The Arabidopsis Malectin-Like Leucine-Rich Repeat Receptor-Like Kinase IOS1 Associates with the Pattern Recognition Receptors FLS2 and EFR and Is Critical for Priming of Pattern-Triggered Immunity^,

This work describes the function of the RLK IOS1 in Arabidopsis immunity against bacteria. IOS1 is required for optimal function of the immune receptors FLS2 and EFR and β-aminobutyric acid-induced resistance and priming. IOS1 associates with FLS2, EFR, and BAK1 and controls the ligand-induced FLS2-BAK1 association.

Abstract

Plasma membrane-localized pattern recognition receptors such as FLAGELLIN SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) recognize microbe-associated molecular patterns (MAMPs) to activate the first layer of plant immunity termed pattern-triggered immunity (PTI). A reverse genetics approach with genes responsive to the priming agent β-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hypersusceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants demonstrated defective PTI responses, notably delayed upregulation of PTI marker genes, lower callose deposition, and mitogen-activated protein kinase activities upon bacterial infection or MAMP treatment. Moreover, Arabidopsis lines overexpressing IOS1 were more resistant to P. syringae and demonstrated a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, coimmunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and FLS2 and EFR. IOS1 also associated with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in a ligand-independent manner and positively regulated FLS2/BAK1 complex formation upon MAMP treatment. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a regulatory protein of FLS2- and EFR-mediated signaling that primes PTI activation upon bacterial elicitation.

INTRODUCTION

Plants possess multilayered recognition systems that detect pathogens at various stages of infection and proliferation. Recognition of microbial invasion is essentially based upon the host’s ability to distinguish “self” and “nonself” components. Early microbial pathogen detection is performed by cell surface–localized pattern recognition receptors (PRRs) that sense pathogen-associated molecular patterns or microbe-associated molecular patterns (MAMPs) (Monaghan and Zipfel, 2012). Major examples of MAMPs are the lipopolysaccharides present in the envelope of Gram-negative bacteria, eubacterial flagellin, eubacterial elongation factor Tu (EF-Tu), peptidoglycans from Gram-positive bacteria, methylated bacterial DNA fragments, and fungal cell wall–derived chitins (Girardin et al., 2002; Cook et al., 2004; Boller and Felix, 2009). MAMP recognition promptly triggers the activation of the pattern-triggered immunity (PTI) response (Tsuda and Katagiri, 2010). Early PTI responses, such as calcium influx, production of reactive oxygen species (ROS), and activation of mitogen-activated protein kinases (MAPKs), induce transcriptional reprogramming mediated by plant WRKY transcription factors as well as calmodulin binding proteins (Boller and Felix, 2009; Tena et al., 2011). In addition, Arabidopsis thaliana plants in contact with bacteria close stomata in a MAMP-dependent manner (Melotto et al., 2006; Singh et al., 2012). Callose deposition and PTI marker gene upregulation are usually observed later (Zipfel and Robatzek, 2010). Activation of PTI leads to broad resistance to pathogens (Nicaise et al., 2009; Tsuda and Katagiri, 2010; Zeng et al., 2010; Desclos-Theveniau et al., 2012). Virulent bacterial pathogens inject proteins some of which suppress PTI (Deslandes and Rivas, 2012; Feng and Zhou, 2012). Often, recognition of microbial effectors by plant resistance proteins activates effector-triggered immunity (ETI). ETI is a rapid and robust response, usually associated with a hypersensitive reaction (Maekawa et al., 2011; Gassmann and Bhattacharjee, 2012).

In Arabidopsis, the most extensively studied PRRs are the leucine-rich repeat receptor-like kinases (LRR-RLKs) FLAGELLIN SENSING2 (FLS2) and EF-TU RECEPTOR (EFR). FLS2 and EFR recognize bacterial flagellin (or the derived peptide flg22) and EF-Tu (or the derived peptides elf18/elf26), respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006). Upon ligand binding, FLS2 and EFR rapidly associate with another LRR-RLK, BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR-LIKE KINASE1/SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE3 (BAK1/SERK3), forming a ligand-inducible complex that triggers downstream PTI responses (Chinchilla et al., 2007; Heese et al., 2007; Roux et al., 2011). In addition to associating with FLS2, BAK1 recognizes the C terminus of the FLS2-bound flg22, thus acting as a coreceptor (Sun et al., 2013). BAK1-LIKE1/SERK4 also cooperates with BAK1 to regulate the PRR-mediated signaling pathway (Roux et al., 2011). Recently, the BAK1-INTERACTING RECEPTOR KINASE2 (BIR2) was shown to prevent BAK1 interaction with FLS2 before elicitation. Importantly, BIR2 is released from BAK1 upon MAMP perception, allowing FLS2–BAK1 association and PTI activation (Halter et al., 2014). While BAK1 and other SERKs are the primary regulators downstream of FLS2 and EFR, other early PTI signaling components exist. Notably, BOTRYTIS-INDUCED KINASE1 (BIK1) plays a critical role in mediating early flagellin signaling from the FLS2/BAK1 receptor complex (Lu et al., 2010a; Zhang et al., 2010), and the BRASSINOSTEROID-SIGNALING KINASE1 (BSK1) associates with unstimulated FLS2 (Shi et al., 2013). The DENN (for differentially expressed in normal and neoplastic cells) domain protein STOMATAL CYTOKINESIS-DEFECTIVE1 (SCD1) is also necessary for some FLS2- and EFR-mediated responses and associates in a ligand-independent manner with FLS2 in vivo (Korasick et al., 2010). Furthermore, lectin receptor kinases (LecRKs) such as LecRK-VI.2 and LecRK-V.5 modulate early PTI signaling (Desclos-Theveniau et al., 2012; Singh et al., 2012; Singh and Zimmerli, 2013).

In addition to PTI and ETI, other resistance responses, such as systemic acquired resistance and induced systemic resistance, are activated after pathogen challenges (Durrant and Dong, 2004; Van Wees et al., 2008). Organic and inorganic compounds can also induce systemic resistance in plants. The nonprotein amino acid β-aminobutyric acid (BABA) is a potent inducer of resistance against abiotic stress (Jakab et al., 2005; Zimmerli et al., 2008), nematodes (Oka et al., 1999), insects (Hodge et al., 2005), and microbial pathogens (Jakab et al., 2001; Zimmerli et al., 2001; Cohen, 2002; Ton and Mauch-Mani, 2004; Po-Wen et al., 2013). BABA-induced resistance is associated with faster activation of defense mechanisms upon stress perception, a phenomenon known as priming (Conrath et al., 2006; Návarová et al., 2012). Although the accumulation of defense signaling components before stress exposure (Beckers et al., 2009; Singh et al., 2012) and epigenetic modifications (Jaskiewicz et al., 2011; Luna et al., 2012; Rasmann et al., 2012; Slaughter et al., 2012; Po-Wen et al., 2013) are suggested to be critical for priming, the identities of the signaling components involved in priming are still largely unknown.

In an effort to identify novel critical players in Arabidopsis immunity and priming, we used a reverse genetic approach by testing mutants of genes whose expression is induced by the priming agent BABA (Tsai et al., 2011). Three independent insertion lines in the malectin-like LRR-RLK IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) (Hok et al., 2011) were found to be hypersusceptible to bacterial pathogens. Through loss- and gain-of-function analyses and biochemical approaches, we show that IOS1 is an essential priming modulator of Arabidopsis PTI that associates with the LRR-RLKs FLS2, EFR, and BAK1 in a ligand-independent manner, regulating the complex formation between FLS2 and BAK1.

RESULTS

IOS1 Is Required for Resistance to Hemibiotrophic Bacteria

To identify Arabidopsis genes involved in immunity to bacteria, we followed a reverse genetic analysis of genes upregulated by the priming agent BABA (Tsai et al., 2011). One of these genes is the malectin-like LRR-RLK IOS1 (At1g51800) (Hok et al., 2011). For our analyses, we used ios1-1, a transcriptional knockout Ds transposon insertion line in the Landsberg erecta-0 (Ler-0) background (GT_5_22250) recently isolated (Hok et al., 2011), and ios1-2 (Salk_137388) and ios1-3 (SAIL_343_B11), two independent T-DNA insertion lines in the Columbia-0 (Col-0) background (Supplemental Figure 1). To test if IOS1 is required for antibacterial immunity, the three insertion lines were dip-inoculated with the virulent hemibiotrophic bacterium Pseudomonas syringae pv tomato DC3000 or Pseudomonas syringae pv maculicola ES4326. At 3 d after inoculation (DAI), ios1-1, ios1-2, and ios1-3 developed stronger symptoms than wild-type plants, as illustrated by increased chlorosis and necrosis formation (Figure 1A). This phenotype correlated with significantly higher bacterial titers (Figure 1B). Typically, the susceptibility phenotype of ios1-2 to P. s. tomato DC3000 is similar to that of the mutant bak1-5 (Supplemental Figure 2). We also evaluated the susceptibility of ios1 mutants to the P. s. tomato DC3000 hrcC⁻ mutant, a strain defective in delivering type III effectors that cannot repress the PTI response and consequently is mostly nonvirulent on Arabidopsis (Brooks et al., 2004). All three ios1 mutants tested allowed more growth of P. s. tomato DC3000 hrcC⁻ than wild-type plants upon syringe infiltration (Supplemental Figure 3), suggesting a defective PTI response in ios1 mutants.

Figure 1.

A Critical Role for IOS1 in Arabidopsis Resistance to Hemibiotrophic Bacteria.

