Tomato wall-associated kinase SlWak1 acts in an Fls2- and Fls3-dependent manner to promote apoplastic immune responses to Pseudomonas syringae

Wall-associated kinases (Waks) are known to be important components of plant immunity against various pathogens including Pseudomonas syringae pv. tomato (Pst) although their molecular mechanisms are largely unknown. In tomato, SlWak1 has been implicated in immunity because its transcript abundance increases significantly in leaves after treatment with the flagellin-derived peptides flg22 and flgII-28, which activate the receptors Fls2 and Fls3, respectively. We generated two SlWak1 tomato mutants (Δwak1) using CRISPR/Cas9 and investigated the role of SlWak1 in tomato-Pst interactions. PTI activated in the apoplast by flg22 or flgII-28 was compromised in Δwak1 plants but PTI at the leaf surface was unaffected. The Δwak1 plants developed fewer callose deposits than wild-type plants but retained the ability to generate reactive oxygen species and activate MAPKs in response to flg22 and flgII-28. The induction of Wak1 gene expression by flg22 and flgII-28 was greatly reduced in a tomato mutant lacking Fls2 and Fls3 but induction of Fls3 gene expression by flgII-28 was unaffected in Δwak1 plants. After Pst inoculation, Δwak1 plants developed disease symptoms more slowly than Δfls2.1/fls2.2/fls3 mutant plants, although both plants ultimately were similarly susceptible. SlWak1 co-immunoprecipitated with both Fls2 and Fls3 independently of flg22/flgII-28 or Bak1. These observations suggest that SlWak1 acts in a complex with Fls2/Fls3 and plays an important role at later stages of the PTI in the apoplast.

To overcome PTI, pathogens deliver virulence proteins (effectors) into the plant cells to interfere with MAMP detection or PTI signaling and promote disease development (Dou & Zhou, 2012).
AvrPto and AvrPtoB, two effectors from Pseudomonas syringae pv. tomato (Pst), suppress the early PTI response by interfering with the interaction of Fls2 with Bak1 (Xiang et al., 2008;Martin, 2012;Hind et al., 2016). In response to bacterial effectors, plants have evolved genes encoding NLRs (nucleotide-binding oligomerization domain-like receptors) which recognize specific effectors and activate NTI (Martin et al., 2003;Jones & Dangl, 2006). In tomato, the Pto kinase protein interacts with AvrPto or AvrPtoB and forms a complex with the NLR protein Prf resulting in the induction of NTI and inhibition of pathogen growth (Martin et al., 1993;Salmeron et al., 1996;Pedley & Martin, 2003).
Although Wak proteins have been identified as important contributors to disease resistance against various pathogens (Hu et al., 2017;Bacete et al., 2018), much remains to be learned about the molecular mechanisms they use to activate immune responses. The best-studied Wak protein, the Arabidopsis AtWAK1, recognizes cell wall-derived oligogalacturonides (OGs) and activates OG-mediated defense responses against both fungal and bacterial pathogens (Brutus et al., 2010;Gramegna et al., 2016). In maize, the ZmWAK-RLK1 protein (encoded by Htn1) confers quantitative resistance to northern corn leaf blight (NCLB) by inhibiting the biosynthesis of secondary metabolites, benzoxazinoids (BXs), that suppress pathogen penetration into host tissues (Yang et al., 2019). Another ZmWAK protein located in a major head smut quantitative resistance locus qHSR1 enhances maize resistance to Sporisorium reilianum by arresting the fungal pathogen in the mesocotyl (Zuo et al., 2015). One wheat Wak protein encoded by the Stb6 gene recognizes an apoplastic effector (AvrStb6) from Zymoseptoria tritici and confers resistance to the fungal pathogen without a hypersensitive response (Saintenac et al., 2018). In rice, three OsWAKs act as positive regulators in resistance to the rice blast fungus by eliciting ROS production, activating defense gene expression, and recognizing chitin by being partially associated with the chitin receptor CEBiP (Delteil et al., 2016). Wak proteins therefore appear to exhibit extensive functional diversity and have different mechanisms to defend against pathogen infection in different plant species. The functional characterization of Wak proteins in tomato has not been reported and their possible contributions to PTI or NTI are not well understood in this species.
Tomato is an economically important vegetable crop throughout the world and its production is threatened by many pathogens including Pseudomonas syringae pv. tomato which causes Page 5 of 26 bacterial speck disease and can result in severe crop losses (Jones, 1991;Kimura & Sinha, 2008).
Understanding the functions of Wak proteins in tomato could therefore provide fundamental information for breeding tomato cultivars that are resistant to various pathogens. Tomato contains seven Wak and sixteen Wakl genes (Zheng et al., 2016). The SlWak1 (Solyc09g014720) gene is clustered together with another three SlWak genes (Solyc09g014710, Solyc09g014730 and Solyc09g014740) on chromosome 9; however, the expression of only the SlWak1 gene (hereafter Wak1) is significantly induced after MAMP treatment or Pst inoculation (Rosli et al., 2013). Knock down of Wak1 gene expression in N. benthamiana leaves using virus-induced gene silencing (VIGS) compromised resistance to the bacterial pathogen Pst. However, three closelyrelated NbWak genes were simultaneously silenced in these experiments, making it unclear if one or a combination of NbWak genes contributed to the enhanced susceptibility to Pst (Rosli et al., 2013). To gain a deeper insight into the role of Wak1 in tomato-Pst interactions, we generated two homozygous Wak1 mutant lines (Dwak1) in tomato using CRISPR/Cas9. Characterization of these Dwak1 mutants indicated that Wak1 protein acts as an important positive regulator in later stages of flagellin-mediated PTI response in the apoplast and associates in a complex with Fls2 and Fls3 to trigger immune signaling.

