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The bacterial effector HopM1 suppresses PAMP-triggered oxidative burst and stomatal immunity Rosa Lozano-Duran1, Gildas Bourdais1, Sheng Yang He2 and Silke Robatzek1 1

The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK; 2Department of Energy Plant Research Laboratory, Howard Hughes Medical Institute-Gordon and Betty Moore

Foundation, Michigan State University, East Lansing, MI 48824, USA

Summary Author for correspondence: Silke Robatzek Tel: +44 (0)1603 450408 Email: [email protected] Received: 20 May 2013 Accepted: 8 November 2013

New Phytologist (2014) 202: 259–269 doi: 10.1111/nph.12651

Key words: 14-3-3, effector, HopM1, immunity, oxidative burst, PAMP/MAMP, stomata, TFT1.

 Successful pathogens counter immunity at multiple levels, mostly through the action of effectors. Pseudomonas syringae secretes c. 30 effectors, some of which have been shown to inhibit plant immunity triggered upon perception of conserved pathogen-associated molecular patterns (PAMPs). One of these is HopM1, which impairs late immune responses through targeting the vesicle trafficking-related AtMIN7 for degradation.  Here, we report that in planta expressed HopM1 suppresses two early PAMP-triggered responses, the oxidative burst and stomatal immunity, both of which seem to require proteasomal function but are independent of AtMIN7. Notably, a 14-3-3 protein, GRF8/AtMIN10, was found previously to be a target of HopM1 in vivo, and expression of HopM1 mimics the effect of chemically and genetically disrupting 14-3-3 function.  Our data further show that the function of 14-3-3 proteins is required for PAMP-triggered oxidative burst and stomatal immunity, and chemical-mediated disruption of the 14-3-3 interactions with their client proteins restores virulence of a HopM1-deficient P. syringae mutant, providing a link between HopM1 and the involvement of 14-3-3 proteins in plant immunity.  Taken together, these results unveil the impact of HopM1 on the PAMP-triggered oxidative burst and stomatal immunity in an AtMIN7-independent manner, most likely acting at the function of (a) 14-3-3 protein(s).

Introduction Plants have evolved a multifaceted immune system, the first layer of which is PAMP-triggered immunity (PTI). PTI is the result of the recognition of pathogen-associated molecular patterns (PAMPs) by cognate pattern-recognition receptors located at the surface of the plant cell, and is sufficient for resistance against most microbes (Zipfel & Robatzek, 2010). Successful pathogens need to overcome PTI, which they are able to achieve through the action of effectors or toxins that interfere with defence responses at multiple levels. PTI comprises different cellular responses that fall into early or late categories (Boller & Felix, 2009). Upon ligand binding, the receptor for the bacterial PAMP flagellin (or the elicitor-active peptide flg22), FLAGELLIN SENSING 2 (FLS2), a leucine-rich repeat receptor kinase, forms a complex with the BRI1-Associated Receptor Kinase 1 (BAK1) within seconds (Boller & Felix, 2009). Downstream early PTI responses occur within minutes to hours and include the rapid and transient production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases, transcriptional reprogramming, and stomatal closure – an event also referred to as stomatal immunity (Melotto et al., 2006). Late PTI responses such as callose deposition or seedling growth inhibition develop Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

within longer periods of time, ranging from hours to days (Nicaise et al., 2009). A well-studied key virulence mechanism of Gram-negative bacteria is the Type III secretion system, which enables bacteria to deliver effector proteins into the host cell (Collmer et al., 2000; Cunnac et al., 2009). The tomato and Arabidopsis thaliana pathogen Pseudomonas syringae pathovar (pv) tomato DC3000 (Pto DC3000) translocates c. 30 Type III-secreted effectors (TTEs). The virulence functions of these TTEs are often overlapping, and therefore mutants in single TTEs usually lack a phenotype due to functional redundancies (Kvitko et al., 2009). However, a partial deletion in the conserved effector locus CEL, which contains the effector genes hopM1 and avrE, results in a severe virulence defect (Badel et al., 2006; Nomura et al., 2006; Kvitko et al., 2009). The reduction in Pto DC3000 DCEL virulence can be reverted by complementation with HopM1 and its cognate chaperone, or by transgenic expression of HopM1 in planta (DebRoy et al., 2004; Nomura et al., 2006); HopM1 has also been shown to suppress basal resistance against bacteria in Nicotiana benthamiana, measured as reduced vascular flow (Oh & Collmer, 2005). The aforementioned results indicate that this effector plays an important role in the promotion of Pto DC3000 virulence; in line with this idea, HopM1 belongs to the minimal New Phytologist (2014) 202: 259–269 259 www.newphytologist.com