(A) Disease symptoms in ios1 mutants. Five-week-old Arabidopsis plants were dip-inoculated in a bacterial solution of 10⁶ cfu/mL P. s. tomato DC3000 or 5 × 10⁵ cfu/mL P. s. maculicola ES4326. Symptoms were evaluated at 3 DAI. This experiment was repeated at least three times with similar results.

(B) Bacterial growth in ios1 mutants. Five-week-old Arabidopsis plants were dip-inoculated as in (A), and bacterial titers were evaluated at 3 DAI. Data represent four independent biological replicates each with three technical repeats (n = 12). Asterisks indicate significant differences from the wild-type control based on a t test (P < 0.01).

(C) Growth of P. s. tomato DC3000 in lines overexpressing IOS1. Bacterial titers in 5-week-old Col-0 and IOS1 overexpression lines OE1, OE2, and OE3 were determined at 3 DAI with 10⁶ cfu/mL P. s. tomato DC3000. Values are means ± sd of three independent biological replicates each with three technical repeats (n = 9). Asterisks indicate significant differences from the Col-0 wild type based on a t test (*P < 0.05, **P < 0.01).

(D) Stomatal innate immunity in lines overexpressing IOS1. Stomatal apertures in leaf epidermal peels from 5-week-old Col-0 and IOS1 overexpression lines OE1, OE2, and OE3 were analyzed after 1.5 or 3 h of exposure to MgSO₄ (Mock) or 10⁸ cfu/mL P. s. tomato DC3000. Values are means ± se of three independent replicates each consisting of at least 60 technical repeats (n > 180 stomata). Asterisks indicate significant differences from the respective mock controls based on a t test analysis (P < 0.001).

To test if IOS1 is also required for immunity to pathogens other than bacteria, the susceptibility of ios1 mutants to the necrotrophic fungal pathogen Botrytis cinerea was evaluated by droplet inoculation. Mutants ios1-1, ios1-2, and ios1-3 were as susceptible as wild-type plants to B. cinerea (Supplemental Figure 4), indicating that IOS1 is critical for immunity to virulent hemibiotrophic bacteria but not to necrotrophic fungi such as B. cinerea.

The role of IOS1 in antibacterial immunity was further evaluated by analyzing the susceptibility to P. s. tomato DC3000 of transgenic Arabidopsis lines overexpressing IOS1 at high (OE1 and OE3) or moderate (OE2) levels (Supplemental Figure 5). All the IOS1-OE lines were significantly less susceptible to P. s. tomato DC3000, with the increased resistance phenotype correlating with IOS1 expression levels (Figure 1C). Notably, although ios1 mutants did not show any defect in stomatal innate immunity (Supplemental Figure 6), overexpression of IOS1 inhibited the bacteria-mediated reopening of stomata (Figure 1D). Together, these data are consistent with a positive role of IOS1 in antibacterial immunity.

IOS1 Is Critical for Late PTI Responses

To analyze whether IOS1 is involved in PTI responses, we first monitored IOS1 mRNA expression levels by quantitative RT-PCR (qRT-PCR) after treatment of seedlings with 1 μM flg22 or elf26. Both MAMPs induced a strong upregulation of IOS1 transcript accumulation at 1 h after treatment (Supplemental Figure 7). To evaluate the role of IOS1 in late PTI responses, we measured callose deposition in ios1 mutants after infiltration with P. s. tomato DC3000 hrcC⁻ or the MAMP flg22 or elf26. Aniline blue staining and image analysis indicated lower levels of callose deposition in ios1-1 and ios1-2 than in wild-type leaves (Figure 2A). These results indicate that IOS1 is critical for PTI-induced callose deposition. To further evaluate late PTI responses, we monitored the expression levels of the PTI marker genes FRK1, CYP81F2, and WRKY53 (Xiao et al., 2007; Boudsocq et al., 2010) in time-course experiments upon infiltration of P. s. tomato DC3000 hrcC⁻, flg22, or elf26. At early time points, ios1-1 and ios1-2 demonstrated a delayed upregulation of FRK1, CYP81F2, and WRKY53 upon bacteria or MAMP treatment (Figure 2B; Supplemental Figure 8A). However, at later time points, the expression of these marker genes in ios1-1 and ios1-2 mutants was essentially at wild-type levels (Figure 2B; Supplemental Figure 8A). These data suggest that upregulation of PTI marker genes is delayed in ios1 mutants upon MAMP perception.

Figure 2.

Altered Late PTI Responses in ios1 Mutants and IOS1-OE Lines.

(A) and (C) Callose deposition. Leaves of 5-week-old ios1-1 and ios1-2 (A) were syringe-infiltrated with 10⁸ cfu/mL P. s. tomato DC3000 hrcC⁻ or with 1 μM flg22 or elf26, and samples were collected 9 h (P. s. tomato DC3000 hrcC⁻ and flg22) or 24 h (elf26) later for aniline blue staining. For IOS1-OE lines (C), leaves were syringe-infiltrated with 10⁸ cfu/mL P. s. tomato DC3000 hrcC⁻, 1 μM flg22, or 5 μM elf26, and samples were collected 6 h (P. s. tomato DC3000 hrcC⁻ and flg22) or 21 h (elf26) later for aniline blue staining. Mock samples were infiltrated with MgSO₄. Numbers under the panels are averages ± sd of the number of callose deposits per square millimeter from three independent biological replicates each consisting of nine technical repeats (n = 27). Bars = 200 μm.

(B) and (D) PTI-responsive gene upregulation. Relative FRK1 expression levels were evaluated at 1, 2, and 3 h after inoculation (hpi) with 10⁸ cfu/mL P. s. tomato DC3000 hrcC⁻ or 15, 30, and 60 min after treatment (mpt) with 1 μM flg22 or elf26 in ios1-1 and ios1-2 mutants (B) or at 30, 60, and 90 min after P. s. tomato DC3000 hrcC⁻ inoculation (mpi) or 10, 20, and 30 min after treatment with 10 nM flg22 or elf26 in IOS1-OE lines (D). EF-1 and UBQ10 were used for normalization. Relative gene expression levels were compared with the wild type (Ler-0 or Col-0) at time 0 (defined value of 1) by qRT-PCR analyses. The values are means ± sd of three independent biological replicates each consisting of three technical repeats (n = 9). Asterisks indicate significant differences from the wild-type controls based on a t test (P < 0.01).

Furthermore, we analyzed late PTI responses in the IOS1-OE lines. Interestingly, these lines did not exhibit constitutive callose deposition, while more callose deposits were observed in OE1 and OE3 upon elicitation with P. s. tomato DC3000 hrcC⁻ or the MAMP flg22 or elf26 (Figure 2C). Similarly, constitutive upregulation of PTI marker genes was not observed in IOS1-overexpression lines, but FRK1, CYP81F2, and WRKY53 expression levels were potentiated in the OE1 and OE3 lines upon P. s. tomato DC3000 hrcC⁻ inoculation or flg22 or elf26 treatment (Figure 2D; Supplemental Figure 8B). These data suggest that high overexpression levels of IOS1 prime late PTI responses. Collectively, these data indicate that IOS1 is a positive regulator of several late PTI responses.

IOS1 Modulates Several Early PTI Responses

To test if IOS1 is required for early PTI events, we analyzed ROS production in response to 10 nM flg22 or elf26 for 30 min in wild-type, ios1-1, ios1-2, and IOS1-OE leaves. Both mutants and overexpression lines displayed wild-type levels of ROS production, while MAMP-mediated ROS production was strongly reduced in the negative control bak1-4 (Figures 3A and 3B; Supplemental Figures 9A and 9B). Treatment with MAMPs rapidly activates the Arabidopsis MAPKs MPK3 and MPK6 (Nühse et al., 2000). Notably, both ios1-1 and ios1-2 mutants demonstrated weaker activation of MPK3 and MPK6 than the wild type following treatment with flg22 or elf26 (Figure 3C; Supplemental Figure 9C). On the other hand, MPK3 and MPK6 activation was stronger than the wild type in the OE1, OE2, and OE3 transgenic lines (Figure 3D; Supplemental Figure 9D). Together, these results suggest that IOS1 is required for full MAPK activation but not for the ROS burst after flg22 or elf26 perception. This observation is consistent with these responses being uncoupled (Segonzac et al., 2011; Xu et al., 2014a).

Figure 3.

Early PTI Responses.

(A) ROS production in ios1 mutants. The responsiveness of 5-week-old Ler-0 and Col-0 wild-type controls and respective mutants ios1-1 and ios1-2 to 10 nM flg22 was determined. bak1-4 was used as a negative control. Production of ROS in Arabidopsis leaf discs is expressed as relative light units (RLU) for a period of 30 min after elicitation. Values are means ± se of three independent biological replicates each with six technical repeats (n = 18). Differences between ios1 mutants and the wild type were not statistically significant based on a t test (P < 0.01).

(B) ROS production in IOS1-OE lines. The responsiveness of 5-week-old overexpression lines OE1, OE2, and OE3 and the Col-0 wild-type control to 10 nM flg22 was determined. Production of ROS in Arabidopsis leaf discs is expressed as relative light units for a period of 30 min after elicitation. Values are means ± se of three independent biological replicates each with six technical repeats (n = 18). Differences between overexpression lines and the wild type were not statistically significant based on a t test (P < 0.01).

(C) MAPK activation in ios1 mutants. Nine leaves from three 5-week-old Ler-0 and ios1-1 plants or Col-0 and ios1-2 plants were syringe-infiltrated with 100 nM flg22 for 5 min. Immunoblot analysis using phospho-p44/42 MAPK antibody is shown in the top panels. Lines indicate the positions of MPK3 and MPK6. FastBlue staining was used to estimate equal loading in each lane (bottom panels). This experiment is one of two independent replicates.

