing the response of simple ati-2 mutants to infection by WT Pst DC3000 in order to determine whether this gene is involved in the immune response. I also performed flg22 assays, comparing the growth inhibition of plants in presence of flg22.

Material and methods

Plant and bacterial material

All plants from this study are in the Columbia background. The three alleles of the ati-2 mutant: ati-2-1 (SALK_110571, [START_REF] Hou | Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling[END_REF], ati-2-2 (GABI_425D10), and ati-2-3 (WiscDsLoxHs092_09H) were ordered from the NASC. Transgenic pATI-2::ATI-2-TAP/ati-2-1 and pATI-2::ATI-2-TAP/ati-2-2 plants were generated previously by the STRESS team. Briefly the ATI-2 endogenic locus including a 500 nt fragment upstream of the ATG and a 400 nt fragment downstream of the stop codon was cloned in pGREEN 0229. By PCR, a restriction site was created to allow the insertion of a TAP tag just before the stop codon. Plants from the ati-2-1 and ati-2-2 backgrounds were transformed using the floral dip method. For the pathoassays, plants were grown in soil in growth rooms at 20 °C day, 18 °C night and 65 % relative humidity in short day conditions (8 h day, 16 h night).

For in vitro assays, seeds were sterilized 10 min in a sterilization solution (50 % ethanol, 37.5 % water, 12.5 % bleach 9.6 %) and washed three times with ethanol 100 %. They were sown on ½ Murashige and Skoog (MS) medium (0.22 % (w/v) MS (Sigma, M6899) basal salts, 0.05 % MES, and 0.5 % agar, pH 5.6) and stratified in darkness at 4 °C for 3 days. They were then transferred in growth chamber at 20 °C day, 18 °C night under long day condition (16 h day, 8 h night) and at 50 % relative humidity.

The wild-type strain of Pseudomonas syringae pv. tomato (Pst) DC3000 was grown on Luria-Bertani (LB) medium supplemented with rifampicin 50µg/mL, at 30°C.

Plant Infection

Bacteria were incubated overnight at 30 °C in liquid LB under agitation and then washed with MgCl 2 10 mM and diluted to obtain an OD 600 = 0.2. Prior to the infection, Silwet 77 was added to the inoculum at a final concentration of 0.04 %. 6 weeks old plants were spray-inoculated with Pst DC3000, watered and kept covered for one day to favor stomata opening.

Samples were harvested at 3 hpi and 72 hpi and were typically composed of two leaves coming from single independent plants. The leaves were surface sterilized with three successive washes: in ethanol 70 %, and in sterile water. Samples were frozen in liquid nitrogen and kept at -80 °C.

Genomic DNA extraction

Plant material was ground in Tissue Lyser (26 Hz, 2x30 sec) and powder was resuspended in extraction buffer (Tris HCl pH = 7.5 200 mM, NaCl 250 mM, EDTA 25 mM, SDS 0.5 %),. After centrifugation (13000 rpm, 1 min), DNA was precipitated by adding an equal volume of isopropanol. After a second centrifugation (13000 rpm, 5 min), the pellet was washed with ethanol 75 %, let to dry and DNA was diluted in sterile water.

Bacterial quantification by qPCR

DNA concentration for each sample was quantified by NanoDrop spectrometer and quantitative PCRs (qPCRs) were carried out using 10 ng of DNA with LightCycler® 480 SYBR Green I Master mix (Roche) and a LightCycler® 480 System (96 wells), following the manufacturer's standard instructions. Multiplication of the bacteria was measured using the 2 -ΔΔCt method where Ct stands for cycle threshold and corresponds to the number of cycles from which the amplification is above the noise signal. The 2 -ΔΔCt method compares the ΔCt = Ct(At)-Ct(Pst) of each sample to the mean of ΔCt for a genotype chosen as reference. [START_REF] Ross | A DNA-based real-time PCR assay for robust growth quantification of the bacterial pathogen Pseudomonas syringae on Arabidopsis thaliana[END_REF]Somssich, 2016, Livak and[START_REF] Livak | Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method[END_REF]. The reference used is indicated in the text when required. Ct(At) and Ct(Pst) correspond to the Ct of fragments specific to A. thaliana and to Pst DC3000 respectively. Primers used for the amplification of those fragments are in Table S1.

Genotyping

Plants were genotyped using the primers listed in Table S1 and the Terra PCR Direct Polymerase®, following the manufacturer's instructions and PCR visualization was done with 1 % agarose gel electrophoresis.