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functional effector repertoire of Pto DC3000, promoting growth when in combination with the effectors AvrPto or AvrPtoB (Cunnac et al., 2011). Exploitation of the eukaryotic ubiquitination pathway by effectors is a common strategy employed by animal and plant pathogens (Angot et al., 2007; Spallek et al., 2009). HopM1 is able to bind and trigger proteasome-dependent degradation of several host targets in Arabidopsis (Nomura et al., 2006, 2011). One such target is the ADP ribosylation factor (ARF) guanine nucleotide exchange factor (GEF) AtMIN7, which is required for both PTI and effector-triggered immunity (ETI), a second layer in plant immunity (Nomura et al., 2011); HopM1 reduces callose deposition at least partially through the degradation of AtMIN7 (Nomura et al., 2006). However, the biological relevance of the other described HopM1 interactors for the promotion of virulence, as well as whether this bacterial effector also has an impact on early PTI responses, are open questions that remain to be addressed. We report that HopM1 expression in planta suppresses two early PTI responses – the PAMP-triggered ROS burst and stomatal immunity – in both Arabidopsis and N. benthamiana. The suppression of these responses by the effector is independent of AtMIN7 but may involve another of the previously identified targets of HopM1, one of which is AtMIN10, the 14-3-3 protein GRF8 (Nomura et al., 2006). GRF8 binds BZR1, a major transcription factor in brassinosteroid signalling, and causes its cytoplasmic retention (Gampala et al., 2007; Ryu et al., 2007); and likewise HopM1 expression in planta resulted in nuclear hyperaccumulation of BZR1-YFP, mimicking the effect of disrupting 14-3-3 function. Chemically disrupting 14-3-3 protein interactions with their client proteins reduces the PAMP-triggered ROS burst, stomatal immunity and, importantly, restores virulence of a P. syringae DCEL deletion strain. In agreement, silencing of the GRF8 orthologue TFT1 in tomato and N. benthamiana compromises the PAMP-triggered ROS burst. Taken together, our results show that HopM1 suppresses two early PTI outputs, the PAMP-triggered ROS burst and stomatal immunity, and unveil a role of 14-3-3s in these immune responses.

Materials and Methods Plant materials and growth conditions Transgenic HopM1 and T-DNA insertional atmin7 are described in Nomura et al. (2006). For stomatal assays, Arabidopsis genotypes (Col-0, HopM1, atmin7) were grown on MS plates under long-day conditions (16 h : 8 h, light : dark). For ROS assays and bacterial growth assays, Arabidopsis plants were grown on soil in a controlled-environment chamber under short-day conditions (8 h : 16 h, light : dark) for 4–5 wk. Nicotiana benthamiana L. and tomato (Solanum lycopersicum L.). Money Maker plants were grown in soil in a controlled-environment chamber under longday conditions (16 h : 8 h, light : dark). Before all experiments, samples from HopM1 transgenic Arabidopsis lines and control lines were treated with a 30 lM dexamethasone (DEX) solution or mock solution for 2 h, unless otherwise indicated. Because the New Phytologist (2014) 202: 259–269 www.newphytologist.com

DEX-inducible HopM1 construct conferred constitutive expression in N. benthamiana, visible as cell death at later stages (Supporting Information Fig. S1C, D), samples of transiently transformed N. benthamiana plant were not treated with DEX. Plasmids and cloning The binary vector to express full-length HopM1 is described in Nomura et al. (2006); the binary vector to express AvrPtoB is described in de Torres et al. (2006). BZR1 full-length coding sequence was PCR amplified (using primers 5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA CAT GAC TTC GGA TGG AGC TAC G-3′ and 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG ACC ACG AGC CTT CCC ATT TCC-3′) and gateway-cloned into pDONR201. For C-terminal fusion to the yellow fluorescent protein (YFP), BZR1 was gateway-cloned into pUb-CYFP-Dest (Grefen et al., 2010), and correct cloning was confirmed by sequencing. The binary vector to express FLS2-GFP (FLS2p::FLS2-3xmyc-GFP) is described in Robatzek et al. (2006). To generate the binary vector for RBOHD-YFP expression, the AtRBOHD full-length coding sequence was PCR amplified (using primers 5′-G GGG ACA AGT TTG TAC AAA AAA GCA GGC TAC ATG AAA ATG AGA CGA GGC AAT T-3′ and 5′-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG GAA GTT CTC TTT GTG GAA GTC A-3′) and gateway-cloned into pDONR201. For C-terminal fusion of YFP, AtRBOHD was gateway-cloned into pGWB41 (Nakagawa et al., 2007). The tomato rattle virus (TRV)-based construct to silence tomato TFT1 has been described elsewhere (Taylor et al., 2012). Transient transformation For transient transformation, cultures of Agrobacterium tumefaciens strain GV3101 carrying respective binary vectors were grown overnight in LB medium with the appropriate antibiotics; cells were washed and resuspended in a 100 lM solution of acetosyringone in water, and the OD600 was adjusted to 0.1 before infiltration into the abaxial leaf surface of 2- to 3-wk-old N. benthamiana plants. Samples were taken 1 d after infiltration for ROS measurements and 2 d after infiltration for stomatal assays and quantification of BZR1-YFP signal. ROS measurements Five-week-old plants were used for ROS measurements as previously described (Segonzac et al., 2011). ROS was elicited with 100 nM flg22 (EZBiolab, Carmel, IN, USA) or 1 mg ml 1 chitin oligosaccharide (Yaizu Suisankagaku Industry, Yaizu, Japan); a negative control without PAMP elicitation was included in all experiments. Fourteen (for the AICAR treatments) or 28 (for all other experiments) leaf discs from 5-wk-old plants were used per genotype and treatment. Luminescence was measured over time using an ICCD photon-counting camera (Photek, E. Sussex, UK); the total photon count between 2 and 30 min after elicitation was used for the measurements. For MG132 treatments, Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