(D) MAPK activation in IOS1-OE lines. Nine leaves from three 5-week-old Col-0 and IOS1 overexpression lines OE1, OE2, and OE3 were syringe-infiltrated with 1 nM flg22 for 5 min. Immunoblot analysis using phospho-p44/42 MAPK antibody is shown in the top panel. Lines indicate the positions of MPK3 and MPK6. FastBlue staining was used to estimate equal loading in each lane (bottom panel). This experiment is one of two independent replicates.

IOS1 Localizes to the Plasma Membrane

IOS1 is a predicted transmembrane receptor-like kinase (Hok et al., 2011). We analyzed IOS1 subcellular localization by transiently expressing the IOS1-GFP (for green fluorescent protein) fusion protein driven by the cauliflower mosaic virus 35S promoter in Arabidopsis mesophyll protoplasts. The fluorescence signal was mainly confined to the cell surface with a pattern similar to that of the plasma membrane marker pm-rk CD3-1007 (Nelson et al., 2007), while the control protoplasts expressing GFP alone showed a nuclear/cytoplasmic localization (Figure 4A). IOS1 subcellular localization was also analyzed in root cells stably expressing IOS1-GFP. Consistent with the protoplast data, the IOS1-GFP signal was confined to the cell surface (Figure 4B). Taken together, these data suggest that, similar to the PRRs FLS2 and EFR (Robatzek et al., 2006; Häweker et al., 2010), IOS1 is localized at the plasma membrane.

Figure 4.

IOS1 Localizes to the Plasma Membrane.

(A) Subcellular localization of the IOS1-GFP fusion protein in Arabidopsis mesophyll protoplasts. IOS1-GFP expression was driven by the cauliflower mosaic virus 35S promoter and transiently expressed in Arabidopsis mesophyll protoplasts. Shown are bright-field images ([a] and [f]), images of the GFP fluorescence (green) only ([b] and [g]), images of the chlorophyll autofluorescence (red) only ([c] and [h]), plasma membrane marker (pm-rk CD3-1007)-mCherry fluorescence localization (d), and combined images ([e] and [i]). Bars = 10 μm.

(B) Subcellular localization of IOS1-GFP in mature roots of transgenic Arabidopsis. Confocal microscopy results of 35S-IOS1-GFP ([a] and [b]) and 35S-GFP ([c] and [d]) transgenic root cell expression are shown in bright-field images ([a] and [c]) and GFP expression only ([b] and [d]). Arrows indicate the nucleus. Bars = 100 μm.

IOS1 Associates with FLS2, EFR, and BAK1 in a Ligand-Independent Manner

IOS1 acts upstream of MAPK in flg22- and elf26-triggered PTI signaling cascades. We thus evaluated whether IOS1 associates with PRRs such as FLS2 or EFR. We first used pull-down analysis to show that a Trx-6xHis-tagged IOS1 kinase domain (KD) interacted with MBP-tagged FLS2 and EFR in vitro (Figure 5A). Next, interactions were evaluated by bimolecular fluorescence complementation (BiFC) assays (Walter et al., 2004) in Arabidopsis protoplasts. To test whether our experimental conditions were appropriate, we first analyzed the interactions between BAK1 and FLS2 or EFR that occur only upon elicitation (Chinchilla et al., 2007; Heese et al., 2007; Roux et al., 2011). As expected, yellow fluorescent protein (YFP) signal was clearly observed after flg22 or elf26 treatment (Figure 5B; Supplemental Figure 10). YFP fluorescence was detected before and after elicitation with flg22 or elf26 when testing IOS1 interaction with FLS2 or EFR, respectively (Figure 5B; Supplemental Figure 10). Similarly, IOS1 interacted with BAK1 in a ligand-independent manner (Figure 5B; Supplemental Figure 10). IOS1 is not necessary for resistance to the fungal pathogen B. cinerea (Supplemental Figure 4), which produces the MAMP chitin. We thus used the LysM domain receptor-like kinase CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) that recognizes chitin (Miya et al., 2007) as a negative control. CERK1 is known to dimerize (Liu et al., 2012), and YFP fluorescence was indeed observed when Arabidopsis protoplasts were transfected with CERK1-YFP^N and CERK1-YFP^C, indicating that both constructs were functional. Importantly, no YFP fluorescence at the plasma membrane was observed when testing IOS1 interaction with CERK1, even after elicitation with chitin (Figure 5C). Together, these data suggest that IOS1 interacts at the plasma membrane with the PRRs FLS2 and EFR and the coreceptor BAK1 in a ligand-independent manner.

Figure 5.

Pull-Down and BiFC Analyses of IOS1 Interaction with PRRs.

(A) In vitro MBP pull-down assay of IOS1 interaction with FLS2 and EFR. E. coli–expressed MBP (negative control), MBP-FLS2KD, or MBP-EFRKD was incubated with Trx-6xHis-IOS1KD and pulled down with amylose resin beads. Input and bead-bound proteins were analyzed by immunoblotting with specific antibodies. The experiments were repeated three times with similar results.

(B) BiFC analyses of IOS1 interactions with FLS2 and BAK1. Arabidopsis protoplasts were cotransfected with FLS2-YFP^N + BAK1-YFP^C, IOS1-YFP^N + FLS2-YFP^C, and IOS1-YFP^N + BAK1-YFP^C and treated with (+) or without (−) 1 μM flg22 for 10 min. The bright-field, YFP fluorescence (yellow), chlorophyll autofluorescence (red), and combined images were visualized with a confocal microscope 16 h after transfection. Images are representative of multiple protoplasts. The experiment was repeated twice with similar results. Bars = 10 μm.

(C) BiFC of CERK1 and IOS1 interaction. Arabidopsis protoplasts were cotransfected with CERK1-YFP^N + CERK1-YFP^C or IOS1-YFP^N + CERK1-YFP^C and treated with (+) or without (−) 0.1 mg/mL chitin for 10 min. The bright-field, YFP fluorescence (yellow), chlorophyll autofluorescence (red), and combined images were visualized with a confocal microscope 16 h after transfection. Images are representative of multiple protoplasts. The experiment was repeated twice with similar results. Bars = 10 μm.

To test whether IOS1 associates with FLS2 in vivo, we transiently coexpressed FLS2-GFP with HA₃ epitope-tagged IOS1 in Nicotiana benthamiana. Equal amounts of FLS2 were pulled down with GFP-Trap beads and analyzed for the presence of IOS1-HA₃ using anti-HA immunoblotting. IOS1 could be detected in mock- and flg22-treated samples (Figure 6A). As a control, FLS2-GFP was coexpressed with HA₃ epitope-tagged BAK1. As expected (Chinchilla et al., 2007; Heese et al., 2007; Roux et al., 2011), BAK1 could be detected mostly in the FLS2 immunoprecipitate after flg22 elicitation (Figure 6A). By contrast, BAK1 was not detected in the IOS1 immunoprecipitate even after elicitation with flg22. These data suggest that IOS1 associates with FLS2 in a ligand-independent manner but not with BAK1 in N. benthamiana. Similarly, we analyzed the possible association of IOS1with EFR before and after elicitation with elf26. For that purpose, EFR-GFP was transiently coexpressed with IOS1-HA₃ in N. benthamiana, and EFR was pulled down with GFP-Trap beads. IOS1 could also be detected in the EFR immunoprecipitate from mock- and elf26-treated plants. Similar to the experiment with FLS2, the control BAK1 was only detected in the EFR immunoprecipitate after elf26 treatment, and no BAK1 could be detected in the IOS1 immunoprecipitate even after elicitation with elf26 (Figure 6B). To ensure that IOS1 does not bind aspecifically to anti-GFP magnetic beads, we expressed IOS1-HA₃ alone. No signal was observed in the negative control that expressed IOS1-HA₃ only (Supplemental Figure 11). As an additional negative control, we tested the association of IOS1 with CERK1 by pulling down an equal amount of CERK1 with GFP-Trap beads and by analyzing IOS1-HA₃ presence using anti-HA immunoblotting. IOS1 could not be detected even after elicitation with chitin (Figure 6C). By contrast, dimerization of CERK1 was observed, indicating that the CERK1 constructs used were functional (Figure 6C). These observations suggest that IOS1 associates with both FLS2 and EFR in a ligand-independent manner in N. benthamiana. Of note, IOS1 homodimerized independently of flg22 treatment (Supplemental Figure 12), as reported previously for FLS2 (Sun et al., 2012; Supplemental Figure 12).

Figure 6.

IOS1 Associates with Unstimulated and Stimulated FLS2, EFR, and BAK1.

(A) Coimmunoprecipitation of IOS1, FLS2, and BAK1 proteins. N. benthamiana leaves expressing FLS2-GFP and BAK1-HA₃ (lanes 1 and 2), FLS2-GFP and IOS1-HA₃ (lanes 3 and 4), or IOS1-GFP and BAK1-HA₃ (lanes 5 and 6) constructs were treated (+) or not (−) with 100 nM flg22 for 5 min. Total proteins (input) were subjected to immunoprecipitation (IP) with GFP-Trap beads followed by immunoblot analysis with anti-HA antibodies to detect IOS1-HA₃ and BAK1-HA₃. Anti-GFP antibodies detect FLS2-GFP and IOS1-GFP. These experiments were repeated three times with similar results.