Generation of Wak1 tomato mutants using CRISPR/Cas9
To mutate the Wak1 gene in tomato, we designed two guide RNAs (Wak1-gRNA1: GTTAAGATTAGCATAAAACA; Wak1-gRNA2: GGGGCGGTGGCATTCGTTGG) targeting the first exon of Wak1 using the software Geneious R11 (Kearse et al., 2012). Each gRNA cassette was cloned into a Cas9-expressing binary vector (p201N:Cas9) by Gibson assembly as described previously (Jacobs et al., 2017). Tomato transformation was performed at the biotechnology facility at the Boyce Thompson Institute. Agrobacterium cells containing each gRNA/Cas9 construct were pooled together and used for transformation into the tomato cultivar Rio Grande (RG)-PtoR, which has the Pto and Prf genes. To determine the mutation type, genomic DNA was extracted from cotyledons or young leaves of each transgenic plant using a modified CTAB method (Murray & Thompson, 1980). Genomic regions spanning the target site of the Wak1 gene were amplified with specific primers (Supplemental Table S1) and sequenced  (Brinkman et al., 2014) were used to determine the mutation type and frequency using the sequencing files (ab1. format) as described (Zhang et al., 2020).

Off-target evaluation
To investigate potential off-target mutations caused by gRNAs in the Dwak1 plants, Wak1-gRNA1, which induced target mutations in Wak1 in the transgenic plants, was used as a query to search putative off-target sites across the tomato genome with up to 4 nucleotide mismatches by Geneious R11 or with up to 3 nucleotide mismatches by Cas-OFFinder (Bae et al., 2014). Seven potential off-target sites with the highest similarity to the spacer sequence of Wak1-gRNA1 were chosen for evaluation. Genomic regions spanning the putative off-target sites were amplified with specific primers (Supplemental Table S1) and PCR amplicons were sequenced to determine if off-target mutations were induced at those sites.