Flagellin post-germination growth assays 7-days-old seedlings grown in vitro were transferred into ½ MS liquid medium supplemented or not with 500 mM of flg22 and kept in in vitro growth chamber. Fresh weights were measured at the moment of the transfer and seven days later.

Results

ati-2 mutant shows no phenotype in response to WT Pst DC3000

To test whether ATI-2 plays a role in the immune response, three independent pathoassays were performed with the Pst DC3000 strain on Col-0, ati-2-1 and pATI-2::ATI-2-TAP/ati-2-1 plants. Results for the three independent replicates are shown in Figure S1. We noticed that in the two first replicates, the levels of bacteria in plants at 72 hpi did not really increase compared to those at 3 hpi, while in the last replicate we could observe a strong multiplication of the pathogen up to 80 times the level at 3 hpi. This difference in pathogen virulence might be explained by the fact that for the two first replicates we used a bacterial strain conserved at 4 °C whereas for the last replicate we plated a new strain from the glycerol stock.

We also noticed that globally in the three replicates, the three genotypes seemed to behave similarly, with no clear differences in the levels of multiplication of the pathogen. To highlight this point we integrated the data of the three replicates by normalizing them to the mean values obtained for Col-0 (Figure 2). Non parametric statistical tests confirmed that there were no significant differences between the three genotypes, suggesting that ATI-2 is not involved in the resistance against Pst DC3000. Since for the calculation of the 2 -ΔΔCt we used as a reference the mean values of the 3hpi data for each genotype, our results might miss differences between the genotypes in this early timepoint, which might consequently impact our interpretation. To exclude this possibility, we performed a new analysis using this time as a reference the mean value of Col-0 at 3 hpi. As shown in Figure 3, no difference between the three genotypes could be detected, confirming our interpretation that, based on our pathoassays, ATI-2 does not contribute to defense responses against Pst DC3000. ati-2 mutants show no growth phenotype in response to flg22

To test whether the loss-of-function mutant reacts differently to the presence of a PAMP, we conducted an flg22 post-germination growth assay on three different mutant lines and two complemented lines as well as Col-0. For each genotype and each of the three tested conditions (7 days post germination, after 7 days of mock treatment and after 7 days of flg22 treatment), between 7 and 12 plantlets were analyzed. As we observed growth differences between the genotypes at day 7 post germination as well as in the mock condition (Figure 5), we decided to measure the ratio of the weights in the flg22 condition versus the mean of the weights in the mock condition for each genotype as an indicator of the flg22 effect (Figure 6). Based on those ratios, we first observed that flg22 does indeed provoke a growth inhibition, albeit weak, since the ratio for the Col-0 line is of 0.90. Besides, it seems that the mutant lines have a retarded growth compared to Col-0 when grown in flg22-supplemented medium (0.71, 0.74, 0.63 for ati2-1-1, ati-2-2, ati-2-3 respectively versus 0.90 for Col-0), while the complemented lines tend to behave more like Col-0. However non parametric statistical tests concluded that there was no significant difference between the six genotypes, which suggests that ATI-2 is not involved in the growth response induced by the presence of flagellin.

8 Overall our results indicate that ATI-2 is not involved in response to biotic stress.

However these results must be nuanced for three main reasons.

First our experiments suffered from inconsistencies in the biological replicates.

Indeed, for the pathoassays, the two first replicates were performed with a Pst DC3000 strain drastically less virulent than in the third replicate. Even if we did not take this in account in our interpretation, it is still possible that the discrepancy might affect the defense responses of the various genotypes, and it would be more rigorous to perform new replicates with a same Pst DC3000 strain. In regard to the flg22 assay, we actually carried out only one biological replicate, and therefore more data are required to be fully conclusive.

Second the information regarding the expression levels of ATI-2 in the different lines we used are not completely clear. For instance ati-2-1 is a published line for which a higher sensitivity to salt and ABA was reported [START_REF] Hou | Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling[END_REF]), yet preliminary results in the STRESS group suggest that it still expresses ATI-2 and would not be consequently a real lossof-function line. In the same order of idea, the complemented line in the ati-2-1 background expresses quite more ATI-2 proteins than the one in the ati-2-2 background. Moreover, there are actually two isoforms of ATI-2, and it is possible that some mutant lines are KO for only one of the two. Therefore a comprehensive view of the ATI-2 expression levels in the different alleles we used is still needed to consolidate our analyses.