(B) Coimmunoprecipitation of IOS1, EFR, and BAK1 proteins. N. benthamiana leaves expressing EFR-GFP and BAK1-HA₃ (lanes 1 and 2), EFR-GFP and IOS1-HA₃ (lanes 3 and 4), or IOS1-GFP and BAK1-HA₃ (lanes 5 and 6) constructs were treated (+) or not (−) with 100 nM elf26 for 5 min. Total proteins (input) were subjected to immunoprecipitation with GFP-Trap beads followed by immunoblot analysis with anti-HA antibodies to detect IOS1-HA₃ and BAK1-HA₃. Anti-GFP antibodies detect EFR-GFP and IOS1-GFP. These experiments were repeated three times with similar results.

(C) IOS1 does not associate with CERK1. Coimmunoprecipitation of CERK1 with IOS1 protein in N. benthamiana treated with (+) or without (−) 0.1 mg/mL chitin for 5 min is shown. Total proteins (input) were subjected to immunoprecipitation with GFP-Trap beads followed by immunoblot analysis with anti-HA antibodies to detect CERK1-HA₃ and IOS1-HA₃. Anti-GFP antibodies detect CERK1-GFP. This experiment was repeated two times with similar results.

(D) Coimmunoprecipitation of FLS2, BAK1, and IOS1 proteins in Arabidopsis. Transgenic Arabidopsis seedlings overexpressing IOS1-GFP (OE3) were treated (+) or not (−) with 100 nM flg22 for 10 min. Total proteins (input) were subjected to immunoprecipitation with anti-GFP magnetic beads followed by immunoblot analysis with anti-FLS2 antibodies, anti-BAK1 antibodies, or anti-GFP antibodies to detect FLS2, BAK1, and IOS1-GFP. Untransformed Col-0 Arabidopsis tissue was used as a control to show that FLS2 and BAK1 do not adhere nonspecifically to anti-GFP magnetic beads (lane 1). LTI6B-GFP, a known plasma membrane protein, was used as a control to illustrate that FLS2 and BAK1 do not associate with GFP at the plasma membrane (lane 2). This experiment is one of two independent replicates.

To test whether IOS1-GFP associates with FLS2 in Arabidopsis as well, we performed coimmunoprecipitation experiments using transgenic lines overexpressing IOS1-GFP. IOS1-GFP was immunoprecipitated with anti-GFP magnetic beads and analyzed for the presence of endogenous BAK1 and FLS2 using anti-BAK1 and anti-FLS2 immunoblotting. As negative controls, anti-GFP magnetic beads were incubated with protein extracts of untransformed Col-0 and the transgenic plants expressing LTI6B (for low temperature- and salt-responsive protein 6B) fused to GFP, which is known to localize at the plasma membrane (Cutler et al., 2000). Signals for FLS2 and BAK1 upon LTI6B-GFP immunoprecipitation were largely weaker than those observed upon IOS1-GFP, suggesting that FLS2 and BAK1 do not aspecifically bind to anti-GFP magnetic beads, nor do they interact with GFP itself at the plasma membrane (Figure 6D). By contrast, we could detect a clear association of IOS1-GFP with native FLS2 and BAK1 (Figure 6D). Treatment with flg22 did not affect significantly or reproducibly the associations of IOS-GFP with FLS2 and BAK1 (Figure 6D). Notably, an association between IOS-GFP and BAK1-HA could not be observed upon transient expression in N. benthamiana (Figures 6A and 6B). The presence of the HA tag at the C terminus of BAK1 has been shown previously to affect its function in PTI signaling (Ntoukakis et al., 2011) and therefore may affect the association of IOS1-GFP with BAK1, otherwise observable in native conditions.

Moreover, we found IOS1 as part of the in vivo EFR complex in an unbiased manner while searching for EFR-associated proteins in planta by proteomics analysis (Roux et al., 2011). In these experiments, anti-GFP immunoprecipitates were prepared from untreated and elf18-treated transgenic efr-1/EFRp:EFR-eGFP seedlings, as well as from untreated efr-1 null mutant or Col-0 seedlings, in order to reveal proteins that nonspecifically bind to GFP beads. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis identified eight different peptides matching IOS1 in the EFR-eGFP immunoprecipitates but none in the negative controls (Supplemental Table 1). The IOS1 peptides were found in both untreated and elf18-treated samples, corroborating the fact that IOS1 associates with the PRRs FLS2 and EFR in a ligand-independent manner in vivo.

IOS1 Is Required for Optimal flg22-Induced FLS2-BAK1 Association

To test whether associations between IOS1 and both FLS2 and BAK1 impact other biochemical events within the FLS2 complex, we analyzed the ligand-induced FLS2–BAK1 association (Chinchilla et al., 2007; Heese et al., 2007). Toward this goal, BAK1 was first immunoprecipitated from ios1-2 plants treated or not with 100 nM flg22 and associated FLS2 was revealed by anti-FLS2 immunoblotting. After flg22 treatment, the mutant ios1-2 displayed significantly less FLS2 coimmunoprecipitated with BAK1 than the wild-type control (Figures 7A and 7B). By contrast, a significant increase in coimmunoprecipitated FLS2 was observed in Arabidopsis overexpressing IOS1 treated with two different concentrations of flg22 (Figures 7C and 7D). These data show that the active kinase IOS1 (Supplemental Figure 13) positively regulates the association of BAK1 with FLS2. However, flg22-mediated phosphorylation of BIK1, which is a direct substrate of FLS2 (Lu et al., 2010a; Zhang et al., 2010), was not affected in ios1-2 and the OE3 line (Figures 8A to 8D). Together, these results indicate that IOS1 modulates the FLS2–BAK1 association upon elicitation but is not critical for BIK1 phosphorylation. We then evaluated IOS1 dependency to BAK1 and BIK1 by analyzing callose deposition in lines overexpressing IOS1 in the bak1-5 and bik1 mutant backgrounds. bak1-5 and bik1 mutants are largely defective in flg22-mediated callose deposition (Figure 9A; Zhang et al., 2010). While overexpression of IOS1 strongly primed callose deposition in the Col-0 wild-type control, the bak1-5 mutation completely abolished IOS1-mediated priming of callose deposition (Figure 9A). However, lines overexpressing IOS1 in the bik1 mutant background still displayed a large increase in callose deposits after flg22 treatment (Figure 9A). To further evaluate whether IOS1 function is linked with BAK1, we analyzed MPK3/6 activities upon elicitation with the MAMP chitin or with the damage-associated molecular pattern AtPep1. Fungal chitin recognition is mediated by LysM domain receptor-like kinases (Miya et al., 2007; Wan et al., 2008, 2012), and BAK1 is not required for chitin perception and signaling (Shan et al., 2008; Kemmerling et al., 2011; Ranf et al., 2011). By contrast, AtPep1 recognition through PEP1 RECEPTOR1 (PEPR1) is BAK1 dependent (Krol et al., 2010; Schulze et al., 2010; Ranf et al., 2011; Roux et al., 2011). As expected, MPK3/6 activities were not affected after chitin treatment but were reduced after AtPep1 elicitation in bak1-4 (Figure 9B). The mutant ios1-2 responded similarly to bak1-4 (Figure 9B), suggesting that IOS1 plays a role in those PRR complexes that recruit BAK1 upon elicitation, further pointing to a possible functionality of IOS1 together with BAK1. Collectively, these data suggest that IOS1 functions in a BAK1-dependent but BIK1-independent manner.

Figure 7.

IOS1 Regulates the Ligand-Induced FLS2–BAK1 Association.

(A) and (B) The ligand-dependent association of FLS2 to BAK1 is reduced in the ios1-2 mutant. Col-0 or ios1-2 seedlings were treated (+) or not (−) with 100 nM flg22 for 10 min. Total proteins (input) were subjected to immunoprecipitation (IP) with anti-BAK1 antibodies and IgG beads followed by immunoblot analysis using anti-FLS2 and anti-BAK1 antibodies. For (A), Coomassie blue (CBB) was used to estimate equal loading (bottom panel). The experiment shown in (A) is one of three independent replicates pooled together in (B).

(C) and (D) The ligand-dependent association of FLS2 to BAK1 is augmented in the IOS1-OE3 line. Col-0 or OE3 seedlings were treated with MgSO₄ (0) or 10 or 50 nM flg22 for 10 min. Total proteins (input) were subjected to immunoprecipitation with anti-BAK1 antibodies and IgG beads followed by immunoblot analysis using anti-FLS2 and anti-BAK1 antibodies. The experiment shown in (C) is one of three independent replicates pooled together in (D). For (B) and (D), signals were evaluated with ImageJ software. Values are means ± sd of three independent biological replicates (n = 3). Different letters denote significant differences based on ANOVA (P < 0.05).

Figure 8.

IOS1 Does Not Modulate flg22-Mediated BIK1 Phosphorylation.

Immunoblot analysis of BIK1 phosphorylation revealed by gel mobility shift. Nonphosphorylated (BIK1) and phosphorylated (pBIK1) BIK1 signals are indicated. Protoplasts from Col-0 leaves and ios1-2 ([A] and [C]) or OE3 ([B] and [D]) were treated 4 h after transfection using 0.75 μM flg22 for 3.5, 7, and 10 min. The reaction was stopped by immersion in liquid nitrogen following concentration by low-speed centrifugation. The experiments were repeated at least five times with similar results. For (C) and (D), phosphorylated over nonphosphorylated BIK1 fractions were calculated by measuring digital signals with ImageJ software. Values are means ± sd of five independent biological replicates (n = 5). Differences were not statistically significant based on ANOVA (P < 0.05).

Figure 9.

IOS1 Function Is BAK1 Dependent but BIK1 Independent.

