Analysis of Hyaloperonospora arabidopsidis-induced immunity in Arabidopsis thaliana plants deploying a non-toxic NEP1-like protein.

Molecular and Cellular Life Sciences

By: Pier Paolo Posata

Supervision: Guido van den Ackerveken

Daily supervision: Tom Raaymakers

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Laymen`s summary

Hyaloperonospora arabidopsidis is a model pathogen causing downy mildew disease in Arabidopsis thaliana plants. This model pathosystem is highly relevant in agriculture where downy mildew disease has a great economic impact on large-scale production of several crops, causing significant economic losses. Data revealed that H. arabidopsidis secretes over 800 proteins and many of these are believed to be involved in infection process. A group of these secreted proteins are Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP) whose function is still unknown. This class of proteins has been described for other oomycete species as a highly cytolytic group of proteins. However, the NLPs from H. arabidopsidis (HaNLPs) are non-cytolytic, and act like MAMPs, stimulating MTI immune response. This research sets itself the goal to find H. arabidopsidis mutants which have mutations in the HaNLPs receptor (RLP23) and co-receptor (SOBIR1) genes in order to better understand MTI downstream responses in A. thaliana. The results show that two already known MTI responses take two different downstream pathways at a certain point after the HaNLP detection. Furthermore, the generation of a copious number of mutants constitutes the starting point for further tests in order to unravel NLP functions in H. arabidopsidis infection process.

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Table of contents

-Introduction …………………………………………………………………………………………………………………….……………… 4

Oomycete Hyaloperonospora arabidopsidis and downy mildew disease in Arabidopsis thaliana ….. 4

The plants immune system and their strategies against pathogens ……………..…………………….….……… 5

MTI receptors ………………………………………………………………………………………….………………………..…..……… 6

MTI downstream responses ……………………………………………………………………………………………….….……… 6

Nep1 like proteins (NLPs) ……………………………………………………………………….......................................... 6

NLPs like MAMPs ………………………………………………………..………………………..…………………………….….…….. 7

-Research aim and approaches …………………………………………………………………………………………….…….……. 8

-Results ………………………………………………………………………………………………………………………………….….…….. 9

EMS mutagenesis yields normal phenotype mutants ………………………………………………………….…..……. 9

Ethylene production as evidence for plant immunity activation ………………..………………………………… 10

RLP23 and SOBIR1 sequencing …………………………………………………………………………………………..….…….. 12

H. arabidopsidis infection screen ……………………………………………………………………………….……….………. 13

-Conclusions and discussion …………………………………………………………………………………………………..………. 15

-Materials and methods ……………………………………………………………………………………………………….…..……. 18

EMS dni screen …………………………………………………………………………………………………………….……………… 18

Nucleic acid extraction and PCR analysis …………………………………………………………………………….….….… 18

Ethylene measurement ……………………………………………………………………………………………………….……… 18

Sequencing analysis …………………………………………………………………………………………………….……….…..…. 18

Hya infection experiment ………………………………………………………………………………………………………….… 18

-References ……………………………………………………………………………………………………………………….….……….. 20

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Introduction

Oomycete Hyaloperonospora arabidopsidis and downy mildew disease in Arabidopsis thaliana.

The downy mildew Hyaloperonospora arabidopsidis is an obligate biotrophic parasite belonging to the oomycetes (Slusarenko & Schlaich 2003). Oomycetes are fungus-like heterotrophic microorganisms (Beakes et al., 2009). Nevertheless they have a different phylogenetic lineage than fungi. Oomycetes belong to the kingdom Stramenopila which includes photosynthetic group like brown algae (Stassen et al., 2011). The biology of these two filamentous microorganisms is not the same. Oomycetes have diploid nuclei, their cell wall is composed by cellulose and they do not form septa like fungi do (Latijnhouwers et al., 2003).

H. arabidopsidis causes downy mildew disease in Arabidopsis thaliana. This plant species has become a model system for genetic and molecular biological studies during the early 90s. Therefore, the interaction between the oomycete H. arabidopsidis and the plant A. thaliana is studied in order to uncover the mechanisms causing downy mildew disease in plants. As a matter of fact, this model pathosystem is highly relevant in agriculture where downy mildew disease has a great economic impact on large-scale production of several crops, causing significant economic losses (Slusarenko & Schlaich, 2003; Holub, 2008).