Bacterial inoculation assay
Four-week-old Dwak1 and wild-type plants were vacuum infiltrated with various Pst DC3000 strains at different titers, including DC3000∆avrPto∆avrPtoB (DC3000DD) or DC3000∆avrPto∆avrPtoB∆fliC (DC3000DDD) at 5 x 10 4 cfu/mL or DC3000 at 1 x 10 6 cfu/mL. Three to four plants per line were tested with each bacterial strain. Bacterial populations were measured at 3 h and two days after inoculation. Disease symptoms were photographed 4 or 5 days after bacterial infection. Dwak1 and wild-type plants were also spray inoculated with DC3000DD at 1 x 10 8 cfu/mL and photographs of disease symptoms were taken 6 days after inoculation.

PTI protection assay
Four leaflets on the third leaf of 4-week-old plants were first syringe infiltrated with 1 x 10 8 cfu/mL of heat-killed DC3000ΔavrPtoΔavrPtoBΔhopQ1-1ΔfliC (DC3000DDDD) complemented with a fliC allele from DC3000 or ES4326, or no fliC (empty vector; EV). Sixteen hours later, the whole plants were vacuum inoculated with DC3000ΔavrPtoΔavrPtoBΔfliC (DC3000DDD) at 5 x 10 4 cfu/mL. Bacterial populations were measured two days after Page 7 of 26 inoculation. Alternatively, plants were first syringe infiltrated with 1 μM flg22 (GenScript), 1 μM flgII-28 (EZBiolab), or buffer alone (10 mM MgCl 2 ), respectively. Plants were inoculated with DC3000DDD 16 h later and bacterial populations were measured two days after inoculation as described above.

Measurement of stomata number and stomata conductance
Leaf samples were taken from Dwak1 and wild-type plants. Photographs from the abaxial epidermis of the leaves were taken using an epifluorescence microscope (Olympus BX51) and the number of cells and both closed and open stomata were counted manually. The stomata index was calculated as the percentage of stomata number per total number of cells (stomata plus epidermal cells). Stomatal conductance was measured at 2 pm, using a leaf porometer (SC1 Decagon Devices, Inc.) on the abaxial side of two leaflets of the third leaf from four plants per line.

Reactive oxygen species assay
ROS production was measured as described previously (Hind et al., 2016). In brief, leaf discs were collected and floated in water overnight (~16 h). Water was then removed and replaced with a solution containing either 50 nM flg22 (QRLSTGSRINSAKDDAAGLQIA) or 50 nM flgII-28 (ESTNILQRMRELAVQSRNDSNSSTDRDA), in combination with 34 µg/mL luminol (Sigma-Aldrich) and 20 µg/mL horseradish peroxidase. ROS production was then measured over 45 min using a Synergy 2 microplate reader (BioTek). Three to four plants per line and three discs per plant were collected for each experiment.

Callose deposition
Four-week-old plants were vacuum infiltrated with 1 x 10 8 cfu/mL P. fluorescens 55, a strong inducer of PTI (Rosli et al., 2013). Leaf samples were taken 24 h post infiltration, cleared with 96% ethanol and stained with aniline blue for 1 h. Callose deposits were analyzed using an epifluorescence microscope (Olympus BX51). Quantification was performed using ImageJ software. Fifteen photographs per biological replicate were analyzed using four plants per line.

Reverse transcriptase quantitative PCR
Four leaflets from the third leaf of 5-week-old plants were first syringe infiltrated with 1 μM flgII-28 or buffer. Three plants were used for each treatment and two biological replicates were performed. Leaf tissues were collected 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h after infiltration, immediately frozen in liquid N 2 and stored at -80°C until used. Total RNA was isolated using RNeasy Plant Mini Kit (Qiagen). RNA (4 μg) was treated with TURBO DNA-free DNase (ThermoFisher Scientific) twice, each for 30 min at 37°C. First-strand cDNA was synthesized from 2 μg RNA using SuperScript TM III (ThermoFisher Scientific). Quantitative PCR was performed with specific primers (Table S1) using the QuantStudio™ 6 Flex Real-Time PCR System (ThermoFisher Scientific) and cycling conditions for PCR were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 30 s.