(A) Callose deposition upon elicitation with flg22. Leaves from the Col-0 wild type, the IOS1-OE3 line (OE), bak1-5 and bik1 mutants, and IOS1 overexpression in the bak1-5 and bik1 backgrounds were syringe-infiltrated with 100 nM flg22, and samples were collected 9 h later for aniline blue staining. Values are averages ± sd of callose deposits per square millimeter from two independent biological replicates each consisting of nine technical repeats (n = 18). Different letters denote significant differences among different lines based by ANOVA (P < 0.01).

(B) MAPK activation upon elicitation with chitin or AtPep1. Nine leaves from three 5-week-old Col-0 wild-type, bak1-4, and ios1-2 plants were syringe-infiltrated with 0.1 mg/mL chitin or 10 nM AtPep1 for 5 min. Immunoblot analysis using phospho-p44/42 MAPK antibody is shown in the top panel. Lines indicate the positions of MPK3 and MPK6. FastBlue staining was used to estimate equal loading in each lane (bottom panel). This experiment is one of three independent replicates.

[See online article for color version of this figure.]

IOS1 Is Necessary for BABA-Induced Resistance and Priming

Since overexpression of the BABA-responsive IOS1 primes Arabidopsis PTI (Figures 2C, 2D, and 3D), we tested whether IOS1 is required for induced resistance to P. s. tomato DC3000 and P. s. maculicola ES4326 triggered by the priming agent BABA (Zimmerli et al., 2000; Tsai et al., 2011). While BABA treatments protected both Col-0 and Ler-0 wild types against P. s. tomato DC3000 and P. s. maculicola ES4326 infection, ios1-1 and ios1-2 mutants demonstrated a defective BABA-induced resistance toward these hemibiotrophic bacteria (Figure 10A). BABA is known to prime the PTI response in Arabidopsis (Singh et al., 2012; Po-Wen et al., 2013). We thus tested the role of IOS1 in BABA-induced priming of PTI responses. Notably, the priming effect of BABA on flg22-induced callose deposition and FRK1 expression was largely abolished in ios1-1 and ios1-2 in comparison with the wild type (Figures 10B and 10C). IOS1 positively modulates the flg22-mediated FLS2–BAK1 association (Figure 7), and BABA treatment upregulates IOS1 expression (Tsai et al., 2011). We thus asked whether BABA affects the FLS2–BAK1 association upon flg22 elicitation. No clear increase in the FLS2–BAK1 association upon flg22 treatment was observed in BABA-treated Col-0 plants (Supplemental Figure 14).

Figure 10.

BABA Action Is Defective in ios1 Mutants.

(A) BABA-induced resistance. Bacterial titers in 5-week-old Ler-0, ios1-1, Col-0, and ios1-2 were determined at 3 DAI with 10⁶ cfu/mL P. s. tomato DC3000 or 5 × 10⁵ cfu/mL P. s. maculicola ES4326. Two days before bacterial inoculation, plants were soil-drenched with water as a control or 225 μM BABA. Values are means ± sd of three independent biological replicates each with three technical repeats (n = 9). Asterisks indicate significant differences from the respective water-treated control based on a t test (P < 0.01).

(B) BABA priming of PTI-mediated callose deposition. Leaves of water- or BABA-pretreated (225 μM) Ler-0 and ios1-1 or Col-0 and ios1-2 plants were syringe-infiltrated with 1 μM flg22, and samples were collected 6 h later for aniline blue staining. Values are averages ± sd from three independent biological replicates each consisting of nine technical repeats (n = 27). Asterisks indicate significant differences from the respective water + mock–treated wild-type control based on a t test (P < 0.01).

(C) BABA priming of PTI-mediated FRK1 expression. Water- or BABA-pretreated (225 μM) 10-d-old Ler-0 and ios1-1 or Col-0 and ios1-2 seedlings were treated with MgSO₄ (Mock) or 1 μM flg22, and FRK1 expression levels were analyzed 30 min later by qRT-PCR. EF-1 and UBQ10 were used for normalization. Relative gene expression levels were compared with the respective water + mock–treated wild type (defined value of 1). Values are means ± sd of three independent biological replicates each with three technical repeats (n = 9). Asterisks indicate significant differences from the respective water + mock–treated wild-type control based on a t test (P < 0.01).

(D) BABA inhibition of bacteria-mediated stomatal reopening. Stomatal apertures in epidermal peels from water-treated (W) or BABA-treated (225 μM) (B) Ler-0 and ios1-1 or Col-0 and ios1-2 plants were analyzed after 1.5 and 6 h of exposure to MgSO₄ (Mock) or 10⁸ cfu/mL P. s. tomato DC3000. Results are shown as means ± se of three independent replicates each consisting of at least 60 technical repeats (n > 180 stomata). Asterisks indicate significant differences from the respective mock controls based on a t test analysis (P < 0.001).

Since BABA inhibits bacteria-mediated stomatal reopening (Tsai et al., 2011), we tested whether IOS1 is involved in this phenomenon. While BABA inhibited bacteria-mediated stomatal reopenings in the Col-0 and Ler-0 wild types, it did not in ios1-1 and ios1-2 mutants (Figure 10D). Taken together, these data suggest a positive role for IOS1 in BABA-induced resistance and BABA-mediated priming of PTI, including strengthening of stomatal innate immunity.

DISCUSSION

PRRs are critical to elicit PTI responses and to restrict pathogen ingress (Boller and Felix, 2009; Nicaise et al., 2009; Zhang and Zhou, 2010). To date, all known PRRs are modular transmembrane proteins containing ligand binding ectodomains that function as part of multiprotein complexes (Monaghan and Zipfel, 2012). In this work, we analyzed the role of the Arabidopsis malectin-like LRR-RLK IOS1 in innate immunity and priming with genetic and biochemical approaches. The results support the following conclusions.

IOS1 Is Necessary for Full Activation of Antibacteria PTI in Arabidopsis

Our reverse genetic approach identified three independent insertion mutant lines in IOS1 with hypersusceptibility to virulent hemibiotrophic P. s. tomato DC3000 and P. s. maculicola ES4326 bacteria but with wild-type sensitivity to the necrotrophic fungal pathogen B. cinerea. These observations suggest that IOS1 is critical for resistance to bacteria. A similar behavior is found in the lecrk-VI.2-1 mutant, which is highly susceptible to bacteria but shows wild-type susceptibility to the fungal pathogen B. cinerea (Singh et al., 2012, 2013). Accordingly, microarray profiles of IOS1 expression revealed upregulation by MAMPs and nonpathogenic microbial pathogens such as P. s. tomato DC3000 hrcC⁻, but no dramatic increase of expression is observed after B. cinerea inoculation (Postel et al., 2010). In this regard, we confirmed IOS1 responsiveness to the bacterial MAMPs flg22 and elf26 (Supplemental Figure 7). Together, this evidence strongly suggests that IOS1 is critical for Arabidopsis resistance to hemibiotrophic bacteria but not to necrotrophic fungi. On the other hand, ios1-1 is known to be more resistant to the obligate biotrophic oomycete downy mildew pathogen Hyaloperonospora arabidopsidis (Hok et al., 2011). IOS1 could be a direct or indirect target of an H. arabidopsidis effector necessary for its virulence. IOS1 absence in ios1-1 would not allow H. arabidopsidis to fully repress Arabidopsis PTI. This observation suggests that IOS1 is also involved in Arabidopsis immunity to pathogens other than bacteria.

The increased susceptibility of ios1 mutants to virulent bacteria was correlated with a defective PTI response. Typically, bacteria- and MAMP-induced callose depositions were dramatically reduced in ios1 mutants. In addition, upregulation of PTI-responsive genes such as FRK1, CYP81F2, and WRKY53 was delayed in plants with a defective IOS1. In agreement, Arabidopsis overexpressing IOS1 demonstrated increased accumulation of callose and potentiated expression levels of PTI marker genes upon elicitation. MPK3/6 activation was reduced in ios1 mutants and augmented in IOS1 overexpression lines, suggesting that IOS1 acts upstream of MPK3/6 in PTI signaling. These observations point to the fact that IOS1 is necessary for the full activation of both early and late PTI responses. Similarly, LecRK-VI.2 is necessary for the full activation of some early and late PTI responses (Singh et al., 2012a). However, while Arabidopsis overexpressing LecRK-VI.2 demonstrated a constitutive PTI response, IOS1 overexpression lines showed a strengthened PTI only upon elicitation by bacteria or MAMPs, suggesting a different mechanism of action for these two positive regulators of PTI. Importantly, PTI-mediated ROS production was at wild-type levels in ios1 mutants and in IOS1-OE lines, suggesting that IOS1 may not regulate all aspects of the PTI response. The PRR-associated kinase BIK1 directly regulates PTI-mediated ROS production (Kadota et al., 2014; Li et al., 2014). Therefore, the apparent absence of IOS1 regulation of ROS production could be explained by the fact that IOS1 acts largely in a BIK1-independent manner (Figures 8 and 9A). The ios1 mutant demonstrated wild-type bacteria-mediated stomatal closure, while IOS1-OE lines harbored a strengthened stomatal immunity. Redundancy may explain the stomatal innate immunity discrepancy between ios1 mutants and IOS1-OE lines (compare Figure 1D with Supplemental Figure 6). Other malectin-like LRR-RLKs may indeed play a redundant role in stomatal closure (Hok et al., 2011), thus masking the possible function of IOS1 in this early PTI response. Taken together, these data reveal IOS1 as a major positive regulator of Arabidopsis PTI against bacteria, acting upstream of MPK3/6 in FLS2- and EFR-dependent defense signaling pathways.