H. arabidopsidis produces conidiospores that land on the leaf surface and these form a specialized infection structure called the appressorium. This structure enters in the host plant by means of both physical pressure and hydrolytic enzymes. Later, it creates junctions between two epidermal cells and it gradually forms penetration hyphae that grow intercellularly (the apoplast) (Slusarenko & Schlaich 2003) (fig.1). Feeding structures called haustoria penetrate cell walls and they insert into plant cells. In addition, hyphae develop branches that emerge from stomata and they carry conidiospores out of the leaf (fig.2). A new cycle begins and that lasts approximately one week (Coates et al., 2010; Cabral et al., 2012).

Figure 1 | Intercellular hypha penetration. Figure 2 | Downey mildew branch with conidiophores. Hypha spread between walls of cells. After 1-2 weeks of growth, conidiophores grow out of the A haustorium is formed in every adjacent plant cell. stomata to be released.

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The plant immune system and the defense against pathogens.

Plants have developed several ways to defend themselves in order to prevent pathogen infection. Two main strategies can be identified. The first line of defense involves molecules detected from the pathogens into the extracellular spaces of the plant, like flagellin. These molecules are called pathogen/microbe-associated molecular patterns (PAMPs or MAMPs) and they are highly conserved among species. MAMPs are recognized by cell surface pattern recognition receptors (PRRs) and they elicit MAMP-triggered immunity (MTI) (Jones &Dangl, 2006). The second strategy involves plant intracellular receptors that recognize pathogen virulence molecules called effectors which are delivered into plant cellsto enhance microbial fitness (Boller& Felix, 2009). This recognition induces effector-triggered immunity (ETI) (Dodds et al., 2010). Effectors are secreted by different types of pathogens. Fungi and oomycete expel effectors mainly through haustoria (Stassen et al., 2011). One of the functions of these secreted proteins is to alter plant pathways, blocking MTI response in order to reduce plant resistance against pathogens. However, plants have developed specialized intracellular proteins to detect these effectors. Intracellular nucleotide binding (NB)-LRR receptors are able to recognize these effectors and induce the immune system (ETI)(fig.3). On the other hand, evolution allowed plant pathogens to acquired additional effector capable of suppress ETI; thus no defense will be induced by the plant (Jones & Dangl, 2006). Plants evolve new resistance specificities capable of overcoming the effector functions, and eventually trigger ETI again (Jones & Dangl, 2006). This research will be focused on the MTI and the MTI receptors.

Figure 3 |The principles of plant immunity. Simplified explanation of pathogens strategy to infect plants. Bacteria and oomycete prime either MTI response or ETI response (Doddset al. 2010). MAMPS and other apoplastic effectors are detected on plant cell surface by PRRs which trigger the immune system by means of a regulatory element, resulting in MTI response. Effectors are released by pathogen haustorium inside plant cell and they can block MTI response in order to get through pant immune system. However, plants have developed effector specific receptors inside the cell to trigger immune system again (ETI) and prevent pathogen infection.

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MTI receptors

The PRRs include a group of leucine-rich repeat (LRR) receptor-like kinases (RLKs) and a group of LRR receptor-like proteins (RLPs) located in the plasma membrane (Monaghan & Zipfel, 2012). The extracellular LRR element is a structural common element involved in ligand detection (Kobe &Kajava, 2001). The RLKs are the largest cell surface receptors group containing LRRs (Wang, 2008) and they are also composed of a membrane-spanning region, a juxtamembrane (JM) domain, and a serine/threonine kinase domain (Greeff et al., 2012). Over 600 RLKs have been identified in Arabidopsis (Shiu et al., 2004). The RLPs are the second largest group containing LRRs and they are similar to RLKs except for the cytoplasmic kinase domain which is simply lacking. This lack prevents RLPs from phosphorylation which is supposed to activate intracellular signaling (Wang, 2008). However, some putative endocytosis motifs have been found in some RLPs members (Joosten& de Wit, 1999; Kruijt et al., 2005). It has been studied that some RLPs genes have been implicated in disease resistance in tomato (Kawchuk et al., 2001) and other species (Kruijt et al., 2005). More recently, Arabidopsis genome analysis revealed that it contains 57 RLP genes and some Arabidopsis RLP T-DNA insertion lines showed altered susceptibility against pathogens (Wang, 2008). Consequently, the lack of MAMP perception leads to enhanced disease susceptibility, demonstrating the importance of MAMP perception for immunity against pathogens in vivo (Zipfel, 2008). Lately, it has been discovered that RLK protein Suppressor Of BIR1 (SOBIR1) might act as a co-receptor of RLP proteins. This protein interacts with most of the tested RLPs (Liebrand et al., 2013; Zhang et al., 2014) and it seems to be fundamental for the RLP-mediated immunity (Gust & Felix, 2014).