At last our results focused on pathoassays with Pst DC3000 and on growth inhibition assay in response to flg22. But it is not certain that these two kinds of experiments are enough to conclude that ATI-2 is not involved in biotic stress responses. Indeed we can imagine that ATI-2 is involved in the resistance against other pathogens than Pst. Moreover the growth inhibition is only a part of the responses triggered by flg22, and it is still possible that ATI-2 contributes to some of these other responses.

Conclusion and perspectives

Despite the limitations discussed above, our results indicate that ATI-2 is likely not involved in defense resistance against Pst and in the growth inhibition mediated by flg22. However to consolidate this interpretation, it will be important in the near future to evaluate the expression levels of ATI-2 in the different lines we used. In this sense, plants from the flg22 growth assay have been collected and will be processed through RT-qPCR. Likewise, two more biological replicates for this experiment have been set out.

In order to further investigate the MAPK/CDPK phosphocodes of ATI-2, two main perspectives can be followed. In the first one we can continue to look for a phenotype of the ati-2 lines in response to biotic stress, either by using other pathogens than PstDC3000, for instance fungi like Botrytis cinerea, or by analyzing other immune responses triggered by flg22, like the ROS bursts or the transcriptional reprogramming. It could also be interesting to redo the pathoassays with all the available mutant alleles. In the second perspective, we can focus on other phenotypes associated with the ati-2 loss of function. For instance we started to repeat experiments in response to salt to confirm the hypersensitivity of ati-2 compared to Col-0.

Interestingly a recent phosphoproteomic study reported that phosphorylation of ATI-2 on S47 transiently increased during the first steps of germination, suggesting a role for the MAPK phosphocode in this developmental process (Baudoin et al., 2022).

Given the results showed here, the second option is currently favored. Either way obtaining a clear phenotype for an ati-2 loss of function line that is reverted by a complemented line will be a prerequisite to test the relevance of the MAPK/CDPK-dependent phosphocode of ATI-2.

  mutant shows no phenotype in response to WT Pst DC3000The lack of resistance phenotype is not due to a default in the genotype of the plants ati-2 mutants show no growth phenotype in response to

  internship at the Paris Saclay Institute of Plant Sciences (IPS2), in the Stress Signaling (STRESS) team of the physiology and signaling department (DPHYS), under the supervision of Julien Lang. The DPHYS department aims at understanding the fundamental mechanisms implicated in the adaptation of plants to their environment in the context of global warming and increased food demand. The STRESS group tries to develop original approaches to better understand the signaling mechanisms of Calcium-Dependent Protein Kinase (CDPK or CPK) and Mitogen-Activated Protein Kinase (MAPK or MPK) modules notably in the context of stress responses. Their research revolves around several axis: a) the role of CDPKs in response to abiotic stress and especially in the case of salt stress, b) the impact of global warming on kinase signaling during stress perception, c) the role of an atypical MAPK module in integrating various environmental stresses, and d) the implication of MAPKs in the regulation of immune responses. To investigate these aspects the team is especially interested in the identification and characterization of new substrates targeted by the kinase signaling modules in stress conditions.

Figure 1 .

 1 Figure 1. Model for the MAPK/CDPK phosphocode of ATI-2. The notion of phosphocode refers to how the cellular functions of a protein are modulated by its pattern of phosphorylation.

Figure 2 .

 2 Figure 2. Pathoassays on ati-2-1 mutants, wild type and complemented lines. Plants were sprayed with Pst DC3000 and bacterial load was measured at 3 hpi and 72 hpi using the 2 -ΔΔCt method with the mean value at 3 hpi of each genotype as a reference. The dotplot and histogram showing mean values and standard deviations for the different genotypes were generated from integrating data of three independent replicates normalized to the mean of Col-0 values. Different letters above the bars indicate statistically significant differences (Kruskal-Wallis test followed by a non-parametric Tukey post-hoc test, p<0.05, 15<n<28).

Figure 3 .

 3 Figure 3. Difference in infection at 3 hpi between the genotypes. Bacterial load was measured at 3hpi using the 2 -ΔΔCt method with the mean value at 3 hpi of Col-0 as a reference. The dotplot and histogram showing mean values and standard deviations for different genotypes were generated from integrating data of the three independent replicates normalized to the mean value of Col-0. Letters above the bars indicate no statistically significant differences (Kruskal-Wallis test, p<0.05, 15<n<16).

Figure 4 .

 4 Figure 4. Genotyping of the Col-0, mutant and complemented lines. (A, B) Representation of the wild-type ATI-2 locus (A) and the pATI2::ATI2-TAP/ati-2-1 construct (B) with the localization of primers used for genotyping. (C, D, E) PCR results on a selection of samples using the RP4803+LP4802 pair (C), the RP4803+LBb1.3 pair (D), and the P4804+P160 pair of primers (E).A

Figure 5 .