Generation of Wak1 mutants in tomato by CRISPR/Cas9
We reported previously that virus-induced gene silencing of three homologs of Wak1 in N.
benthamiana led to enhanced susceptibility to Pseudomonas syringae pv. tomato (Rosli et al., 2013). In tomato leaves, transcript abundance of the Wak1 gene (Solyc09g014720) is significantly increased after treatment with flg22, flgII-28, or csp22, suggesting Wak1 might play a role in tomato-Pst interactions (Rosli et al., 2013;Pombo et al., 2017). To study the possible role of Wak1 in plant immunity, we generated mutations in Wak1 using CRISPR/Cas9 with a guide RNA, Wak1-gRNA1 (GTTAAGATTAGCATAAAACA; Fig. 1a), which targets the first exon of the Wak1 gene. After transformation of the cultivar Rio Grande-PtoR (RG-PtoR, which has the Pto and Prf genes), we obtained a biallelic mutant (Dwak1 4) from which two Wak1 homozygous mutant lines (Dwak1 4-1; Dwak1 4-2) were derived (Fig. 1a). Line 4-1 has a 10-bp deletion in Wak1, resulting in a premature stop codon at the 17 th amino acid (aa) of the protein, whereas line 4-2 has a 1-bp deletion in Wak1, causing a premature stop codon at the 18 th aa (Fig.   1a). The growth, development and overall morphology of both Dwak1 mutants were indistinguishable from wild-type RG-PtoR plants (Fig. S1).
To determine if the gRNA designed for Wak1 editing inadvertently caused mutations in other genomic regions of the Dwak1 plants, we selected seven putative sites with the highest off-target scores using Geneious R11 and Cas-OFFinder, although all of these sites had at least three mismatches compared with the spacer sequence of the Wak1 gRNA ( Fig. 1b). Of the seven potential off-target sites, two are located in the coding region of a gene, three are in the untranslated region of genes, and another two are in intergenic regions. For each site, we tested 10 to 20 independent T1 or T2 plants, with or without Cas9, and did not detect any off-target mutations. This is not unexpected as the gRNA we designed for Wak1 was highly specific, with little possibility to target Wak1 homologs or other genes in tomato, considering that even one mismatch in the seed sequence (the last 12 nucleotides of a gRNA spacer sequence) can severely To test whether Wak1 contributes to NTI, Dwak1, RG-PtoR, and Rio Grande-prf3 plants (RG-prf3, which contains a mutation in Prf that makes the Pto pathway nonfunctional) were inoculated with DC3000. Six days after inoculation, the Dwak1 and RG-PtoR plants had no disease symptoms, whereas the RG-prf3 control showed severe disease symptoms (Fig. 2c).
Bacterial populations were about 30-fold less in the Dwak1 and RG-PtoR plants compared to RG-prf3. Wak1 therefore appears to have no observable role in NTI.
The two Dwak1 mutant lines were derived from the same primary transformant and it was formally possible that another mutation induced during tissue culture is responsible for the enhanced susceptibility to Pst. We therefore developed F1 hybrids by crossing the Dwak1 plants to RG-PtoR plants (Fig. S2). Sequencing confirmed that all F1 hybrids were heterozygous for the Wak1 mutation. F1 hybrids that were vacuum-infiltrated with DC3000DD developed disease symptoms and supported bacterial populations similar to RG-PtoR plants (Fig. S2a), indicating Wak1 is a dominant allele. Four F1 plants (two were -10 bp/WT and two were -1 bp/WT; WT, wild type) were selfed to develop F2 populations. After inoculation of 117 F2 plants with DC3000DD we observed a segregation ratio of 3 resistant to 1 susceptible (Fig. S2b). Sequencing revealed all resistant plants were either homozygous wildtype or heterozygous, while the susceptible plants were homozygous for the wak1 mutation (Fig. S2c). Combined with the lack Page 11 of 26 of off-target mutations, these disease assays with F2 populations strongly support that the susceptibility to Pst of Dwak1 plants is due to the CRISPR/Cas9-induced loss-of-function mutations in the Wak1 gene.