MTI downstream responses

Depending on the specific MAMP, the activation of MTI is related to different immune responses which are thought to give resistance against pathogens. These downstream responses include ethylene biosynthesis (Chang et al., 2013), callose deposition (Luna et al., 2011), production of reactive oxygen species and release of antimicrobial compounds (Tsuda & Katagiri, 2010). In addition, a hypersensitive response and a form of programmed cell death were also identified upon MAMP detection (Thomma et al., 2011). These downstream responses help us to identify MAMPs, like Nep1 like proteins (NLPs) are.

Nep1 like proteins (NLPs)

The genome of H. arabidopsidis has been sequenced and analyzed (Baxter et al., 2010). The data revealed that H. arabidopsidis secretes over 800 proteins and many of these are believed to be involved in infection process (Seidl et al., 2011). A group of these secreted proteins are Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP) (Baxter et al., 2010). The NLPs are present in fungi, stramenopiles, gram-positive and gram-negative bacteria (Qutob, 2006). They are also widely distributed in plant pathogens. Previous studies reported that NLP proteins from different kind of plant pathogens induce necrosis (Qutobet al., 2002; Mattinen et al. 2004). On the other hand, it has been reported that H.arabidopsidis NLPs (HaNLPs) do not induce cell death (Cabral et al., 2012).

Three NLP types have been uncovered. Type 1 and type 2 are characterized by N-terminal signal peptide, conserved heptapeptide motif “GHRHDWE” in the central region of the protein, the existence of either two or four conserved cysteine residues (Gijzen&Nurnberger, 2006) and the presence of a cation-binding pocket. Nevertheless, they differ mostly in an exposed region which

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contains a putative calcium-binding domain in type 2 but not in type 1. Type 3 NLPs have been recently discovered and they are more divergent from type 1 and type 2, although they also contain the cation-binding pocket (Oome & Van den Ackerveken, 2014). Type 1 NLPs is the only one present in oomycetes, while fungi and bacteria either have type 1, type 2 or both (Cabral et al., 2012).

Recent studies revealed that the crystal structure of a Pythium aphanidermatum NLP is similar to actinoporins, suggesting that cytotoxic NLPs induce necrosis through a permeabilization of the plasma membrane (Ottmann et al., 2009). On the other hand, the role of non-cytotoxic NLPs, like the HaNLPs, is still unknown. It was observed that non-cytotoxic NLPs proteins were expressed during the biotrophic stage, suggesting they might have a function in early infection (Kanneganti et al., 2006; Cabral et al., 2012). Furthermore, they were able to induce ethylene production and trigger the immune system in Arabidopsis thaliana (Oome et al., submitted).

HaNLPs like MAMPs

10 HaNLPs proteins have been identified (Cabral et al. 2012) and 10 dfferent HaNLPs-overexpression lines in A. thaliana showed that the expression of 7 NLPs causes severely plant reduced growth (Oome et al., submitted). HaNLP3 protein was the one which has been studied in most detail owing to its high similarity to necrosis-inducing NLPs (Cabral et al., 2012). As activation of the plant immunity affects plant growth and development (Zhu et al., 2013), the HaNLPs likely trigger the plant immune system. This hypothesis was also confirmed by gene expression analysis of an A. thaliana line in which theHaNLP3 gene sequence was cloned and expressed by means of an estradiol-inducible expressing construct (Zuo et al., 2000; Oome et al., submitted). The HaNLP3 expression in A. thaliana is capable of giving resistance to H. arabidopsidis; when the HaNLP3 estradiol-inducibile expression was activated 24 hours prior H. arabidopsidis inoculation, the plants were less infected (Oome et al., submitted). As mentioned previously, the non-cytotoxic NLPs are also expressed early during infection (Cabral et al., 2012) and HaNLP recognition appears to trigger a signaling cascade involving calcium and MAP kinases, like MAMPs do (Fellbrich et al., 2002). In addition, the HaNLP proteins induce ethylene production in dicotyledonous plants (Bailey, 1995; Veit et al., 2001), besides stimulating expression of 31 transcripts related to ethylene biosynthesis (Bae et al., 2006). A 24 amino acid region of HaNLP is also able to lead to ethylene production in A. thaliana (Oome et al., submitted). These HaNLP3 effects strongly suggest that HaNLP proteins act like MAMPs. For this reason, the HaNLPs are believed to be detected by the PRRs on the cell surface, stimulating the MTI immune response.