 5 Figure 5. Fresh weights of plantlets from the six studied genotypes. (A) Before transfer in flg22 at day 7 post-germination and (B) after 7 days of mock treatment at day 14 postgermination (Kruskal-Wallis test followed by a non-parametric Tukey post-hoc test, p<0.01, 6<n<12).

Figure 6 .

 6 Figure 6. Flagellin post-germination growth assay on ati-2-1, ati-2-2, ati-2-3 mutants, wild type and complemented lines. The results are presented as the ratio between the fresh weight of plants grown in the flg22 condition versus the mean of weight in the mock condition for each genotype (Kruskal-Wallis test, p<0.05, 7<n<12).

This work has been partially supported by the EUR SPS-GSR (ANR-17-EUR-0007).


Les plantes sont capables de reconnaitre des motifs moléculaires conservés chez les pathogènes pour induire ces réponses immunitaires. Ces motifs sont appelés Pathogen-associated molecular pattern (PAMP), Ils sont reconnus par les récepteurs Pattern recognition receptor (PRR) pour induire une réponse de type pattern-triggered immunity (PTI). L'établissement de cette réponse implique l'activation des voies de signalisation immunitaires et s'accompagne d'une multitude de réaction tel que l'accumulation d'espèces réactives d'oxygène et l'accumulation de calcium. Les modules Mitogen-activated protein kinases (MAPK) sont des acteurs clé de cette voie. Dans le cadre des signalisation immunitaires deux modules MAPK « classiques » sont particulièrement étudies : MAPKKK3/5-MKK4/MKK5-MPK3/MPK6 et MEKK1-MKK1/MKK2-MPK4. Ces modules sont activés rapidement (15 min après l'infection) en aval de la perception du PAMP, flg22, par son récepteur FLAGELLIN SENSING2(FLS2). Dans le cade de ce stage, je me suis intéresse à caractérisation d'un module MAPK atypique dans la voie de signalisation immunitaire déclenché par le PAMP et plus particulièrement en réponse à la flg22. Ce module été caractérisé précédemment dans des contextes de blessure et de sècheresse, il est dit atypique car il présente des cinétiques d'activation tardives par rapport aux modules « classiques » et dont l'activation est dépendante de la néosynthèse de MAP3K du clade 3. Au cours de ce stage, Par des approches de biochimie et de biologie moléculaire, j'ai eu l'occasion de faire des immunoprécipitations, des tests d'activité kinase, des western blot ainsi que des RTqPCR pour répondre aux différentes questions. A l'issue de ce projet, j'ai pu caractériser pour la première fois l'activation module MKK3-MPK7 en réponse à la flg22 ainsi que l'indépendance du module MKK4/MKK5-MPK3/MPK6 pour cette activation, j'ai également identifié deux MAP3Ks candidats pour médier l'activation du module en réponse à la flg22. Par ailleurs, j'ai montré également la convergence des deux modules pour l'expression du gène marqueur de la voie de l'acide salicylique, PR1.

Remerciement

Je tiens à remercier toutes les personnes qui m'ont apporté de l'aide au cours de mon stage.

Premièrement, j'adresse mes sincères remercîments à mes encadrants Julien Lang et Jean Colcombet pour le temps qu'ils m'ont consacré, par un suivi quotidien et des réunions hebdomadaires, pour m'encadrer au cours de ce stage. Je les remercie de m'avoir accompagné le long de ce stage pour la prise en mains des expériences, la compréhension du sujet ainsi que la rédaction de ce manuscrit. J'aimerai les remercier également pour les explications et les conseils qu'ils m'ont fourni avec bienveillance et bonne humeur.

Je tiens à remercier également les membres de mon équipe Sarah Regnard et Jean Bigerad pour leur aide et leur précieux conseils qu'ils ont fourni de plein coeur.

Je voudrais aussi remercier tous les membres de l'équipe pour les moments de détente et de plaisanterie que nous avons partagé, et notamment, au cours des pauses café et au bureau avec Sarra, Sarah et Axelle.

Je suis très heureux d'avoir fait partie de cette équipe qui m'as tant apporté au niveau des connaissances qu'au niveau moral.

Table des matières

IFigure 1 :

 1 Figure 1 : Modes de perception des pathogènes et les réponses immunitaires qu'ils induisent.

Figure 3 :

 3 Figure 3 : Les modules MAPK sont impliqués dans la transduction d'un signal extracellulaire pour l'établissement d'une réponse cellulaire adaptée au signal perçu.