Wak1 mutant plants are compromised in PRR-triggered immunity induced by flg22 and flgII-28
The observation that Dwak1 plants are more susceptible to DC3000DD but show no differences compared to wild-type plants for their response to DC3000DDD which lacks flagellin, suggests that Wak1 is involved in immune responses mediated by flg22 and/or flgII-28. To further test this, we performed a 'PTI protection' assay using a heat-killed Pst strain lacking flagellin and three type III effectors (DC3000ΔavrPtoΔavrPtoBΔhopQ1-1ΔfliC; DC3000DDDD) complemented with a construct expressing fliC from either DC3000 (which has active flg22 and flgII-28) or P. cannabina pv. alisalensis ES4326 (only flgII-28 is active) (Hind et al., 2016), or an empty vector (EV) as a control (Fig. 3a). Since both of the Dwak1 lines were similarly susceptible to DC3000DD, most subsequent experiments were focused on the 4-1 line. Dwak1 4-1 plants were first infiltrated with the various suspensions of heat-killed bacteria to induce PTI and then challenged with DC3000DDD 16 h later. Wild-type plants pretreated with Pst DC3000DDDD with an empty vector supported a significantly higher bacterial population than plants pretreated with the heat-killed bacterial suspensions containing either DC3000 fliC or ES4326 fliC (7.5-fold and 3.3-fold, respectively), indicating that pretreatment of wild-type plants activated PTI defenses due to recognition of flg22 and/or flgII-28. The Dwak1 plants, however, supported higher bacterial populations regardless of the pretreatment indicating the PTI response was compromised (Fig. 3a).
We next performed the PTI protection assay using the synthetic peptides flg22 and flgII-28.
Plants were first syringe-infiltrated with buffer alone, 1 μM flg22, or 1 μM flgII-28, and then challenged with DC3000DDD 16 h later as described above (Fig. 3b). Two days later, wild-type plants that were pretreated with either flg22 or flgII-28 had significantly lower bacterial populations compared to the buffer-only treatment. In contrast, no significant differences in bacterial populations regardless of pretreatment were observed in Dwak1 plants. Collectively, Page 12 of 26 these experiments demonstrate that Wak1 plays an important role in PTI that is activated by two flagellin-derived MAMPs.
Dwak1 plants are not compromised in PRR-triggered immunity responses on the leaf surface, or in stomatal numbers or conductance Pst inoculation experiments using vacuum infiltration assess PTI responses primarily in the apoplast. To test if Wak1-mediated immunity also plays a role in PTI on the leaf surface, we spray inoculated Dwak1 and wild-type RG-PtoR plants with DC3000DD. This inoculation method requires the pathogen to enter the apoplastic space through stomata or natural openings.
Interestingly, in contrast to experiments using vacuum infiltration, both wild-type and Dwak1 plants developed disease symptoms after spray inoculation that were indistinguishable both in the amount of time until they developed and in their ultimate severity (Fig. 4a). Thus, Wak1 does not appear to play an important role in PTI responses on the leaf surface. Measurements of stomatal numbers and of stomatal conductance as an indicator of stomatal activity revealed no differences between wild-type and Dwak1 plants, further indicating that Wak1 does not play a role at the leaf surface (Figs. 4b,c).

Dwak1 plants are unaffected in MAMP-induced ROS production or MAPK activation but have significantly reduced callose deposition
Generation of reactive oxygen species (ROS) and activation of mitogen-activated protein kinase (MAPK) cascades are two typical PTI-associated responses in plants (Nguyen et al., 2010;Zipfel, 2014). To investigate whether Wak1 participates in these responses we performed ROS assays and MAPK activation assays using flg22 or flgII-28. We observed no differences in ROS production in Dwak1 plants compared to wild-type plants when treated with either of these flagellin-derived MAMPs (Fig. 5a,b). Similarly, we observed no difference between wild-type and Dwak1 plants for their ability to activate MAPKs in response to these two MAMPs (Fig. 5c).
Callose deposition is a response associated with later stages of PTI, and one which is regulated Dwak1 plants showed significantly reduced callose deposition one day after vacuum infiltration of Pf 55 (Fig. 5d). These observations therefore indicate that Wak1 functions at a later stage of the PTI response in a flagellin-induced signaling pathway independent of ROS production and MAPK activation.