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Research aim and approaches

Research on the non-cytotoxic HaNLPs is an important starting point in order to understand pathogen infection processes in A. thaliana and, most importantly, to clarify the function of these H. arabidopsidis proteins. The recent discovery that HaNLPs act as MAMPs gives the opportunity to unveil mechanisms and proteins involved in the MTI responses. HaNLPs receptor has been lately recognized by testing ethylene production of RLP T-DNA A. thaliana insertion mutants with HaNLP peptide. Only one mutant did not show an ethylene response meaning that the immune system was not triggered. The mutant concerned RLP23 gene and it belongs to the family of RLPs. This gene is probably the HaNLPs receptor. Mutations in RLP23/SOBIR1 genes may affect HaNLPs detection.

The goal of this research consists in finding A. thaliana mutants which have mutations in RLP23/SOBIR1 genes to better understand both MTI downstream responses and HaNLPs function in H. arabidopsidis infection process.

In order to verify this, transgenic HaNLP3 estradiol-inducible expressing lines were created and they were used to make EMS (ethyl methanesulfonate)-mutagenized transgenic A. thaliana plants. These plants are sprayed with estradiol and checked for insensitivity to HaNLP peptide. Plants with a normal phenotype have possibly mutations in genes involved in HaNLPs recognition (RLP23 or SOBIR1). Seeds of these decreased NLP-triggered immunity (dni) plants are used for a second screen; a synthetic HaNLP peptide is employed to check whether the immune system of plants is triggered (ethylene assay). To find mutations, completely insensitive mutants to the HaNLP peptide are selected and their RLP23 and SOBIR1 genes are sequenced. Furthermore, another purpose of the project is to provide several dni putative mutants which could be used to unveil other genes involved in the HaNLP3 MTI pathway. In addition, as plant immune system is not triggered by the HaNLP peptide, the dni putative mutants should show a more susceptible phenotype to H. arabidopsidis. To investigate that, the dni putative mutants are infected by H. arabidopsidis and sporulation is measured.

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Results

EMS mutagenesis yields normal phenotype mutants

EMS driven mutagenesis was performed on Estradiol-inducible HaNLP3 A. thaliana lines (Zuo et al., 2000; Oome et al., submitted). 390 pools containing 16 M1 plants which seeds have been EMS mutagenized where used to find dni putative mutants. M2 plants growth was compared with two control lines est::YFP and est::NLP3 to look for plants which show no-reduced growth phenotype upon estradiol treatment. 718 M2 plants have been selected from 202 pools. These plants had normal phenotype after being sprayed with estradiol, suggesting that the immune system of these plants could not be triggered by HaNLP3 protein, due to EMS mutation in the HaNLPs MTI pathway. The presence of HaNLP3 gene was tested on the dni plants DNA, using HaNLP3 specific primers, and HaNLP3 gene was found in 588 plants (fig.5). The normal phenotype could also be caused by lack or mutations in the estradiol-inducible HaNLP3 transgene, therefore HaNLP3 expression was also tested on 125 HaNLP3 transgenic plants, proving that 81 dni plants had the mRNA normally expressed (fig. 6). In consequence, the obtained dni putative mutants might possibly have mutations in genes involved in HaNLPs MTI response and thereby, they were brought to the next generation (M3) in order to check their response when treated with HaNLP3 peptide.

Figure 5 | HaNLP3 presence in 718 dni plants. Figure 6 | HaNLP3 expression in 125 dni plants. 130 plants (18%) carry no HaNLP3 transgene. HaNLP3 is expressed in 81 plants (65%) and not expressed in The HaNLP3 transgene is present in 588 plants (82%) 44 plants (35%).

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Ethylene production as evidence for plant immunity activation

To investigate whether the immune system of the dni putative mutants was activated or not, these dni plants were treated with a synthetic 24 amino acid NLP peptide (NLP3_24), based on the HaNLP3 sequence. This peptide was able to trigger plant immune system inducing ethylene production in A. thaliana. M3 seeds from 320 dni putative mutants have been sown out. Seeds from 31 mutants did not grow properly but the other 289, one plant for each dni mutant, was tested for ethylene production. Ethylene peaks of the mutants were compared to controls (Col-0 and est::NLP3) mock treatments (Fig. 7). 237 plants showed either high or low ethylene production, yet 52 plants did not show any ethylene production (Fig. 8). The plant immune system was not triggered by the HaNLP peptide in 18% of M3 dni putative mutants. These 52 mutants descend from 18 different transgenic EMS mutagenized lines of the 202 pools used for the screen. Pools 110, 245 and 335 contain 27 of the 52 no ethylene response mutants (Fig. 9). Some of these mutants are probably genetically identical because they belong to the same pool and they might have interesting mutation in genes involved in the HaNLP3 MTI response, like RLP23 or SOBIR1.