Figure 2 :

 2 Figure 2 : Modèl en zigzag représentant le dialogue moléculaire qui s'opère entre la plante et le pathogène (Jones and Dangl, 2006). PAMP: Pathogen-Associated Molecular Pattern; Avr-R : reconnaissance de l'effecteur du pathogène par le récepteur de la plante; HR: Réponse Hypersensible

Figure 4 :Figure 5 :

 45 Figure 4 : Implication des modules « classiques », à activation rapide, dans la voie de signalisation déclenché par la flg22. SA : Acide Salicylique; SAR : Systemic Acquired Resistance; CDPK : Calcium-dependent protein kinase; TF : Transcription factors, PR1 : pathogenesis-related protein 1

  Afin d'extraire les protéines, le broyat est suspendu dans un tampon d'extraction non dénaturant (10% Laccus Buffer 10X, 20 mM NaF, 20 mM β-glycerophosphate, 5 mM DTT and EDTA-free protease inhibitors cocktail (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail -Roche). Ce tampon est constitué du tampon Laccus-Ortiz-Masia 10X (annexe 3), des inhibiteurs de protéases, des inhibiteurs de phosphatases (NaF et β-glycerophosphate, DTT) pour maintenir l'état natif des protéines kinases. Les extraits sont ensuite centrifugés à 14000 rpm pour faire précipiter les débris cellulaires, puis on récupère le surnagent.La quantité de protéines est mesurée à partir d'une dilution au 20 ième du surnagent par une coloration au Bradford. Dans les puits d'une plaque à 96 puits, on dépose 10µl d'échantillons dilués ainsi que série de la gamme de concentration de BSA puis on rajoute le même 250µl de Bradford (Coomassie (Bradford) Protein Assay reagent (Sigma aldrich N°23200)). Ensuite on relève les DO grace au spectromètre Tecan à 595 nm et suivant le Protocol standard du constructeur. Apres quantification, on calcule les concentrations en protéines dans les échantillons grâce à la gamme d'étalonnage de BSA. Ces concentrations vont servir à la normalisation des échantillons destinés à l'immunoprécipitation et aux western blots. Pour l'immunoprécipation on utilise entre 100 et 150µg de protéines en fonction de la disponibilité en protéinés dans les extraits dans un même volume final. Alors que pour les westernblot on prépare classiquement des échantillons à 1 µg/µl mais si la concentration de certains extraits est plus faible on se met à la concentration la plus faible. On rajoute à un alicot un volume de leamli 6X (30% glycerol, 10% SDS, 0.6M DTT, 0.012% Bromophenol blue, 20% 1.75M Tris-HCl (pH 6.8)) de tel sorte à avoir une concentration finale de1X leamli. 4. Immunoprécipitation, test d'activité kinase, western blot a. Immunoprécipitation L'immunoprécipitation de MPK7 consiste à faire précipiter spécifiquement la protéine MPK7 à l'aide d'un système de billes de sépharose couplé à l'anticorps spécifique à MPK7. A partir des échantillons normalisés, on rajoute un volume équivalent d'un mélange de billes de séphoase et de l'acticorps contre MPK7 puis on incube les échantillons sur une roue à 4°C toute la soirée. Le lendemain, les échantillons sont centrifugés a 10000 rpm à 4°C pour faire précipiter les billes couplé à l'anticorps dirigé contre l'épitope de MPK7 (LYYHPEAEISNA). Avant de procéder au test d'activité kinase, les billes sont lavées trois fois. Les deux premiers lavages sont réalisés avec du tampon LOB 1X alors que le dernier est réalisé par un tampon kinase ((30 mM Tris-HCl (pH 7.5), 1 mM EGTA, 10 mM MgCl2, 1 mM DTT and 20 mM βglycerophosphate)), un lavage consiste en premier à centrifuger les échantillons, pour la précipitation des billes, retirer le surnagent en gardant les billes au fond du tube puis rajouter le tampon. Le liquide résiduel est ôté avec une pipette. b. Test d'activité kinase Les tests d'activités kinases ont été réalisés par mon superviseur Julien LANG car ils nécessitent une habilitation pour manipuler de la radioactivité. Apres le séchage les billes sont reprises dans du tampon kinase((30 mM Tris-HCl (pH 7.5), 1 mM EGTA, 10 mM MgCl2, 1 mM DTT and 20 mM β-glycerophosphate)) contenant le substrat de phosphorylation Myelin basic protein (MBP), de l'ATP froid puis de l'ATP radioactif ( 33 P). Au bout de 30 minutes d'incubation la réaction est arrêtée par ajout de leamli X6 et chauffage 5 minutes à 95 °C puis les échantillons sont déposés sur gel SDS PAGE (en gel de polyacrylamide contenant du dodécysulfate de sodium) à 15% d'acrylamide à 30mA par gel . Ensuite, les gels sont séchés puis mis dans une cacette d'autoragraphie pendant 24h. Le jour suivant, le signal de radioactivité est révélé par autoragraphie à l'aide d'un scanner Typhoon. c. Western blots Les échantillons sont déposé et mis à migrer par électrophorèse SDS-PAGE 10% avec ampérage constant (30 mA par gel pendant 1h). Apres migration, les protéines sont transférées sur des membranes