New Phytologist samples were submerged in a 100 lM MG132 solution or a mock solution for 6 h before elicitation. For AICAR treatments, samples were submerged in a 20 mM AICAR solution or a mock solution for 3 h before elicitation. Stomatal aperture measurements For flg22 (25 lM), chitin (1 mg ml 1), ABA (50 lM), H2O2 (1 mM), MG132 (100 lM) or AICAR (20 mM) treatments, 2wk-old seedlings grown in MS plates were used for Arabidopsis, and leaf discs of 2- to 3-wk-old soil-grown plant were used for N. benthamiana. For bacterial suspension-triggered stomatal closure experiments, either 2-wk-old seedlings grown in MS plates or 4- to 5-wk-old soil-grown Arabidopsis plants were used with similar results. Bacterial (wild-type Pto DC3000 or the COR mutant) cultures grown overnight were washed and diluted in water to 5 9 107 CFU ml 1. Silwet L-77 was added to a final concentration of 0.025%. Samples were kept in water under light for at least 2 h to ensure maximum stomatal aperture. When applicable, samples were treated with 100 lM MG132 for 2 h. Samples were then treated with the different chemicals or the bacterial suspensions for 2 and 1 h, respectively. The lower epidermis of at least six independent samples was imaged with the Opera confocal microscope (Perkin Elmer, Waltham, MA, USA), using the 405 nm laser (see example images in Fig. S2). Five fields were imaged per sample; images of consecutive series of 25 planes with a distance of 1 lm were taken per field. Generation of maximal projections of the images was performed as described in Salomon et al. (2010). Stomatal apertures (width/ length) of at least 30 stomata per sample were determined using the Image J software (NIH, Bethesda, MD, USA). Statistical analyses were performed using the SigmaPlot software (Systat, San Jose, CA, USA). Quantitative real-time PCR Quantitative real-time PCR analyses were performed as previously described (Mersmann et al., 2010). UBQ10 (At5g15400), Actin and FBOX were used as internal controls for Arabidopsis, tomato and N. benthamiana, respectively. Primer pairs to amplify CPD, DWF4, UBQ10, tomato TFT1, tomato Actin and N. benthamiana FBOX are described elsewhere (Nemhauser et al., 2004; Gampala et al., 2007; Liu et al., 2012; Taylor et al., 2012). TFT1 from N. benthamiana was amplified with primers 5′-TGT TGA ATT AGC CCC TAC CC-3′ and 5′-GGC GAG ATT ACA AGC ACG AT-3′. Statistical analysis was done using the SigmaPlot software. HopM1 transgenic Arabidopsis plants were treated with DEX for 2 h before RNA extraction. Quantification of BZR1-YFP fluorescent signal Pictures of BZR1-YFP expressing tissues were acquired on the Opera confocal microscope (Perkin Elmer) using a 488 nm laser (for quantification of signal) or a Leica DM6000B/TCS SP5 microscope (for figure images), using the preset settings for YFP. When using the confocal microscope, manipulation of the Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

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samples was performed as described in Salomon et al. (2010); five fields were imaged per sample, and images of consecutive series of 21 planes with a distance of 1 lm were taken per field. For AICAR treatments, samples were submerged in a 20 mM AICAR solution or a mock solution for 1 h before imaging. For MG132 treatments, samples were submerged in a 100 lM MG132 solution or a mock solution for 2 h before imaging. Images were analysed using Image J. To measure the ratio between nuclear and cytoplasmic signals, a small area of fixed size (10 pixels) was drawn, measurements were made in nuclear and cytoplasmic areas of the same cell, and the nuclear/cytoplasmic signal ratio was calculated in each case. Measurements were performed for at least 15 cells per sample. Statistical analyses were done using the SigmaPlot software. Bacterial growth assays Arabidopsis leaves were infiltrated with 1 9 106 CFU ml 1 of bacteria (wild-type Pto DC3000 or Pto DC3000 ΔCEL mutant), detached, and placed in microtitre plates with the petioles immersed in a 20 mM AICAR solution or a mock solution. Bacterial growth was determined 24 h post inoculation as previously described (Zipfel et al., 2004). Western blot Plant samples were ground in liquid nitrogen and solubilized in 19 Laemli buffer containing 5 ll ml 1 protease inhibitor cocktail (Sigma) and 1 mM PMSF and boiled for 10 min. Proteins were separated on 7% or 10% SDS-PAGE gels and transferred onto PVDF membranes using a semi-dry transfer system followed by blocking in 5% milk. Antibodies were diluted as follows: anti-FLS2 (Gohre et al., 2008; 1 : 5000), anti-GFP (Roche; 1 : 1000), AP-conjugated anti-rabbit (Sigma; 1 : 30 000) or APconjugated anti-mouse (Sigma; 1 : 30 000). Alkaline-phosphatase activity was detected using CDP-Star (Roche). Virus-induced gene silencing Virus-induced gene silencing (VIGS) experiments in N. benthamiana and tomato were performed as previously described (Lozano-Duran et al., 2011, and Taylor et al., 2012, respectively).