NLP3_24 NLP3_24 MOCK MOCK NLP3_24 NLP3_24 MOCK MOCK

Col-0 Est::NLP3

Figure 7 | Ethylene production in the two controls Col-0 and Est::NLP3. The picture represents four different measurements for the two controls: two with the peptide (NLP3_24) and two without (mock). The highest peak is probably carbon dioxide and it is detected after 30 seconds. Ethylene peak appears after 45 seconds (blue arrow) and it is much smaller. Ethylene peaks is detected in Col-o and est::NLP3 only when the HaNLP3 peptide is present in the solution.

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NLP3_24 NLP3_24 MOCK MOCK NLP3_24 NLP3_24 MOCK MOCK

294-2 245-3

Figure 8 | Ethylene production in two dni putative mutants. These two pictures represent the difference between mutants which show ethylene production upon HaNLP3 detection and mutants which do not show it. On the left, mutant 294-2 shows ethylene production: ethylene peaks (blue arrows) are detected when the peptide is present (NLP3_24) but not when it is absent (mock). On the other side, ethylene is not detected in mutants 245-3.

Figure 9 | No ethylene reponse dni putative mutants. The graph shows the 18 pools where the no ethylene response mutants belong to. Most of the no ethylene response mutants were located in three different pools: 110, 254, 335. 52 no ethylene response mutants were found in total and 27 belong to pools 110, 254 or 335.

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RLP23 and SOBIR1 sequencing

As described in the introduction, RLP23 and SOBIR1 are the receptor and the co-receptor of HaNLP3 MTI response in A. thaliana. Both genes could have been mutated due to EMS and the mutation might cause the loss of MTI activation. Nine mutants, which show no ethylene response and which have the HaNLP3 transgene present in the genome, were checked for mutations in both RLP23 and SOBIR1 genes. These nine mutants belong to six different pools (212, 226, 245, 270, 317, 335). Mutants from the same line could be genetically identical except for line 270 in which only two plants of eight did not show ethylene response [table 1].

A single point DNA mutation was found on mutant 226-5, corresponding to the 66th amino-acid and another one was found on the 859th a. a. of mutant 317-1, both regarding the RLP23 gene. No mutation was found in the remaining plants. Both mutations changed a Guanine to an Adenine (transition). As a consequence, the codon for a Tryptophan became a stop codon in the mutant 226-5, and the codon for Glycine turned into a Glutamic acid in the mutant 317-1 [table2]. Moreover, a single point mutation in SOBIR1 gene was found in mutant 245. Two mutants have been sequenced from this line and they both showed the same mutation: a Cytosine was replaced by a Thymine (Transition). Consequently, a Glutamine codon changed to a stop codon at position 232. The remaining mutants did not carry mutations in the SOBIR1 gene [table2].

Pool No of dni putative mutants

No of no ethylene responsemutants No of mutants sequenced

212 2 2 1 226 5 4 1 245 11 11 3 270 8 2 1 317 1 1 1 335 10 8 2

Table 1 | Dni putative mutants used for sequencing. This table shows where mutants, sent for sequencing, belong to. 226,245 and 335 pools have many plants which do not have ethylene response. 24 of the 52 no ethylene response mutants belong to these three pools.

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H. arabidopsidis infection screen

As it is described in literature, plants are more susceptible to pathogens if the immune system is not activated (Jones & Dangl, 2006). The important issue regarding non-cytotoxic NLPs function in the infection process can be tested with H. arabidopsidis infection assay. As it was mentioned previously, HaNLPs are expressed early during infection (Cabral et al., 2012) and this suggests they serve a function at that stage of infection. Furthermore it has been reported that HaNLP3 activates plant immune system causing resistance to H. arabidopsidis (Oome et al., submitted). Therefore, the reasonable idea is that dni putative mutants which do not recognize HaNLP3 should show a more susceptible phenotype towards H. arabidopsidis compared to the controls. In order to investigate that, M3 dni mutants were tested for H. arabidopsidis infection 24 hours post estradiol treatment. Sporulation was verified on A. thaliana leaves. 279 dni mutants have been tested: 153 showed H. arabidopsidis sporulation, 126 showed no sporulation. 47 no ethylene response dni mutants have been tested: 35 showed H. arabidopsidis sporulation, 12 showed no sporulation. These 47 mutants belong to 16 M2 lines [table 3] and these lines could become very useful for further studies.