PVDF préalablement activées à l'éthanol 100% suivant les recommandation du fabricant (BioRad). Les membranes sont ensuite bloquées, pendant 1h sous faible agitation, dans une solution de Tris-buffered saline with 0.1% Tween® 20 detergent (TBS-T) + 5% BSA pour l'anticorps α-pTpY, ou de TBS-T + 5% lait pour l'anticorps α-MPK7. Apres le blocage, les anticorps primaires dilué dans du TBS-T+5% BSA pour l'anticorps α-pTpY ou dans TBS-T pour MPK7 sont ajouté aux membranes puis laissé incuber toute la nuit à 4°C sous faible agitation. Les solutions de blockage et de dilution des anticorps primaires et secondaire ainsi que les dilutions utilisées sont résumées dans (annexe 4). Le lendemain les membranes sont lavées 3 fois 10 min au TBS-T puis incubées pendant 1h avec l'anticorps secondaire couplé à l'enzyme peroxydase de raifort (HRP) dilué dans une solution de TBS-T+5% BSA pour l'anticorps α-pTpY ou dans TBS-T pour MPK7. Apres 3 derniers lavages au TBS-T, l'activité HRP de la membrane est révélée grâce aux reactif de chemiluminescence (Clarity Western ECL Substrate -Biorad). Le signal de chemiluminescence est ensuite acquis à l'aide du système d'imagerie ChemiDoc (Biorad). Après revelation, les membranes sont colorées par incubation avec du bleu de Coomassie pour vérifier la charge totale des protéines. 5. RTqPCR L'extraction d'ARN est réalisée à partir d'un broyat des différents échantillons, à l'aide du kit Nucleospin™ RNA Plus, en suivant les instructions du fabricant. J'ai ensuite quantifié à l'aide d'un spectrophotomètre Nanodrop, puis normalisé les ARN pour avoir une concentration de 100ng/µl destinée à la transcription inverse. La synthèse du premier brin d'ADNc a été réalisé grâce à la transcriptase inverse SuperScript™ II en suivant les instructions du fabricant (annexes 5). J'ai utilisé des amorces Oligo(dT) pour la transcription inverse de tous les ARNm. A la fin de la réaction les ADNc sont dilués avec 80 µl d'eau (sans DNAase) pour avoir une concentration finale de 10ng/µl. Les PCR quantitatives (qPCR) ont été réalisées avec un système LightCycler® 480 (96 puits), en utilisant le mélange prêt à l'emploi LightCycler® 480 SYBR Green I (Roche) et en suivant les instructions du fabricant. L'actin2 (AT3G18780) a été utilisée comme gène de normalisation. A l'issue de cette analyse, les Ct ont été mesurés pour chaque échantillon et chaque couple de primer. Le ct correspond au nombre de cycles nécessaires pour que le niveau de fluorescence atteigne le seuil de référence. Dans chaque échantillon et pour chaque couple d'amorces l'abondance est calculée grâce à l'équation 10 (ct-24.313)/-3.4 . L'expression relative pour chaque gène est ensuite obtenue par le rapport entre son abondance et celle de l'actine dans le même échantillon. J'ai réalisé ces qPCR sur trois réplicats biologiques. Les moyennes des niveaux d'expression entre les trois réplicats biologiques a été calculé pour les MAP3K13 à 21 ainsi que pour PR1 et ont été exploités pour les représentations graphiques des expressions relatives. III. Résultats 1. Caractérisation du module atypique en réponse la flg22 a. Vérification de l'activation des modules « classiques » dans nos conditions expérimentales Afin de vérifier le bon fonctionnement de notre système expérimental, j'ai réalisé des western blot pour détecter les formes phosphorylées sur la boucle d'activation de MPK3, MPK6 et MPK4 (fig. 5). Cette phosphorylation est connue pour provoquer l'activation des MAPKs et représente un bon indicateur, facilement détectable, de l'activation des modules. Par ailleurs, MPK3/4 et 6 n'ont pas le même poids moléculaire et migrent à différents niveaux, les trois bandes détectées en partant du haut correspondent à MPK6, MPK3 et MPK4. J'ai testé l'activation des modules classiques pour leur activation en réponse à la qui est bien connue (Bigeard, Colcombet, and Hirt 2015). Mes western blot confirment une activation des modules classiques en réponse à la flg22. En effet, à 15 min on détecte une forte phosphorylation de MPK3, MPK4, MPK6 chez Col-0 traité à la flg22 par rapport au temps 0 qui diminue fortement dès 1h. Le traitement à la seringue seul active également les MAPKs, est observée chez le Col-0 mock à 15 min et 1h pour MPK3 et MPK6 mais sans activation de MPK4 est spécifique à la flgg22. L'activation à 15 min n'est pas affectée chez le mutant mkk3 par rapport au Col-0 alors que pour les mutants mkk4mkk5 et mkk4mkk5mkk3, on détecte une réduction de l'activation du module par rapport au Col-0. Ces résultats montrent que phosphorylation MPK6, MPK3 et MPK4 est indépendante de MKK3. Il est connu que le mutant mkk4mkk5 est un mutant partiel du module ce qui peut expliquer la forte activation résiduelle dans le background.

Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :

 678910 Figure 6: Activation des modules classiques en réponse à la flg22 dans nos conditions expérimentales. L'activation de MPK3, MPK6 et MPK4 à été testé par Western blot avec un anticorps anti-pTpY qui reconnait les formes phosphorylés de ces MAPK, à partir de feuilles prétraitées à la flg22 dans le fond génétique sauvage, Col0, et les fonds mutants mkk3, mkk4mkk5 et mkk3mkk4mkk5 au temps de cinétiques indiqués (panneau du haut) Le contrôle de charge à été réalisé par une coloration au bleu de Coomassie (panneau du bas) mkk45: mkk4mkk5, mkk345: mkk3mkk4mkk5

  Associated Molecular Pattern-Triggered (DAMP) déclenche la réponse PTI pour fournir un niveau basal de résistance. Dans la deuxième phase, cette résistance peut être contournée par des pathogènes qui produisent puis injectent dans le cytosol des protéines appelées effecteurs qui vont perturber l'établissement de la réponse PTI, cette réponse est appelée Effector-Triggered Susceptibility (ETS). Au cours de la troisième phase, certaines plantes on acquit un système de défense basé sur la reconnaissance de certains effecteurs par des récepteurs de type NLR. Cette perception induit la réponse ETI, qui fournit un état de résistance plus fort que celui de la PTI et induit une réponse dite "hypersensible" (HR) se traduisant par une mort cellulaire programmée des cellules au site d'infection. Dans la quatrième phase, les pathogènes qui ont perdu ou modifié l'effecteur déjà reconnu par la plante vont induire, via la suppression de ETI,

	l'interaction va dépendre en fin de compte de la coévolution entre la plante et le pathogène.
	2. Les modules MAPK sont des acteurs clé des signalisations immunitaires
	a. Qu'est-ce qu'un module de signalisation MAPK chez les plantes ?
	Les Mitogen-Activated Protein kinases (MAPK) sont des proteines kinases, c'est-à-dire,
	des enzymes capables de catalyser la phosphorylation des protéines sur des groupements OH
	des acides aminés: serine, thréonine et tyrosine. En effet, cette modification post-
	traductionnelle est la plus abondante chez les eucaryotes, elle permet de réguler de la fonction
	des protéines par la modification de leurs propriétés, telles que l'activité enzymatique, la
	localisation subcellulaire ou la stabilité (Bigeard et al., 2015).
	Un module de signalisation MAPK est composé de trois protéines kinases : une
	MAPKKKinase (MAP3K), MAPKKinase (MAP2K), MAPK qui s'activent les unes les autres,
	de manière séquentielle, par une cascade de phosphorylations (fig. 3). Ces cascades
	fonctionnent généralement en aval des récepteurs et sont activées suite à la perception d'un
	signal par ces récepteurs. Une fois la dernière kinase de la chaine active, elle phosphoryle ses
	substrats pour induire une réponse cellulaire adaptée au signal perçu (Xu and Zhang, 2015).
	Les modules MAPK sont des acteurs ubiquitaires de la signalisation chez les eucaryotes.
	A cause de leur implication dans les différents processus biologiques, ils sont indispensables à
	la survie de ces organismes. Chez les plantes, ils sont impliqués dans la croissance, le