Results The bacterial effector HopM1 suppresses stomatal immunity and PAMP-triggered ROS burst HopM1 suppresses basal resistance in N. benthamiana, measured as reduced vascular flow into minor veins (Oh & Collmer, 2005), as well as the late PTI-associated callose deposition in Arabidopsis (Nomura et al., 2006). However, the impact of HopM1 on early PTI responses remains to be determined. One such response is the PAMP-triggered production of ROS, or oxidative burst. We first investigated if transgenic Arabidopsis plants expressing HopM1 (Nomura et al., 2006) or N. benthamiana New Phytologist (2014) 202: 259–269 www.newphytologist.com

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leaves transiently expressing this effector display normal PAMPtriggered oxidative burst. In planta expression of HopM1 hampered ROS production triggered by either bacterial flg22 or the fungal PAMP chitin (Figs 1a, S1A). Because PAMP-induced stomatal closure – which impedes pathogen entry through the stomata and therefore plays a crucial 2000

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role in pre-invasive immunity (Melotto et al., 2006) – is abolished in the PAMP-triggered ROS burst deficient mutant rbohD (Mersmann et al., 2010), which lacks the NADPH oxidase RBOHD, we decided to examine the effect of HopM1 on stomatal immunity. In planta expression of HopM1 reduced stomatal closure triggered by flg22 or chitin, but did not affect the stomatal response to abscisic acid (ABA) or H2O2 (Figs 1b, S1B), indicating that in these plants stomatal closure can occur normally upon cues other than PAMP perception. In contrast to flg22 and chitin, the response to ABA or H2O2 was clearly detectable and statistically significant in all measurements, although the stomata of HopM1 transgenic Arabidopsis plants were often smaller in size and therefore appeared as more closed than those in wildtype plants under mock conditions (Fig. 1b, left panel; Fig. S3). Because stomata of plants expressing HopM1 exhibited closure in response to ABA or H2O2, the effect of HopM1 on PAMPtriggered stomatal closure may be a direct consequence of the HopM1-mediated suppression of PAMP-triggered ROS. Given that stomatal immunity is an important mechanism to prevent bacterial entry into the leaf interior, Pto DC3000 has evolved the bacterial toxin coronatine (COR), which triggers the re-opening of stomata closure upon PAMP perception (Melotto et al., 2006). A coronatine-negative mutant Pto DC3000 (COR ) is unable to counteract stomatal immunity at the preinvasive level, and therefore invades the leaf tissues less efficiently. Interestingly, in planta expressed HopM1 was able to complement the virulence deficiency of the Pto DC3000 COR , measured as impairment of bacterial-induced stomatal closure, after 1 h of treatment with a bacterial suspension (Fig. 1c).

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Fig. 1 Suppression of the PAMP-triggered oxidative burst and stomatal closure by HopM1 in Arabidopsis. (a) Flg22- and chitin-induced reactive oxygen species (ROS) burst in the absence and presence of in planta expressed HopM1 in Arabidopsis. ROS generation (indicated as total photon count) was monitored over time for 30 min. Each experiment was repeated at least three times; results from a representative experiment are shown. Error bars,  SE based on n = 28 leaf discs taken from 24 plants. WT, wild-type. (b) Flg22-, chitin-, ABA- or H2O2-mediated stomatal closure in the absence or presence of in planta expressed HopM1 in Arabidopsis. Each experiment was repeated at least three times; results from a representative experiment are presented. Error bars,  SE based on n ≥ 30 stomata from at least three independent plants. (c) Pathogeninduced stomatal closure in the absence or presence of in planta expressed HopM1 in Arabidopsis. This experiment was repeated at least three times; results from a representative experiment are presented. Error bars,  SE based on n ≥ 30 stomata from at least three independent plants. Statistically significant compared to controls according to Student’s t-test, P < 0.05. New Phytologist (2014) 202: 259–269 www.newphytologist.com

HopM1 acts on stomatal immunity and PAMP-triggered oxidative burst in a proteasome-dependent but AtMIN7independent manner HopM1 has been described to mediate the ubiquitination of host proteins, and this activity is required for its virulence function (Nomura et al., 2006). In order to determine whether proteasomal degradation is necessary for the HopM1-mediated suppression of PAMP-triggered oxidative burst and stomatal closure, we tested if the proteasome inhibitor MG132 can revert the effects of HopM1 on these outputs. Interestingly, the HopM1-mediated suppression of flg22-induced ROS production as shown in Fig. 1 was moderately restored upon treatment with the proteasome inhibitor MG132 (Fig. 2a); this suggests that the HopM1-mediated proteasome-dependent degradation of a host target underlies the inhibition of the PAMP-triggered ROS burst. Accordingly, treatment with MG132 partially restored the flg22- or chitintriggered stomatal closure (Figs 2b, S2). AtMIN7 is a confirmed target of HopM1 (Nomura et al., 2006) and is required for PTI (Nomura et al., 2011). We therefore reasoned that degradation of this host protein might underpin the HopM1-mediated suppression of PAMP-triggered ROS production and stomatal immunity. To test this hypothesis, we investigated the PAMP-triggered oxidative burst and stomatal closure in an atmin7 knockout mutant line (Nomura et al., 2006). Surprisingly, our results showed that atmin7 mutant Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