The increase in Wak1 transcript abundance upon flgII-28 treatment is Fls3-dependent
In tomato, the transcript abundance of Wak1 is low in unchallenged conditions, but is significantly higher after Pst inoculation (Rosli et al., 2013). To gain insight into the transcriptional regulation of Wak1 and Fls3 during the immune response, we used RT-qPCR to measure Wak1 and Fls3 transcript abundance after treatment of wild-type leaves with flgII-28 ( Fig. 6a). The relative abundance of Wak1 or Fls3 transcripts at various time points after syringe infiltrating 1μM flgII-28 was compared to a mock treatment (10 mM MgCl 2 ). Both Wak1 and Fls3 transcript abundance increased significantly at 6 and 8 hours after syringe infiltrating flgII-28 compared to the mock control (Fig. 6a).
To investigate possible co-dependence of Wak1 and Fls3 gene expression, we measured the Wak1 transcript abundance in tomato plants that have mutations in the two Fls2 genes and Fls3 (Dfls2.1/2.2/3; (Roberts et al., 2020)) and the Fls3 transcript abundance in Dwak1 plants after treatment with flgII-28. The abundance of Wak1 transcripts was greatly reduced in the Dfls2.1/2.2/3 plants compared to wild-type plants, whereas Fls3 abundance was not significantly different in Dwak1 or wild-type plants (Fig. 6b,c). These results indicate that Wak1 gene expression is regulated by the Fls3 pathway and its function likely occurs downstream of the mechanism inducing Fls3 gene expression.
Wak1 occurs in a complex with Fls2 and Fls3 independent of flg22, flgII-28 or Bak1 The results above indicate that Wak1 plays a major role in flg22-and flgII-28-induced processes that occur in the apoplast later in the PTI response. We considered the possibility that Wak1 acts in a complex with Fls2 and Fls3 similar to what has been reported for FLS2 and FERONIA in Arabidopsis (Stegmann et al., 2017). We therefore used transient expression of proteins in N.
benthamiana leaves and co-immunoprecipitation (Co-IP) to investigate if Wak1 physically associates with Fls2, Fls3, or the co-receptor Bak1 and, if so, whether the interaction is affected by the presence of flg22 or flgII-28. We observed a weak, but reproducible and specific, interaction of Wak1 with both Fls2 and Fls3 with the interactions occurring independently of flg22, flgII-28, or the presence of Bak1 (Fig. 8a and Fig. S3). As expected, Fls3 and Fls2 each interacted strongly with Bak1 only in the presence of flgII-28 or flg22, respectively. No interaction was observed between Wak1 and Bak1 proteins (Fig. 8b). Additionally, Wak1 did not affect the accumulation of the Fls2, Fls3 or Bak1 proteins or vice versa ( Fig. 8 and Fig. S3).