Dni mutants carrying a mutation were susceptible to H. arabidopsidis except for 317-1 [table 4]. Mutations in the HaNLP3 receptor and co-receptor would unable the plant to detect HaNLP3 and to trigger the immune system. For this reason, mutants 226-5, 245-1 and 245-6 were susceptible to the pathogen. On the other hand, the mutant 317-1 shows very interesting results. As the mutant is resistant to the pathogen the immune system has to be triggered, therefore the HaNLP3 peptide is detected by this mutant. This implies that the RLP23 mutation in 317-1 is not essential for HaNLP3 detection and that the downstream resistance pathway is not mutated. In addition, this mutant shows no ethylene production and therefore, it could have mutations in the ethylene pathway. These results bring us to hypothesize that the ethylene pathway and the resistance pathway are separated in the HaNLP3 downstream response.

Dni putative mutants

Mutations in RLP23 gene Mutations in SOBIR1 gene

212-1 No Not Sequenced

226-5 Yes (G>A, W66STOP) No

245-1 No Yes C>T (Q232STOP)

245-6 No Yes C>T (Q232STOP)

245-7 No Not Sequenced

270-5 No No

317-1 Yes (G>A, G859E) No

335-4 No No

335-9 No No

Table 2 | RLP23 and SOBIR1 sequencing results of nine dni mutants. RLP23 and SOBIR1 genes were sequenced in nine different dni mutants. Some of these mutants belong to the same EMS mutagenized pool. Two mutants have a point mutation in RLP23 and two mutants, both from pool 245, have the same point mutation in SOBIR1.

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Pool No of dni putative mutants

No of no ethylene response mutants

No of mutants infected by H. arabidopsidis

No of mutants not infected by H. arabidopsidis

103 3 2 2 0 110 12 8 8 0 212 2 2 1 1 226 5 4 2 2 231 1 1 1 0 241 1 1 1 0 243 4 1 1 0 244 4 1 1 0 245 11 11 9 2 268 8 1 1 0 270 8 2 2 0 292 5 2 No data No data 317 1 1 0 1 335 10 8 4 4 339 5 1 1 0 343 4 1 1 0 345 5 2 0 2 348 5 3 No data No data

Mutants RLP23/SOBIR1 mutation Ethylene response H. arabidopsidis sporulation

226-5 RLP23,(G>A, W66STOP) No Yes 245-1 SOBIR1,(C>T, Q232STOP) No Yes 245-6 SOBIR1,(C>T, Q232STOP) No Yes 317-1 RLP23,(G>A, G859E) No No

Table 3 | H. arabidopsidis infection screen. The table shows results of H. arabidopsidis infection on A.thaliana dni putative mutants. Only mutant lines, in which ethylene is not released upon HaNLP3 detection, are provided in the table. Each row shows the number of the mutant line, how many mutants of that line have a decreased NLP-triggered immunity, how many mutants have no ethylene production and how many of no ethylene response mutants are either infected or not infected by H. arabidopsidis.

Table 4 |RLP23/SOBIR1 EMS mutants. The table provides a summary of H. arabidopsidis infection results, regarding only dni putative mutants which carry EMS mutations. The four mutants at issue show all no ethylene response. In addition, mutants 226-5, 245-1, 245-6 are also susceptible to the pathogen unlike mutant 317-1 which is not.

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Conclusions and discussion

The NLP group of proteins is finally classified as MAMPs but the role of the non-cytolytic NLPs is still unknown. As HaNLP3 has been studied in most detail (Cabral et al., 2012), this protein was chosen to analyze the effect of non-toxic NEP1-like proteins in A. thaliana plants. Estradiol-inducible HaNLP3 A. thaliana lines, coupled with EMS driven mutagenesis, generate dni plants which could lead to find genes involved in HaNLPs recognition pathway. 718 dni plants have been selected in this research and they could carry important mutations in genes involved in HaNLP3 MTI response. The issue is to find out causes which lead to the dni phenotype; they could be either mutations in the MTI genes or lack/mutations in the estradiol-inducible HaNLP3 transgene, causing no expression of the protein in the plants. To answer that, the HaNLP3 presence and its expression have been checked in the dni putative mutants. 588 plants harbor the construct. Mainly due to time consuming reasons, the expression has been checked only on 125 plants. 81 plants show both the expression of the protein and normal growth phenotype, meaning that HaNLP3 is not detected by plant immune system. However, it is quite possible that HaNLP3 gene of the dni putative mutants has been altered in the “GHRHDWE” motif or in another site due to EMS mutations, eliminating the ability of the plant to recognize the protein. Therefore, the activation of the immune system was tested using the HaNLP3 peptide.