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Fig. 2 The proteasome inhibitor MG132 reverts the HopM1-mediated inhibition of PAMP-triggered oxidative burst and stomatal closure. (a) Flg22-induced reactive oxygen species (ROS) burst in the absence and presence of in planta expressed HopM1 upon MG132 treatment. This experiment was repeated at least three times; results from a representative experiment are shown. Error bars,  SE based on n = 28 samples. The Pvalue according to a Student’s t-test is shown. WT, wild-type. (b) Flg22or chitin-induced stomatal closure in the absence or presence of in planta expressed HopM1 upon treatment with the proteasome inhibitor MG132 in Arabidopsis. Each experiment was repeated at least three times; results from a representative experiment are presented. Error bars represent standard error based on n ≥ 30. Statistically significant compared to controls according to Student’s t-test, P < 0.05.

plants were not impaired in either of these PTI responses (Fig. 3), indicating that these phenotypes must rely on the degradation of a different host target. Curiously, the stomata of the mocktreated atmin7 mutant seemed to be more closed than those in wild-type plants under similar conditions, although the responses to flg22, ABA and H2O2 were clearly detectable and statistically significant. At this moment, we cannot exclude the possibility that AtMIN7 may play a role in the regulation of stomatal apertures, for example stomatal opening. HopM1 does not affect components of PTI receptor complexes Some bacterial effectors affecting early PTI responses have been shown to target membrane-localized immune receptors or other components of the receptor complexes (Gohre et al., 2008; Shan et al., 2008; Xiang et al., 2008; Nicaise et al., 2013). To determine whether this is the case for HopM1, we compared the protein accumulation of FLS2, BAK1 and RBOHD in the presence or absence of the effector. As shown in Fig. S4, HopM1 does not Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

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Fig. 3 Loss-of AtMIN7 gene function does not affect PAMP-triggered oxidative burst or stomatal immunity. (a) Flg22- and chitin-induced oxidative burst in wild-type (WT) Col-0 or atmin7 mutant Arabidopsis plants. Reactive oxygen species (ROS) generation (indicated as total photon count) was monitored over time for 30 min. Each experiment was repeated at least three times; results from a representative experiment are shown. Error bars  SE based on n = 28 samples. (b) Flg22-, ABA- or H2O2-mediated stomatal closure in wild-type Col-0 or atmin7 mutant Arabidopsis plants. Error bars,  SE based on n ≥ 30. Statistically significant compared to controls according to Student’s t-test, P < 0.05.

affect the accumulation of these proteins in planta; moreover, formation of the FLS2-BAK1 complex following elicitation with flg22 occurs normally in the presence of the effector (Fig. 4), suggesting that the membrane-based receptor complex formation upon PAMP treatment is not affected by HopM1. HopM1 can alter BZR1 nuclear-cytoplasmic ratio in a similar manner than inhibition of 14-3-3s Because the HopM1-mediated suppression of early PTI responses is independent of AtMIN7 but requires proteasomal activity, the degradation of a different host target must be responsible for these phenotypes. Besides AtMIN7, the other in vivo confirmed target of HopM1 that is destabilized by Pto DC3000 in a CEL-dependent manner is the 14-3-3 protein GRF8/ AtMIN10, which is degraded by this effector in yeast, in transient assays in N. benthamiana, and during Pto DC3000 infection in Arabidopsis (Nomura et al., 2006). GRF8 has been well studied in the control of BZR1, a major transcription factor involved in brassinosteroid (BR) signalling (Gampala et al., 2007; Ryu et al., 2007). Because BZR1 is retained in the cytoplasm by 14-3-3s in the absence of the New Phytologist (2014) 202: 259–269 www.newphytologist.com

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hormone and the nuclear/cytoplasmic ratio of a BZR1-GFP fusion protein has been successfully used as a marker of BZR1 activation (Gampala et al., 2007; Ryu et al., 2007), we used the nuclear/cytoplasmic ratio of a BZR1-YFP fusion protein as a proxy of the functionality of 14-3-3s. Notably, when transiently co-expressed in N. benthamiana, HopM1, but not other bacterial effectors, enhances the nuclear accumulation of BZR1-YFP (Figs 5a,b, S5C). In agreement with the increased nuclear accumulation of BZR1, HopM1 expression results in the downregulation of the BZR1 targets CPD and DWF4 (Sun et al., 2010), indicating an increase in BZR1 function (Fig. 5c). Of note, the HopM1-mediated nuclear accumulation of BZR1-YFP is impaired by the proteasome inhibitor MG132 (Fig. 5b). Taken together, these results suggest that HopM1 interferes with the activity of 14-3-3s in the plant cell, and that this effect requires proteasomal degradation. Stomatal immunity and PAMP-triggered ROS require the function of 14-3-3 proteins 14-3-3 proteins appear to function redundantly in Arabidopsis (Roberts & de Bruxelles, 2002; Gampala et al., 2007; Ryu et al., 2007; Paul et al., 2012), which makes genetic analysis challenging. To overcome this limitation, we used the established method of chemical inhibition with 5-aminoimidazole-4-carboxamide-1b-D-ribofuranoside (AICAR), which disrupts the 14-3-3 interactions with their target proteins (Paul et al., 2005). Because the cytoplasmic retention of BZR1 in Arabidopsis is dependent on 14-3-3 binding (Gampala et al., 2007; Ryu et al., 2007), AICAR increases the nuclear/cytoplasmic ratio of BZR1 in Arabidopsis, resulting in the inhibition of the marker genes CPD and DWF4 (Gampala et al., 2007). Likewise, AICAR triggered an increase in New Phytologist (2014) 202: 259–269 www.newphytologist.com