Discussion
The tomato Wak1 gene was first identified as a FIRE gene (flagellin-induced, repressed by effectors) in the immune response against Pseudomonas syringe (Rosli et al., 2013). When its expression was knocked down by virus-induced gene silencing (VIGS) in N. benthamiana the morphology of the plants was unaffected but their ability to activate PTI was compromised leading to more severe disease symptoms and enhanced growth of a virulent Pst strain (Rosli et al., 2013). The interpretation of these experiments was limited somewhat by the fact that three N.
benthamiana Wak1 homologs were silenced by the tomato Wak1 VIGS construct and, as is typical for VIGS, their transcripts were not completely eliminated (they were reduced by ~50%).
Thus, whether one, or more, of the Wak1 homologs in N. benthamiana play a role in PTI was unclear as was the degree to which a complete knockout of the Wak1 genes might affect PTI or affect plant morphology. Here we have addressed these limitations by developing two Several observations support the hypothesis that Wak1 acts at a later stage of the PTI response in tomato. First, the Dwak1 plants showed no difference from wild-type plants when Pst was sprayinoculated, a method that assays for PTI responses at the leaf surface. The importance of PTI on the leaf surface has been extensively documented in Arabidopsis where a major regulator of this response is the activity of Fls2 in the stomata (Melotto et al., 2006;Melotto et al., 2008;Melotto et al., 2017). Our observations suggest that Wak1 does not act in PTI on the leaf surface but instead exerts its function at a later stage, after Pst enters the apoplastic space as simulated by vacuum infiltration. Second, Dwak1 plants showed no defects in their ability to produce ROS or activate MAPKs in response to flg22 and flgII-28. Both of these responses occur early (within minutes) in leaves that are exposed to MAMPs. Third, The dependence of Wak1-mediated PTI on Fls2 and Fls3 activity could be explained, in part, by the induction of Wak1 gene expression by the Fls2 and Fls3 pathways. However, our observations also raised the possibility that Wak1 resides in a complex that contains Fls2 and Fls3 and its function involves these receptors. We tested this hypothesis and found that Wak1 does co-immunoprecipitate with Fls2 and Fls3 in a MAMP-independent manner and it does not affect accumulation of Fls2/Fls3 proteins. This is reminiscent of the Arabidopsis malectin-like receptor kinase, FERONIA (FER), which was found to weakly associate with Fls2 independent of flg22 treatment and also had no effect on Fls2 accumulation (Stegmann et al., 2017). It is possible that Wak1, like FER, may act as an important cell wall-associated scaffold to regulate immune receptor-complex formation. Tomato Wak1 did not co-immunoprecipitate with Bak1, and Bak1 was not required for the Wak1-Fls2/3 interactions. In contrast, FER weakly associates with Bak1 and the interaction is enhanced upon flg22 treatment, but whether Bak1 is required for the weak association of FER-Fls2 was not investigated (Stegmann et al., 2017).
Based on our observations, we propose a model for the role of Wak1 in PTI (Fig. 9). In this model, Wak1 transcript abundance is greatly increased upon activation of the PRRs Fls2 and Fls3. We hypothesize this gene expression occurs primarily when Pst enters the apoplastic space and that Wak1 is not expressed in leaf surface or stomatal cells. Increased transcript abundance leads to increased Wak1 protein accumulation and subsequent localization to a cell wallassociated protein complex that contains Fls2 and Fls3 and possibly other PRRs. Wak1 might act as a receptor of a damage-associated molecular pattern (DAMP), such as oligogalacturonides.
Binding of such a DAMP might impact the association of Wak1 with the Fls2/Fls3 complex to promote stabilization and accumulation of the PRRs, enhance the interaction of Wak1 with Page 17 of 26 PRRs, or possibly stimulate PRR kinase activity. Whatever the mechanism, the presence of Wak1 in this wall-associated kinase plays a critical role in later stages of PTI including callose deposition and other processes that ultimately inhibit growth of virulent Pst.
This model gives rise to several questions that will need to be addressed in the future. First, why is Wak1 not active in plant cells on the leaf surface, including stomata, but only functions when Pst enters the apoplastic space? This could be due to lack of Wak1 gene expression, protein accumulation, association with the Fls2/Fls3 complex, or kinase activity in leaf surface cells.

