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 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 ΔCEL 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 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 hyper-accumulation 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 ΔCEL 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.