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Fig. 5 HopM1 triggers nuclear accumulation of BZR1. (a) Confocal micrographs show BZR-YFP localization in control BZR1-YFP or BZR1YFP+HopM1 transiently co-transformed Nicotiana benthamiana leaves; bars, 10 lm. (b) Nuclear/cytoplasmic signal ratio in control BZR1-YFP or BZR1-YFP+HopM1 co-transformed N. benthamiana leaves, treated or not with the proteasome inhibitor MG132. This experiment was repeated six times with similar results; in each case, at least 15 cells were measured. Error bars,  SE. (c) Relative expression of the BZR1-target genes CPD (clear bars) and DWF4 (grey bars) in wild-type Col-0 or transgenic HopM1 Arabidopsis plants as determined by quantitative real-time PCR. Values are the mean of three independent biological replicates; bars,  SE. Statistically significant difference compared to controls according to Student’s t-test: *, P < 0.05. WT, wild-type.

the nuclear/cytoplasmic ratio of BZR1-YFP in transiently transformed N. benthamiana plants (Fig. S5A,B), proving that this compound also inhibits 14-3-3s in this plant species. We then examined the effect of AICAR treatment on PAMP-triggered ROS burst and stomatal closure in both Arabidopsis and N. benthamiana, and observed that AICAR treatment was sufficient to suppress both outputs (Fig. 6a,b,c), suggesting an involvement of 14-3-3s in these early PTI responses. Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

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Partial deletion of the Pto DC3000 effector locus CEL has a severe negative impact in bacterial virulence (Badel et al., 2006; Nomura et al., 2006; Kvitko et al., 2009), which can be reverted by expression of HopM1 in trans (DebRoy et al., 2004; Nomura et al., 2006). Moreover, HopM1 is one of the effectors within the minimal functional effector repertoire of Pto DC3000 (Cunnac et al., 2011). Evidently, the HopM1 effector exerts a critical function in the bacterial interaction with the host plant. HopM1 was shown previously to suppress basal resistance in N. benthamiana, measured as reduced vascular flow (Oh & Collmer, 2005), and decrease callose deposition triggered by Pto DC3000 in Arabidopsis (Nomura et al., 2006). Because these outputs are late immune responses, however, the potential effect of HopM1 on early responses was unknown. In this work, we show that HopM1 is able to suppress two early immune responses, namely the PAMP-triggered ROS burst and stomatal immunity (Figs 1, S1), revealing that the impact of HopM1 on immune responses is broader than previously thought. The inhibition of these responses involves proteasomal degradation of host proteins, because the effect of HopM1 can be partially reverted by the proteasomal inhibitor MG132 (Figs 2, S2). HopM1, however, does not seem to affect the protein accumulation of immune receptors or the PAMP-regulated NADPH oxidase RBOHD (Fig. S4), so additional components, involved in PAMP signal transduction or regulation of outputs, must be targeted by this effector.

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Importantly, if one of the virulence functions exerted by HopM1 is to interfere with host 14-3-3s, AICAR treatment should replace HopM1 function, complementing the virulence deficiency of the Pto DC3000 ΔCEL mutant. Indeed, we found that multiplication of Pto DC3000 ΔCEL bacteria in Arabidopsis was significantly enhanced in the presence of AICAR (Fig. 6d). This effect appeared to be specific, as AICAR did not cause any significant changes in the growth of wild-type Pto DC3000. A double knockout mutant in GRF8 and the closely related homologue GRF6 did not result in any noticeable phenotype (Gampala et al., 2007), suggesting functional redundancy within the 14-3-3 gene family in Arabidopsis. By contrast, knock-down of the GRF8 orthologue in tomato, TFT1, by virus-induced gene silencing (VIGS) caused measurable phenotypes in these plants (Taylor et al., 2012). We used VIGS to knockdown GFR8/TFT1 and observed a compromised flg22-triggered oxidative burst in both tomato and N. benthamiana (Fig. 7a,b). The extent of this phenotype correlated with the level of GRF8/TFT1 silencing (Fig. 7c,d). These data are in agreement with previous findings of TFT1 playing roles in plant immunity (Taylor et al., 2012), and indicate that this 14-3-3 protein is also involved in the PAMPtriggered oxidative burst, further supporting our results from the chemical inhibition approach.