IOS1 Associates with FLS2, EFR, and BAK1 in a Ligand-Independent Manner

Having genetically demonstrated the importance of IOS1 in bacteria-, flg22-, and elf26-triggered PTI upstream of MPK3/6, and also considering that IOS1 is an LRR-RLK with only two Leu-rich repeat motifs, a transmembrane domain, and a complete extracellular malectin-like domain (Hok et al., 2011), we further investigated whether IOS1 is part of the PRR complexes recognizing bacterial MAMPs. We first showed that the KD of IOS1 associates in vitro with the KDs of FLS2 and EFR using a pull-down approach. In addition, the in vivo associations of IOS1 with FLS2, EFR, and/or the regulatory LRR-RLK BAK1 were evaluated by BiFC and coimmunoprecipitation analyses. We first performed BiFC assays in Arabidopsis protoplasts and coimmunoprecipitation experiments in N. benthamiana and found that IOS1 constitutively associates with FLS2 and EFR and that elicitation with flg22 or elf26 does not significantly affect the association. The constitutive IOS1 and FLS2 association was further confirmed in Arabidopsis using transgenic lines overexpressing IOS1-GFP, while IOS1 was also found to be part of unstimulated and stimulated EFR complexes by in planta proteomics analysis of EFR-associated proteins (Roux et al., 2011). In addition to associating with PRRs, IOS1 interacts with BRASSINOSTEROID SIGNAL-KINASE3 in Arabidopsis (Xu et al., 2014b). In Arabidopsis, only a few proteins are known to be present in PRR complexes before elicitation by MAMPs. Notably, the cytoplasmic kinases BIK1/PBLs and BSK1 interact constitutively with FLS2 and are released upon elicitation (Lu et al., 2010a; Zhang et al., 2010; Shi et al., 2013). Additionally, the DENN domain protein SCD1, which negatively regulates innate immunity, associates in a ligand-independent manner with FLS2 in vivo (Korasick et al., 2010). Furthermore, the ubiquitin E3 ligases PUB12/13 interact with BAK1 prior to elicitation and ubiquitinate FLS2 upon flg22-induced FLS2/BAK1 complex formation, leading to FLS2 degradation (Lu et al., 2011), and BIR2 negatively regulates Arabidopsis PTI by association before elicitation with BAK1 (Halter et al., 2014). This work thus reveals an additional component of FLS2 and EFR protein complexes.

Heteromerization between epitope-tagged IOS1 and BAK1 was also evaluated in N. benthamiana, where IOS1 was not able to mount a complex with BAK1 before and after elicitation with flg22 or elf26. As already reported (Ntoukakis et al., 2011), the HA epitope tagged at the C terminus of BAK1 used in N. benthamiana may alter its function in PTI signaling and may thus affect the association of IOS1 with BAK1 observable otherwise in Arabidopsis. Nonetheless, our coimmunoprecipitation data undoubtedly demonstrate that IOS1 and the native untagged BAK1 associate in Arabidopsis in a ligand-independent manner (Figure 6D).

IOS1 Positively Regulates FLS2/BAK1 Complex Formation

Since the malectin-like LRR-RLK IOS1 constitutively associates with the PRRs FLS2 and EFR and the regulatory LRR-RLK BAK1, and since ios1 mutants demonstrate a defective PTI, we hypothesized that IOS1 affects early events at PRR complexes. The flg22-mediated association of FLS2 and BAK1 was indeed reduced in ios1-2 and increased in the OE3 line when compared with wild-type Col-0 controls. By contrast, the positive regulator of PTI LecRK-VI.2 does not modulate the flg22-mediated association of FLS2 and BAK1 (Singh et al., 2012). The heteromerization between FLS2 and BAK1 occurs within seconds (Schulze et al., 2010) with BAK1 acting as coreceptor for flg22 (Sun et al., 2013), indicating that both LRR-RLKs most likely exist in close proximity at the plasma membrane, as recently suggested in the case of BAK1 and BRI1 (Bücherl et al., 2013). We thus propose that the plasma membrane-localized IOS1 is required for promoting rapid FLS2/BAK1 complex formation upon flg22 binding. Importantly, the flg22-mediated association between FLS2 and BAK1 was not completely abolished in ios1-2. Other players such as other malectin-like LRR-RLKs may generate the partial FLS2-BAK1 association observed upon flg22 elicitation in ios1-2. IOS1 constitutively interacts with both FLS2 and BAK1; however, FLS2 and BAK1 complex formation only occurs after flg22 treatment. In addition, FLS2-FLS2 and IOS1-IOS1 homodimerization could be observed independently of elicitation (Sun et al., 2012; Supplemental Figure 12). IOS1 monomers or dimers may thus bind both FLS2 and BAK1 in different complexes before PTI elicitation. Upon flg22 treatment, IOS1 may participate in the formation of a new complex that integrates both FLS2 and BAK1. Contrary to IOS1, BIR2 negatively regulates FLS2/BAK1 complex formation (Halter et al., 2014). Thus, BIR2 may directly or indirectly antagonize IOS1.

Treatments with flg22 induce rapid phosphorylation of BIK1, which further increases the phosphorylation of FLS2 and BAK1 (Lu et al., 2010a; Zhang et al., 2010). BIK1 phosphorylation occurs within minutes after flg22 treatment and is thus considered a good marker of PRR activities. Surprisingly, BIK1 phosphorylation was at wild-type levels in the flg22-treated ios1-2 mutant and in the OE3 line. Since MPK3/6 activities were altered in ios1 mutants and in IOS1-OE lines, a BIK1-independent signaling cascade that affects MPK3/6 activities must be present in ios1-2 and in IOS1-OE lines. This observation is in agreement with the findings of Zhou and colleagues, who demonstrated that BIK1 and the closely related PBL1 are not required for flg22-induced MAPK activation (Feng et al., 2012). Therefore, other receptor-like cytoplasmic kinases could play a role in regulating different branches of PTI signaling (Lu et al., 2010b). In addition, our data show that bik1 mutants behave differently from ios1 mutants. Although both are defective in bacterial resistance, bik1 is also more susceptible to B. cinerea (Veronese et al., 2006; Lu et al., 2010a), while ios1 mutants showed wild-type susceptibility. This observation suggests that IOS1 and BIK1 are involved in different branches of PTI signaling. Confirming this hypothesis, Arabidopsis overexpressing IOS1 in the bik1 mutant background still demonstrated a strong priming of callose deposition. By contrast, augmented callose deposition was abolished in lines overexpressing IOS1 in the bak1-5 background. BAK1 is also required for MAPK activation (Ranf et al., 2011; Roux et al., 2011; Schwessinger et al., 2011), and according to our data, MPK3/6 activation in the ios1-2 mutant is only impaired in BAK1-dependent PTI signaling pathways (Figure 9B). Taken together, these data suggest that an altered FLS2-BAK1 association in ios1-2 impacts MPK3/6 activation independently of BIK1 phosphorylation. In addition, IOS1 is likely to act upstream of BAK1.

IOS1 Plays a Critical Role in the Priming of PTI

The accumulation of positive regulators of defense, such as MPK3/6 or LecRK-VI.2, prior to stress challenge is critical for priming (Beckers et al., 2009; Singh et al., 2012). Plants overexpressing IOS1 demonstrated potentiated expression of PTI-responsive genes, primed callose deposition, and increased MPK3/6 activities upon PTI elicitation. These observations further suggest that increased accumulation of positive regulators of PTI before elicitation is sufficient to prime PTI and consequently to increase resistance to bacteria. The BABA-mediated accumulation of IOS1 mRNA (Tsai et al., 2011) may thus be critical for BABA-mediated priming of PTI (Singh et al., 2012; Po-Wen et al., 2013). Therefore, we investigated whether ios1 mutants are defective in BABA-mediated priming. While the lecrk-VI.2-1 mutant is only partially defective in BABA priming (Singh et al., 2012), ios1-1 and ios1-2 mutants were largely deficient in BABA-induced resistance to bacteria, priming of FRK1 expression and callose deposition, and BABA-mediated strengthening of stomatal innate immunity. These results suggest that IOS1 plays a predominant role during the priming of PTI by the nonprotein amino acid BABA. Surprisingly, BABA had no effect on the flg22-mediated FLS2–BAK1 association, suggesting that the reported role of IOS1 in BABA-triggered priming involves another regulatory mechanism.

LRR-RLKs such as FLS2 and EFR or PEPR1/2 are receptors for MAMPs or damage-associated molecular patterns, respectively (Huffaker et al., 2006; Yamaguchi et al., 2006, 2010; Ryan et al., 2007; Krol et al., 2010). Another LRR-RLK, BAK1, functions in several PRR complexes as an adapter or coreceptor (Chinchilla et al., 2007; Heese et al., 2007; Boller and Felix, 2009; Schulze et al., 2010; Sun et al., 2013; Liebrand et al., 2014). Our data identified the malectin-like LRR-RLK IOS1 as a novel member of FLS2 and EFR PRR complexes that also associates in a ligand-independent manner with BAK1. This work further reveals the intricate regulation of the PRR complex dynamics needed for transmitting and regulating PTI signaling, which requires additional components beyond the ligand binding receptor and coreceptor.