Ethylene production of the dni plants was measured in presence of the HaNLP3 peptide to unravel which dni putative mutants could be used to find genes and mutations involved in the HaNLP3 detection pathway. Ethylene assay revealed 52 mutants which do not trigger ethylene production; therefore they could carry mutations in MTI genes. In particular three pools (110, 245, 335) were the most represented. These mutations could prove to be important in order to discover genes involved in the pathway. Meanwhile HaNLP3 surface receptor has been identified. Therefore, primers of the receptor (RLP23) along with its possible co-receptor (SOBIR1) were used in our research to sequence nine no ethylene response mutants. According to the results, no mutations were found in these two genes for mutants 212-1, 245-7, 270-5, 335-4, 335-9, suggesting that they could probably carry mutations in other genes involved in MTI pathway. On the other hand, the same SOBIR1 gene point mutation is present in two mutants (245-1, 245-6) which belong to the same pool and most likely genetically identical. The remaining two mutants (226-5, 317-1) have a different single point mutation in RLP23. Considering the length of RLP23 and SOBIR1 proteins, 890 amino-acid and 641 amino-acid, respectively, the stop codon mutation occurs in position 66 for RLP23 in mutant 226-5 and in position 232 for SOBIR1 in mutant 245-1, 245-6. For this reason, the mutated receptor and co-receptor are truncated and not able to recognize the HaNLP3 peptide, causing no ethylene response and no resistance to the pathogen in mutants 226-5, 245-1 and 245-6 (fig. 10). Regarding mutant 317-1, the whole protein remains intact and only one amino-acid changes in position 859. The mutation is present in the hydrophobic part of the receptor which is located inside the cell membrane. As recognition site of the receptor remains intact, this mutation should not cause the lack of HaNLP3 recognition in the plant. To support this hypothesis, the results show that mutant 317-1 is resistant to H. arabidopsidis [table 4], meaning that the HaNLP3 peptide has to be detected by the receptor to trigger the immune system and to make the plant resistant. In addition, mutant 317-1 shows no ethylene response, meaning that this mutant should have mutations in the ethylene pathway. Assembling together these results, it is possible to hypothesize that the ethylene pathway and the resistance pathway are both activated from the HaNLP3 peptide through MTI upon the

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receptor and the co-receptor interaction. However, these two pathways diverge from each other in a certain point along the HaNLP3 downstream response (Fig. 10).

MTI

RLP23 SOBIR1

HaNLP3

226-5

Ethylene production

No resistance

No ethylene production

No resistance

SOBIR1 RLP23

245-1/245-6

317-1

HaNLP3

No ethylene production

Resistance

Model

No ethylene production Resistance MTI

Mutation

HaNLP3

SOBIR1 RLP23

HaNLP3

SOBIR1 RLP23

Figure 10 |Schematic representation of the HaNLP3 downstream response in different RLP23 and SOBIR1 mutants. The model shows that RLP23 interacts with SOBIR1 upon HaNLP3 binding, triggering MTI response. Mutant 226-5 has RLP23 truncated; therefore it does not detect HaNLP3. Mutants 245-1 and 245-6 have SOBIR1 truncated; therefore SOBIR1 does not bind to RLP23 to trigger MTI when HaNLP3 is detected by RLP23. Both 226-5 and 245-1/-6 show no ethylene production and no resistance to the pathogen. Mutant 317-1 has only one RLP23 point mutation which does not affect RLP23 integrity. Strikingly, this mutant shows no ethylene production and resistance to the pathogen, implying that there is mutation in the ethylene pathway which diverges from the resistance pathway in the downstream response.

Membrane Membrane

Membrane

Membrane

Extracellular space Extracellular space

Extracellular space Extracellular space

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Dni mutants with no mutations in RLP23 and SOBIR1 genes might be less resistant to the pathogen even if ethylene is produced, due to possible mutations in the downstream resistance pathway. Regarding the HaNLP3 studies, it is known that resistance of the plant to the pathogen is related to the activation of the immune system and that the activation of the immune system triggers ethylene production (Oome et al., submitted). However, the activation of the immune system might cause resistant to the pathogen but not activate ethylene production, due to EMS mutation (Fig. 10). On the other hand, the immune system could be still activated leading to ethylene production but not to resistance if there is a mutation in the downstream resistance pathway. Therefore all the dni mutants, in which the HaNLP3 peptide is expressed (Fig. 6) could have interesting mutations in the HaNLP3 downstream pathway even if they showed ethylene production. Furthermore, as no ethylene response mutants have surely mutations in the HaNLP3 downstream pathway, they constitute the starting point for further tests in order to unravel HaNLPs infection process. In effect, mutants, which do not carry any mutations in RLP23 or SOBIR1, could be used for genetic mapping and backcrossing with parental lines in order to locate mutations causing a lack of response upon HaNLP3 detection. Indeed, all the dni putative mutants coupled with RLP23 and SOBIR1 sequencing analysis provide a tool to discover others genes involved in the HaNLP3 MTI pathway.