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Fig. 6 Disruption of 14-3-3 interactions with their client proteins is sufficient to suppress PTI responses and restore virulence of the Pto ΔCEL mutant. (a) Flg22-triggered oxidative burst in AICAR-treated or mocktreated plants. Reactive oxygen species (ROS) generation (indicated as total photon count) was monitored over time for 30 min. (b) Flg22-, ABAor H2O2-mediated stomatal closure in AICAR-treated or mock-treated Arabidopsis plants. (c) Flg22-, ABA- or H2O2-mediated stomatal closure in AICAR-treated or mock-treated Nicotiana benthamiana plants. All experiments were repeated at least twice with similar results; results from one representative experiment are shown. Bars,  SE for n = 16 (ROS measurements) or n ≥ 30 (stomatal measurements). Statistically significant difference compared to controls according to Student’s t-test: *, P < 0.05. (d) Bacterial growth in AICAR- or mock-treated Arabidopsis leaves at 24 h post infiltration. This experiment was repeated twice with similar results; results from one representative experiment are shown. Values are the mean of three independent biological replicates; error bars,  SD. The P-value according to a Student’s t-test is shown. New Phytologist (2014) 202: 259–269 www.newphytologist.com

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bacteria successfully invading the leaf tissues could aid those living epiphytically to penetrate through stomata. Further studies will be required in order to determine whether this is the case.

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Fig. 7 Knockdown of the 14-3-3 TFT1 in tomato (Solanum lycopersicum) and Nicotiana benthamiana compromises the PAMP-triggered reactive oxygen species (ROS) burst. (a) Flg22-triggered oxidative burst in control (TRV; open bars) or TFT1-silenced (TRV-TFT1; closed bars) tomato plants; and (b) in N. benthamiana plants. ROS generation (indicated as total photon count) was monitored over time for 30 min. This experiment was repeated four times with similar results; results from a representative experiment are shown. Error bars,  SE based on n = 21 leaf discs taken from five plants. (c) Relative TFT1 expression in control (TRV) or TFT1silenced (TRV-TFT1) tomato plants, and (d) N. benthamiana plants as determined by quantitative real-time PCR. Values are the mean of four independent biological replicates; bars,  SE. Statistically significant difference compared to controls according to Student’s t-test: *, P < 0.05.

The PAMP-triggered oxidative burst is required for stomatal closure upon pathogen detection, and hence our data suggest that HopM1 may compromise stomatal immunity through the suppression of ROS production. However, HopM1 has also been found to suppress salicylic acid-dependent basal defences (DebRoy et al., 2004) and, as this hormone has been shown to play roles in stomatal aperture regulation (Melotto et al., 2006), we cannot exclude the possibility that such activity might also affect immunity at this level. Interestingly, in planta expressed HopM1 could complement the lack of COR in the Pto DC3000 COR strain in triggering stomatal re-opening (Fig. 1b). Notably, even though Pto DC3000 can produce endogenous HopM1, effectors are not expected to be secreted through the Type-III secretion system at the short time used in this experiment (Thwaites et al., 2004). Together with the inhibition of PAMP-induced stomatal closure by the effector, this finding can give rise to the hypothesis that the role of HopM1 as a suppressor of stomatal immunity could have biological relevance in natural conditions, in which those New Phytologist (2014) 202: 259–269 www.newphytologist.com

HopM1 has been found previously to bind and destabilize the plant ARF-GEF AtMIN7, likely promoting bacterial virulence through the interference with host vesicle trafficking, leading to a suppression of the cell wall-associated defence (Nomura et al., 2006). Supporting this, Brefeldin A (BFA)-mediated inhibition of vesicle trafficking can substitute for HopM1 and restore the virulence of the Pto DC3000 ΔCEL mutant (Nomura et al., 2006). However, HopM1 binds and destabilizes at least another seven plant proteins in yeast (Nomura et al., 2006). Additionally, a knockout mutant of AtMIN7 does not fully complement the Pto DC3000 ΔCEL mutation (Nomura et al., 2006). Taken together, these results suggest that HopM1 is a multifunctional effector that may target several different plant proteins to promote bacterial virulence. Our results show that HopM1 can suppress early PTI responses, and this activity requires proteasomal degradation but is independent of AtMIN7 (Fig. 3). Consequently, the suppression of these responses must rely on the degradation of a different plant target. Several plant proteins have been shown to bind HopM1 in yeast, and one other than AtMIN7, AtMIN10, the 14-3-3 protein GRF8, is destabilized by this effector in yeast, in N. benthamiana, and during Pto DC3000 infection in Arabidopsis in a CEL-dependent manner (Nomura et al., 2006). HopM1, which localizes to endomembranes, was shown to mainly degrade membrane-associated GRF8/AtMIN10 (Nomura et al., 2006, 2011). This is in agreement with the previously described function of GRF8 regulating nucleo-cytoplasmic shuttling of BZR1 in Arabidopsis, of which phosphorylated BZR1 is enriched in membrane fractions (Gampala et al., 2007). Our findings that MG132 can at least partially revert the HopM1-mediated suppression of PAMP-triggered stomatal closure seem to stand in contrast to a previous report showing that inhibition of the proteasome by MG132 and syringoline A counteracts stomatal immunity (Schellenberg et al., 2010). However, in that report the effect of proteasome inhibition was measured after bacterial treatments, thereby primarily investigating the role of proteasome inhibition in stomatal re-opening. By contrast, our experiments addressed the effect of the proteasome during pathogen-triggered stomatal closure. Although we cannot rule out a possible influence of the different experimental settings, it could be that the proteasome can play roles in both stomatal opening and inhibition of stomatal closure. This would also indicate that pathogens deploy different strategies to counter stomatal immunity by inhibiting the PAMP-triggered closure of stomata as well as their active re-opening. An increasing body of evidence points at a role of 14-3-3s in plant immunity: the tomato 14-3-3 protein 7 (TFT7) regulates immunity-associated cell death by enhancing the protein accumulation and signalling ability of MAPKKK alpha, and interacts with Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