METHODS

Biological Materials and Growth Conditions

Arabidopsis thaliana ecotypes Col-0 and Ler-0 were grown in commercial potting soil:perlite (3:2) at 22 to 24°C day and 17 to 19°C night temperatures under a 9-h-light/15-h-dark photoperiod. The lighting was supplied at an intensity of ∼100 μE m^–2 s^–1 by fluorescent tubes. The Ds transposon insertion line (Ler-0) ios1-1 (GT_5_22250) and T-DNA insertion mutants (Col-0) ios1-2 (Salk_137388) and ios1-3 (SAIL_343_B11) were obtained from the ABRC. The mutant bak1-4 (Salk_116202) has been described elsewhere (Chinchilla et al., 2007). Bacterial strains Pseudomonas syringae pv tomato DC3000 and the P. s. tomato DC3000 hrcC⁻ mutant were provided by B.N. Kunkel (Washington University), while Pseudomonas syringae pv maculicola ES4326 was a gift from J. Glazebrook (Minnesota University). All bacteria were cultivated at 28°C and 340 rpm in King’s B medium with 25 mg/mL rifampicin (P. s. tomato DC3000), 25 mg/mL rifampicin and kanamycin (P. s. tomato DC3000 hrcC⁻ mutant), or 50 mg/mL streptomycin (P. s. maculicola ES4326). The fungus Botrytis cinerea was obtained from C.Y. Chen (National Taiwan University) and grown at room temperature (18 to 25°C) on PDA-agar plates (Zimmerli et al., 2001).

Pathogen Infection Assays

Five-week-old Arabidopsis plants were dipped in 10⁶ colony-forming units (cfu)/mL P. s. tomato DC3000 or 5 × 10⁵ cfu/mL P. s. maculicola ES4326 in 10 mM MgSO₄ containing 0.01% Silwet L-77 (Lehle Seeds) for 15 min. After inoculation, plants were kept at 100% relative humidity, and symptoms were evaluated 3 d later. Bacterial titers were determined as described previously (Zimmerli et al., 2000). For B. cinerea infection, spores were diluted to 10⁵ spores/mL in half-strength potato dextrose broth (Zimmerli et al., 2001) medium. Droplets (10 μL) of half-strength potato dextrose broth with B. cinerea spores were deposited on leaf surfaces of 5-week-old plants (three leaves per plant). Leaves of the same age were used for droplet inoculation. Disease symptoms and lesion diameters were determined at 3 DAI. At least 18 lesion diameters were evaluated for each biological replicate (six plants).

IOS1 Overexpression Plants

The DNA plasmids (pH35GWG) expressing IOS1 protein fused with GFP at the C terminus under the control of the cauliflower mosaic virus 35S promoter were obtained from the ABRC (Gou et al., 2010) (ABRC stock S1G51800HGF). Agrobacterium tumefaciens GV3101 was used for the transformation of Col-0 plants. Successful transformation was determined by screening on 1.5% Murashige and Skoog agar plates containing 50 μM glufosinate-ammonium (Fluka) and raised to homozygous T3 lines. For the generation of IOS1-OE lines in the bak1-5 or bik1 mutant background, mutant plants were dip-inoculated with Agrobacterium strain GV3101 carrying Pro35S-IOS1-GFP (pFAST-R05) using OLE1-GFP as a screenable marker (Shimada et al., 2010) and raised to T2 for analyses.

BABA and MAMP Treatments

For bacterial titer, callose deposition, and stomatal aperture evaluations, 5-week-old Arabidopsis plants were soil-drenched with BABA (Fluka) at a final concentration of 225 μM 2 d before bacteria inoculation or MAMP treatment. BABA was dissolved in water, and controls were soil-drenched with water only. For FRK1 expression, seedlings grown on half-strength Murashige and Skoog (MS) plates for 10 d were transferred to liquid half-strength MS medium containing 1% Suc one night before the addition of 225 μM BABA (final concentration). Treatments with 1 μM flg22 were performed 2 d later, and samples for RNA extraction were collected 30 min after flg22 treatment.

The flg22 and elf26 peptides were purchased from Biomer Technology and dissolved in 10 mM MgSO₄; MgSO₄ only was used as a control. Both Atpep1 (obtained from Y.R. Chen, Academia Sinica) and chitin from shrimp shells (Sigma-Aldrich) were dissolved in water. Water-only treatments were used as controls. MAMPs were syringe-infiltrated into leaves, and samples were harvested at the time points indicated in the respective figure legends.

Callose Deposition

Five-week-old Arabidopsis leaves were syringe-infiltrated with 10⁸ cfu/mL P. s. tomato DC3000 hrcC⁻, 1 μM flg22, or 1 μM elf26 in 10 mM MgSO₄. Control plants were infiltrated with 10 mM MgSO₄ only. Nine leaf discs from three different plants were selected for analyses at the indicated time points. Callose deposits were visualized as described (Singh et al., 2012).

RT-PCR

For qRT-PCR, Arabidopsis seedlings grown on half-strength MS plates for 10 d were transferred to liquid half-strength MS containing 1% Suc one night before treatment with 10⁸ cfu/mL P. s. tomato DC3000 hrcC⁻, 1 μM flg22, or 1 μM elf26, and samples were collected at the indicated time points. For PTI-responsive gene expression analyses in IOS1 overexpression lines, 5-week-old plants were syringe-infiltrated with 10⁸ cfu/mL P. s. tomato DC3000 hrcC⁻, 10 nM flg22, or 10 nM elf26, and samples were collected at the indicated time points. Total RNA isolation, cDNA biosynthesis, and real-time PCR analyses were performed as described (Wu et al., 2010). Normalization of gene expression was conducted with At5g60390 (EF-1) and At4g05320 (UBQ10). For RT-PCR, 1 μL of cDNA was used as a template, and standard PCR conditions were applied as described (Singh et al., 2012). At4g05320 (UBQ10) was used as a loading control. Primers used are listed in Supplemental Table 2.

MAPK Assay

Nine leaves of three 5-week-old plants (three leaves per plant) were syringe-infiltrated with 100 nM flg22, 10 nM elf26, or 10 mM MgSO₄ (control) or with 10 nM Atpep1, 0.1 mg/mL chitin, or water (control) for 5 min before being pooled for harvest. MAPK assays were performed as described (Singh et al., 2012).

ROS Burst

ROS assays were performed as described previously (Huang et al., 2013). Briefly, six leaf discs (10 mm diameter) from three 5-week-old Arabidopsis plants (two discs per plant) were incubated in double-distilled water on 96-well plates overnight. The following day, the water was replaced by 10 nM flg22 or 10 nM elf26 in 10 mM MgSO₄ buffer or by 10 mM MgSO₄ buffer only for the mock controls containing 2 μM luminal (Sigma-Aldrich) and 10 μg/mL peroxidase (Sigma-Aldrich). The plates were analyzed every 2 min after the addition of MAMPs for a period of 30 min using the CentroLIApc LB 692 plate luminometer (Berthold Technologies).

Stomatal Assay

Five-week-old plants were kept under light (100 μE m^–2 s^–1) for at least 3 h to open stomata before the beginning of the experiments. For each biological replicate, stomatal apertures were evaluated from 12 epidermal peels from four plants (three epidermal peels per plant) as described (Tsai et al., 2011).

Subcellular Localization in Protoplast and Root

For transient expression of the GFP fusion proteins, constructs expressing 35S-IOS1-GFP (plasmid pH35GWG; ABRC stock S1G51800HGF) or vector alone were cotransfected into Arabidopsis mesophyll protoplasts according to He et al. (2007). The GFP fusion constructs were cotransfected with the plasma membrane marker pm-rkCD3-1007 (Nelson et al., 2007). For stable transformant analyses, IOS1 overexpression line OE3 and a vector-only control (pEarlyGate103) were grown on 1.5% Murashige and Skoog agar plates for 10 d. Transformed protoplasts or roots of IOS1 transgenic OE3 were visualized using a confocal laser scanning microscope (LSM 780; Carl Zeiss) with excitation at 488 nm and emission at 490 to 515 nm for protoplast and 490 to 551 nm for roots; autofluorescence was observed at 650 to 700 nm. The plasma membrane marker was detected with excitation at 594 nm and emission at 595 to 650 nm.

BIK1 Phosphorylation

Mesophyll protoplasts were obtained as described by He et al. (2007). BIK1 phosphorylation assays on protoplasts treated with 0.75 μM flg22 for 3.5, 7, and 10 min were as described (Singh et al., 2013).

Cloning, Expression, and Purification of Recombinant Proteins

In order to generate a Trx-6xHis N-terminal fusion of the IOS1 KD, the sequence coding for the IOS1 cytosolic domain was amplified from the pH35GWG vector expressing the IOS1-GFP fusion using primers carrying BamHI and XhoI restriction sites (Supplemental Table 2) and introduced into the polylinker of the pET-32a+ expression vector (Novagen). To produce an inactive kinase fusion protein, a point mutation in the kinase activation site (D710N) was introduced into the expression vector by primer extension (Supplemental Table 2) using the Phusion polymerase (New England Biolabs) followed by DpnI (New England Biolabs) digestion according to the manufacturer’s instructions. The Trx-6xHis-IOS1KD and Trx-6xHis-IOS1KDm (for kinase dead) fusion proteins were expressed in Escherichia coli Rosetta (DE3) pLysS (Novagen). After overnight induction at 16°C with 0.4 mM isopropyl β-d-1-thiogalactopyranoside, the bacteria were pelleted by centrifugation, resuspended in 100 mL of binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, and 0.04% 2-mercaptoethanol), and sonicated. The soluble His-tagged proteins were affinity-purified using a HisTrap FF column (GE Healthcare) according to the manufacturer’s instructions. All constructs were confirmed by Sanger sequencing, and the purified Trx-6xHis-IOS1KD and Trx-6xHis-IOS1KDm were analyzed by LC-MS/MS.