à leur tour le retour à état de sensibilité ETS. Dans la cinquième phase, grace à la selection certaines plantes vont acquérir de nouveau allèles de gènes de résistance capable de reconnaitre un des effecteurs nouvellement acquis pour induire de nouveau une ETI. La finalité de

  ', 15 min, 1h, 2h, 8h (annexe 2). A chaque point de cinétique, les feuilles infiltrées sont rapidement récoltées, mises dans des tubes puis plongé dans l'azote liquide. Pour chaque condition, on collecte dans deux tubes différents, l'un destiné à l'extraction de protéine et

	2. Culture & Traitement
	a. In vitro
	Avant le semis, les graines sont stérilisées dans une solution de javel à 9,6 % pendant 15 l'autre à l'extraction d'ARN.
	min sous agitation, puis lavées 3 fois 5 min dans l'éthanol 70%, ensuite elles sont séchées sur
	du papier wathman stérile. Une quinzaine de graines sont semées à la volée sur du milieu 3. Extraction des protéines & normalisation
	MS/2 (1% sucrose, annexe 1) dans des boites de pétri 60x15mm. Afin de permettre une
	germination homogène, les graines sont mises 3 jours à l'obscurité à 4°C pour lever la L'extraction des protéines, l'immunoprécipitation de MPK7, les lavages des billes ainsi que le
	dormance. Ensuite, elles sont mises à pousser une semaine dans une chambre de culture en test d'activité kinase sont réalisés en deux jours. L'extraction des protéines et
	condition de jours longs (16 heures de lumière/8h obscurité) à 20°C le jour et à 18 °C la nuit l'immunoprécipitation sont réalisés le premier jour puis la suite de l'immunoprécipitation et le
	et à une hygrométrie de 50% le jour et la nuit. J'ai travaillé au départ avec des plantules test d'activité kinase sont réalisés le deuxième.
	b. Plantes en pot « in vivo »
	Quatre à cinq graines sont semées dans des pots sur du terreaux puis mises à pousser dans
	une chambre de culture en condition de jours court (8 heures de lumière) à 20°C le jour et
	18°C la nuit et une HR à 50 pendant 6 semaines. Dans les premiers jours (4 à 5j) suivant le
	semis, on garde les deux plantes les plus espacées dans le pot et on élimine les autres. Les

). Le double mutant mkk4mkk5 a été obtenu par croisement des simples mutants généré par mutagène EMS et identification par TILLING

(Su et al., 2017)

.

Le triple mutant a été généré au sein de l'équipe par croissement des mutants mkk3 et mkk4 mkk5 préalablement rétrocroisés. cultivées in vitro, mais avec ce système j'ai eu beaucoup de contaminations que je n'est pas pu supprimées malgré plusieurs essais de protocole de stérilisation. Par conséquent, j'ai dû passer à un système de culture avec des plantes en pot qui a permis d'éviter ces problèmes.

Pour réaliser le traitement in vitro, il faut le préparer la veille du traitement. On sort les boites de pétri de la chambre de culture, on les dispose sur paillasse puis on rajoute un volume de milieu MS/2 liquide de manière à ce que toutes les plantules soient immergés. Ce volume doit être le même pour toutes les boites. Le lendemain on applique les traitements ABA, flg22 et mock. Les concentrations finales utilisées pour ABA et flg22 sont respectivement 10µM et 1µM. J'ai réalisé deux réplicas in vitro pour lesquels j'ai testé 5 points de cinétiques de traitement flg22 : 0', 15 min, 1h, 2h, 8h ou 24h. Le point 8h pour le premier réplicas et 24h pour le deuxième. A chaque point de cinétique, les plantes sont rapidement collectées à la pince, mises dans du papier aluminium puis plongé dans l'azote liquide. traitements sont réalisés par infiltration des feuilles à la seringue, on infiltre 2 feuilles par plante. La concentration utilisée pour le traitement flg22 est la même que celle in vitro. J'ai réalisé 3 réplicats biologiques et pour lesquels j'ai testé 5 points de cinétiques de traitement flg22 : 0Avant d'entamer l'extraction des protéines, le matériel végétal est broyé. Le broyage est réalisé différemment pour les plantes cultivées in vitro et celles in vivo. Pour les échantillons in vitro j'ai broyé au mortier alors que les échantillons in vivo ont été broyés congelés grâce aux broyeurs à billes TissueLyser (deux fois 30 secondes à 26 Hrz).