New Phytologist this MAPKKK and the MAPKK SlMKK2 (Oh et al., 2010; Oh & Martin, 2011); the Pto DC3000 effector HopQ1 targets TFT5 and TFT7 and interacts with these 14-3-3s in a phosphorylationdependent manner (Giska et al., 2013; Li et al., 2013); in rice, the 14-3-3 protein GF14e acts as a negative regulator of cell death and disease resistance (Manosalva et al., 2011); and the effector XopN from Xhantomonas campestris pv vesicatoria targets the tomato 14-3-3 isoform TFT1, the Arabidopsis GRF8 orthologue, to promote bacterial pathogenesis (Taylor et al., 2012). The full extent of the role of different 14-3-3s in defence and immunity, however, still needs to be further explored. We found that chemical inhibition of 14-3-3s results in suppression of PAMP-triggered ROS burst and stomatal immunity, mimicking the effects of HopM1 on these early immune responses (Fig. 6), and, strikingly, could complement the lack of the CEL locus in promoting bacterial virulence (Fig. 6c). Although results based on the use of potent chemical inhibitors, such as AICAR, should be considered cautiously, our findings are supported by genetic data that independently point at an involvement of 14-3-3 proteins in early PTI responses, thus providing validity to our chemical approach. Taking advantage of the use of the nuclear/ cytoplasmic ratio of BZR1-YFP as a proxy for 14-3-3 activity, we could show that HopM1 is likely negatively affecting 14-3-3s (Fig. 5); because this effect can be counteracted by proteasomal inhibition (Fig. 5b), HopM1-mediated degradation of a host protein must underpin the observed inhibition of 14-3-3 activity. Additionally, we showed that knockdown of the tomato GRF8 orthologue, TFT1, suppresses the flg22-triggered oxidative burst; and a similar phenotype was observed in N. benthamiana. This result unveils an involvement of the 14-3-3 protein isoform TFT1 in the regulation of this early PTI response. Taking all of these results together and considering the destabilizing effect of HopM1 on the 14-3-3 GRF8 described by Nomura et al. (2006), we hypothesize that HopM1 may trigger the degradation of GRF8/ TFT1, and likely other redundant members of the family in Arabidopsis, to suppress early PAMP-triggered responses (see model in Fig. S6). Considering that HopM1 can affect the nucleo/cytoplasmic shuttling of BZR1, we could also speculate that HopM1 may suppress flg22 responses through the described negative crosstalk with BR (Albrecht et al., 2012; Belkhadir et al., 2012). Given that RBOHD seems to be required for PAMP-triggered stomatal immunity (Mersmann et al., 2010), the HopM1-mediated suppression of PAMP-triggered stomatal closure may be a consequence of the effect of this effector on PAMP-triggered ROS. Interestingly, a 14-3-3 protein, Nt14-3-3 h/omega1, binds the C-terminus of the tobacco NADPH oxidase NtrbohD in yeast, and expression of an antisense construct of this 14-3-3 abolishes PAMP-triggered ROS accumulation (Elmayan et al., 2007). However, the direct or indirect nature of this effect, as well as the isoform specificity of the antisense construct, remains to be clarified. The results presented here expand on the role of HopM1 in virulence described to date: HopM1 suppresses both early and late immune responses by promoting the degradation of at least two different host proteins, making HopM1 a multifunctional effector. Because we could show that 14-3-3s have a role in early Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

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PTI responses, and at least one member of this family is targeted by HopM1, these proteins are good candidates to mediate the HopM1-triggered suppression of early immune responses upon destabilization by the effector. Further experiments, however, will be required to unravel the exact molecular mechanisms underlying these novel effects of HopM1.

Acknowledgements We thank Heidrun Haweker for excellent technical assistance, Ji Zhou for advice on statistics, members of the Robatzek group for fruitful discussions, Cyril Zipfel for providing the anti-BAK1 antibody and Mary Beth Mudgett for sharing the TRV-TFT1 constructs. This work was supported by the Gatsby Charitable Foundation, the European Research Council (ERC Young Investigator to S.R.), and a Fundacion Ramon Areces postdoctoral fellowship to R.L.-D. S.Y.H. is a Howard Hughes Medical Institute-Gordon and Betty Moore Foundation Investigator.

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Suppression of the PAMP-triggered oxidative burst and stomatal closure by HopM1 in N. benthamiana and characterization of HopM1 expression. Fig. S2 The proteasome inhibitor MG132 reverts the HopM1mediated inhibition of PAMP-triggered stomatal closure in N. benthamiana. Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust

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Fig. S3 Confocal imaging of stomata. Fig. S4 HopM1 does not affect the accumulation of the receptor FLS2 or the NADPH oxidase RBOHD. Fig. S5 AICAR inhibits 14-3-3 proteins in N. benthamiana, and HopM1, but not other bacterial effectors, exerts a similar effect.

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Fig. S6 Proposed model for the effect of HopM1 on PAMPtriggered ROS burst and stomatal closure. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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