The production of MBP-tagged FLS2 and EFR KD constructs was performed as described by Schwessinger et al. (2011). MBP was expressed from pMAL-c5X (New England Biolabs). MBP and the two MBP-tagged proteins were expressed as described above, but using the E. coli strain BL21 (DE3) pLysS (Novagen), and purified using amylose resins (MBPTrap HP; GE Healthcare) following the manufacturer’s instructions. Finally, MBP and the two MBP-tagged proteins were dialyzed against dialysis buffer (20 mM Tris-HCl and 100 mM NaCl, pH 7.4).

In Vitro Pull-Down Assay

One microgram of MBP, MBP-FLS2KD, or MBP-EFRKD was incubated with 2 μg of Trx-6xHis-IOS1KD in a binding buffer (20 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA, pH 7.4) under agitation at 4°C. After 2 h, 50 μL of amylose resin beads (GE Healthcare) was added, and the incubation continued for another 2 h. The beads were then washed five times with the washing buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, and 0.6% Triton X-100, pH 7.4). Input and pulled down proteins were resolved by 8% SDS-PAGE and detected by immunoblotting using appropriate antibodies.

BiFC Assay

Full-length coding sequences of FLS2, EFR, BAK1, CERK1, and IOS1 without stop codons amplified from cDNA of Arabidopsis Col-0 were inserted into entry vector pCR8/GW/TOPO and subcloned into YN (pEarleyGate201-YN) or YC (pEarleyGate202-YC) vector (Lu et al., 2010c) through LR reaction (Invitrogen). The constructs were transformed into Arabidopsis protoplasts by polyethylene glycol (Sigma-Aldrich) for transient expression (Yoo et al., 2007). Transfected cells were treated with or without 1 μM flg22, 1 μM elf26, or 1 mg/mL chitin for 10 min before being imaged using a confocal laser scanning microscope (LSM 780; Carl Zeiss).

Transient Expression in Nicotiana benthamiana

For the coimmunoprecipitation, the whole coding sequences without stop codons of IOS1 and CERK1 were amplified using the primers described in Supplemental Table 2. The resulting PCR products were directly cloned by the TOPO cloning reaction into the pCR8/GW/TOPO vector (Invitrogen) following the manufacturer’s instructions and recombined through the LR reaction (Invitrogen) into the Gateway-compatible destination vector pEarlyGate103 (35S-IOS1-GFP and 35S-CERK1-GFP) or pGWB14 (35S-IOS1-HA₃, 35S-CERK1-HA₃, and 35S-FLS2-HA₃). Every construct was confirmed by DNA sequencing. The 35S-FLS2-GFP-His, 35S-EFR-GFP-His, and 35S-BAK1-HA₃ constructs were as described (Schwessinger et al., 2011). The final constructs were electroporated into Agrobacterium competent cell strain GV3101. Transient expression was performed according to Roux et al. (2011).

Protein Extraction and Immunoprecipitation in N. benthamiana

Protein extraction and immunoprecipitation were performed as described (Roux et al., 2011).

Protein Extraction and Immunoprecipitation in Arabidopsis

The protocol for protein extraction was described by Roux et al. (2011). Arabidopsis seedlings grown in liquid half-strength MS medium containing 1% Suc were used for immunoprecipitation assays. For immunoprecipitation of endogenous BAK1, supernatants were incubated with 25 μL of true-blot anti-rabbit Ig beads (Ebioscience) and 20 μL of anti-BAK1 antibody (Schulze et al., 2010) for 4 h at 4°C. For immunoprecipitation of IOS1-GFP, supernatants were incubated with 50 to 200 μL of anti-GFP magnetic beads (Miltenyi Biotec) for 2 h at 4°C (Kadota et al., 2014). Following incubation, beads were washed three to five times with extraction buffer before adding SDS buffer (Schwessinger et al., 2011).

SDS-PAGE and Immunoblotting

Eight percent to 10% SDS-PAGE gels were run at 80 to 140 V for 2 h before electroblotting on a polyvinylidene difluoride membrane (Millipore) at 100 V for 1 h at 4°C. Membranes were rinsed in Tris-buffered saline (TBS) and blocked in 5% (w/v) nonfat milk powder in 0.1% (v/v) TBS-Tween for 2 h. Primary antibodies were diluted in TBS-Tween solution to the following concentrations and incubated overnight: anti-His (Santa Cruz), 1:1000; anti-MBP, 1:4000 (Sigma-Aldrich); anti-GFP (Santa Cruz), 1:3000; anti-HA-HRP (Sigma-Aldrich), 1:3000; anti-BAK1, 1:500; and anti-FLS2, 1:1000. Membranes were washed three times in TBS-Tween before 1 h of incubation with the secondary antibodies anti-mouse-HRP (Santa Cruz) diluted 1:3000 or anti-rabbit-HRP (Sigma-Aldrich) diluted 1:3000. Signals were visualized using an enhanced chemiluminescence system (Immobilon Western; Millipore) and a LAS-3000 (Fujifilm) scanner following the manufacturer’s instructions.

Mass Spectrometry

Proteins were separated by SDS-PAGE (Nupage precast gel system; Invitrogen), and after staining with Coomassie Brilliant Blue (SeeBlue Safe Stain; Invitrogen), the proteins were cut out and digested by trypsin as described previously (Ntoukakis et al., 2009). LC-MS/MS analysis was performed using an LTQ-Orbitrap mass spectrometer (Thermo Scientific) and a nanoflow-HPLC system (nanoAcquity; Waters) as described previously (Ntoukakis et al., 2009). The entire TAIR10 (www.arabidopsis.org) and E. coli O157 databases were searched using Mascot (with the inclusion of sequences of common contaminants, such as keratins and trypsin). Parameters were set for 65 ppm peptide mass tolerance and allowing for Met oxidation and two missed tryptic cleavages. Carbamidomethylation of Cys residues was specified as a fixed modification, and oxidized Met and phosphorylation of Ser or Thr residue were allowed as variable modifications. Scaffold (version 2_06_01; Proteome Software) was used to validate tandem mass spectrometry–based peptide and protein identifications.

In Vitro Kinase Assay

The in vitro kinase assay was performed as described previously (Singh et al., ‎2013). Briefly, 2 μg of purified Trx-6xHis-IOS1KD and Trx-6xHis-IOS1KDm was incubated for 30 min at 28°C in 30 μL of kinase buffer (50 mM Tris-Cl, pH 7.5, 50 mM KCl, 2 mM DTT, 10% [v/v] glycerol, 5 mM MnCl₂, and 5 mM MgCl₂). Phosphorylation was initiated with the addition of 10 mM ATP and terminated by adding 30 μL of 2× SDS-PAGE loading buffer. Of these, 30 μL was separated on an 8% polyacrylamide gel, and the phosphorylation level of the proteins was detected using the Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen) according to the manufacturer’s instructions. The fluorescent signal was imaged using a Typhoon 9400 scanner (Amersham Biosciences), and the same gel was subsequently stained for total protein with Coomassie Brilliant Blue.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: IOS1 (At1g51800), FRK1 (At2g19190), CYP81F2 (At5g57220), WRKY53 (At4g23810), EF-1 (At5g60390), and UBQ10 (At4g05320).

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure 1. The Mutant ios1-1 Is a Knockout While ios1-2 and ios1-3 Produce Some IOS1 Transcripts.

• Supplemental Figure 2. Susceptibility Phenotypes of ios1-2 and bak1-5 to P. s. tomato DC3000.

• Supplemental Figure 3. ios1 Mutants Demonstrate Increased Susceptibility to P. s. tomato DC3000 hrcC⁻.

• Supplemental Figure 4. Resistance of ios1 Mutants to B. cinerea.

• Supplemental Figure 5. IOS1 mRNA Expression Levels in Three IOS1 Overexpression Lines.

• Supplemental Figure 6. Stomatal Innate Immunity in ios1 Mutants.

• Supplemental Figure 7. Expression of IOS1 Is Upregulated by Bacterial MAMPs.

• Supplemental Figure 8. PTI-Responsive CYP81F2 and WRKY53 Upregulation.

• Supplemental Figure 9. Early PTI Responses in ios1 Mutants and IOS1-OE Lines upon elf26 Elicitation.

• Supplemental Figure 10. Bimolecular Fluorescence Complementation Analyses of IOS1 Interactions with EFR and BAK1.

• Supplemental Figure 11. IOS1-HA₃ Does Not Aspecifically Bind to Anti-GFP Magnetic Beads.

• Supplemental Figure 12. FLS2-FLS2 and IOS1-IOS1 Homodimerization.

• Supplemental Figure 13. IOS1 in Vitro Autophosphorylation.

• Supplemental Figure 14. BABA Does Not Regulate Ligand-Induced FLS2–BAK1 Association.

• Supplemental Table 1. Identification of IOS1 Tryptic Peptides by HPLC-ESI-MS/MS Analysis of EFR Immunoprecipitates.

• Supplemental Table 2. Primer Sequences Used in This Study.

Glossary

PRR

pattern recognition receptor

MAMP

microbe-associated molecular pattern

PTI

pattern-triggered immunity

ROS

reactive oxygen species

MAPK

mitogen-activated protein kinase

ETI

effector-triggered immunity

LRR-RLK

leucine-rich repeat receptor-like kinase

BABA

β-aminobutyric acid

Ler-0

Landsberg erecta-0

Col-0

Columbia-0

DAI

days after inoculation

qRT-PCR

quantitative RT-PCR

KD

kinase domain

BiFC

bimolecular fluorescence complementation

LC-MS/MS

liquid chromatography–tandem mass spectrometry

cfu

colony-forming units

MS

Murashige and Skoog

TBS

Tris-buffered saline