From another point of view, an A. thaliana RLP23 knock-out line may be used in order to assert more about the importance of non-cytotoxic NLPs in H. arabidopsidis infection process. It is tempting to hypothesize that HaNLPs could have been evolved from cytotoxic proteins into non-cytotoxic proteins in order to give to H. arabidopsidis the opportunity to exploit plant resources as a biotrophic pathogen. For this reason, the HaNLPs could have been previous cytotoxic proteins, which had lost their function and plants, like A. thaliana, could have evolved immune system specific receptors in order to protect themselves against their infection. To conclude, cloning of the HaNLP receptor in A. thaliana is of great importance because it could enhance resistant to infection by members of Peronosporales and this could lead to generating crop species with broad resistance towards this kind of pathogens. Indeed, as it was demonstrated for the A. thaliana MAMP-receptor EFR (Lacombe et al., 2010), the transfer of the receptor into different plant species may be feasible.

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Materials and methods

EMS dni screen HaNLP3 expression was induced in EMS mutagenized M2 A. thaliana plants by spraying with 500mM estradiol in 0.02% Silwet. Seedlings were sprayed with estradiol at intervals of 48 hours, resulting in 3 sprays per week, over the course of several weeks until a clear difference between dni putative mutants and control plants was visible. Two controls were used. Estradiol-inducible expressing control line (est::YFP) to prove that the inducing compound does not cause growth reduction, and an estradiol-inducible HaNLP3 parental line (est::NLP3) which has not been EMS mutagenized.

Nucleic acid extraction and PCR analysis Plant DNA was extracted from A. thaliana dni mutants using the Sucrose Prep method (Berendzen et al., 2012). The RNeasy Plant mini kit (Qiagen) was used for RNA extraction. DNA and RNA concentrations were measured using a NanoDrop 2000. cDNA was made from 1 µg of total RNA with Multi-RV H-minus reverse transcriptase, Ribolock for molecular stabilization, and oligo(dT) (Promega) after being treated with DNase (Qiagen). PCR protocols for HaNLP3 presence and expression were performed using cloning primers. PCRs were performed at 32 cycles using Taq-polymerase. Protocol consists of 3 steps: melting temperature for 30 seconds at 94oC, annealing temperature for 15 seconds at 65oC, and an extension time of 30 seconds at 72oC. NLP3 gene bands obtained were compared to a standard A. thaliana reference gene (actin).

Ethylene measurement To assay ethylene production, leaves of 5-week old dni putative mutants plants were cut in 3mm squares and left in MQ overnight at room temperature. On the next day 3 pieces were transferred to glass tubes containing 400 µl of aqueous solution containing 20 mM of MES buffer and the peptide (1 µM). The same procedure was adopted for control solution (mock treatment) but 0,01% DMSO was used instead of the peptide. Col-0(wild-type) and estradiol-inducible HaNLP3 parental line plant leaves were used as controls for ethylene release. The glass tubes were closed and they were shaken gently on a shaker machine for 3 hours. Ethylene accumulation was measured by gas chromatography. Finally, dni putative mutant results were compared to the mock treatment results and to the controls results.

Sequencing analysis Plant DNA was extracted from A. thaliana no ethylene response dni mutants using the DNeasy plant miniprep kit. DNA concentrations were measured using NanoDrop 2000. PCR protocols were performed at 35 cycles using Taq-polymerase to amplify RLP23 and SOBIR1 genes. Protocol consists of 3 steps: melting temperature for 30 seconds at 94oC, annealing temperature for 15 seconds at 54oC, and an extension time of 2:30 minutes at 72oC. RLP23 and SOBIR1 bands were obtained by electrophoresis in gel of agarose and bands were purified for sequencing using purification kit.

Ha infection experiment NLP3 was induced in dni putative mutants by spraying with 500mM estradiol in 0.02% Silwet. Infection assay was performed by spraying plants with spores of H. arabidopsidis isolate Waco9 (50 spores/µl)24 hours post estradiol treatment. After inoculation, plants were air dried for 30 minutes and subsequently incubated at 100% humidity at 16oC with 10 hours of light/day. The amount of

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sporulation was quantified 5 to 6 days after inoculation by cutting of seedlings, suspending the spores in a known volume of water and determining the amount of spores per mg of plant tissue.

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