Acknowledgments
We thank Robyn Roberts for comments on the manuscript and for sharing seeds of the    Photographs of disease symptoms were taken 4 days (A, B) or 6 days (C) after inoculation.
Bacterial populations were measured at 3 hours (Day 0) and two days (Day 2) after infiltration.
Bars show means ± standard deviation (SD). Different letters indicate significant differences based on a one-way ANOVA followed by Tukey's HSD post hoc test (p < 0.05). ns, no significant difference. Three or four plants for each genotype were tested per experiment. The experiment was performed three times with similar results.    mock treatment). Each treatment included three biological replicates and three technical replicates. SlArd2 (Solyc01g104170) was used as the reference gene for quantification. Bars represent means ± SD. B) RT-qPCR was used to measure transcript abundance of Wak1 6 h after treatment of Δfls2.1/2.2/3 or wild-type leaves with 1 µM flgII-28. Bars represent the mean ± SD.
Two asterisks represent a significant difference using Student's t-test (p <0.01). C) RT-qPCR was used to measure transcript abundance of Fls3 6 h after treatment of Δwak1 (4-1) or wild-type leaves with 1 µM flgII-28. Bars represent the mean ± SD. ns, no significant difference using Student's t-test (p <0.05). plants. Four week-old Δwak1 (4-1), Δfls2.1/2.2/3 or wild-type RG-PtoR plants were vacuum infiltrated with 5 x 10 4 cfu/mL DC3000ΔavrPtoΔavrPtoB. A) Photographs were taken at 3, 4, 5, or 10 days after inoculation. B) Bacterial populations were measured 3 hours (Day 0) and two days after infiltration (Day 2). Bars represent means ± SD. Different letters indicate significant differences based on a one-way ANOVA followed by Tukey's HSD post hoc test (p < 0.05). ns, no significant difference. Three or four plants for each genotype were tested per experiment. This experiment was performed twice with similar results   Wild type Δwak1 (4-1) Leaf conductance (mmole m-2 s-1)

Wild type
Δwak1 (  Transcript abundance of Wak1 and Fls3 genes measured by RT-qPCR at the times shown after treatment with 1 µM flgII-28 compared to a buffer-only control (10 mM MgCl 2 ; mock treatment). Each treatment included three biological replicates and three technical replicates. SlArd2 (Solyc01g104170) was used as the reference gene for quantification. Bars represent means ± SD. B) RT-qPCR was used to measure transcript abundance of Wak1 6 h after treatment of Δfls2.1/2.2/3 or wild-type leaves with 1 µM flgII-28. Bars represent the mean ± SD. Two asterisks represent a significant difference using Student's t-test (p <0.01). C) RT-qPCR was used to measure transcript abundance of Fls3 6 h after treatment of Δwak1 (4-1) or wild-type leaves with 1 µM flgII-28. Bars represent the mean ± SD. ns, no significant difference using Student's t-test (p <0.05). week-old Δwak1 (4-1), Δfls2.1/2.2/3 or wild-type RG-PtoR plants were vacuum infiltrated with 5 x 10 4 cfu/mL DC3000ΔavrPtoΔavrPtoB. A) Photographs were taken at 3, 4, 5, or 10 days after inoculation.

B)
Bacterial populations were measured 3 hours (Day 0) and two days after infiltration (Day 2). Bars represent means ± SD. Different letters indicate significant differences based on a one-way ANOVA followed by Tukey's HSD post hoc test (p < 0.05). ns, no significant difference. Three or four plants for each genotype were tested per experiment. This experiment was performed twice with similar results.   Supplemental information  R-9 WT S-9 -10 R-10 WT/-10 S-10 -1 R-11 WT S-11 -10 Figure S2. Enhanced susceptibility to DC3000ΔΔ co-segregates with the wak1 mutations. A) The Δwak1 lines (4-1 and 4-2) were backcrossed to the wild-type RG-PtoR. Seeds from F1 hybrids were genotyped to confirm the heterozygous genotype (either WT/-1 bp or -WT/-10 bp; WT, wild type). F1 hybrids were tested with DC3000ΔΔ (as in Fig. 2A) and no significant difference was observed in disease symptoms or bacterial populations between F1 and wild-type plants.   Table S1. Primers used in this study.