A bacterial type III effector targets plant vesicle-associated membrane proteins.

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.

A plasma membrane nucleotide-binding leucine-rich repeat receptor mediates the recognition of the Ralstonia pseudosolanacearum effector RipY in Nicotiana benthamiana.

Bacterial wilt disease caused by several Ralstonia species is one of the most destructive diseases in Solanaceae crops. Only a few functional resistance genes against bacterial wilt have been cloned to date. Here, we show that the broadly conserved type III secreted effector RipY is recognized by the Nicotiana benthamiana immune system, leading to cell death induction, induction of defense-related gene expression, and restriction of bacterial pathogen growth. Using a multiplexed virus-induced gene-silencing-based N. benthamiana nucleotide-binding and leucine-rich repeat receptor (NbNLR) library, we identified a coiled-coil (CC) nucleotide-binding and leucine-rich repeat receptor (CNL) required for recognition of RipY, which we named RESISTANCE TO RALSTONIA SOLANACEARUM RIPY (RRS-Y). Genetic complementation assays in RRS-Y-silenced plants and stable rrs-y knockout mutants demonstrated that RRS-Y is sufficient to activate RipY-induced cell death and RipY-induced immunity to Ralstonia pseudosolanacearum. RRS-Y function is dependent on the phosphate-binding loop motif of the nucleotide-binding domain but independent of the characterized signaling components ENHANCED DISEASE SUSCEPTIBILITY 1, ACTIVATED DISEASE RESISTANCE 1, and N REQUIREMENT GENE 1 and the NLR helpers NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH-2, -3, and -4 in N. benthamiana. We further show that RRS-Y localization at the plasma membrane is mediated by two cysteine residues in the CC domain and is required for RipY recognition. RRS-Y also broadly recognizes RipY homologs across Ralstonia species. Lastly, we show that the C-terminal region of RipY is indispensable for RRS-Y activation. Together, our findings provide an additional effector/receptor pair system to deepen our understanding of CNL activation in plants.

The Ralstonia pseudosolanacearum effector RipE1 is recognized at the plasma membrane by NbPtr1, the Nicotiana benthamiana homologue of Pseudomonas tomato race 1.

The bacterial wilt disease caused by soilborne bacteria of the Ralstonia solanacearum species complex (RSSC) threatens important crops worldwide. Only a few immune receptors conferring resistance to this devastating disease are known so far. Individual RSSC strains deliver around 70 different type III secretion system effectors into host cells to manipulate the plant physiology. RipE1 is an effector conserved across the RSSC and triggers immune responses in the model solanaceous plant Nicotiana benthamiana. Here, we used multiplexed virus-induced gene silencing of the nucleotide-binding and leucine-rich repeat receptor family to identify the genetic basis of RipE1 recognition. Specific silencing of the N. benthamiana homologue of Solanum lycopersicoides Ptr1 (confers resistance to Pseudomonas syringae pv. tomato race 1) gene (NbPtr1) completely abolished RipE1-induced hypersensitive response and immunity to Ralstonia pseudosolanacearum. The expression of the native NbPtr1 coding sequence was sufficient to restore RipE1 recognition in Nb-ptr1 knockout plants. Interestingly, RipE1 association with the host cell plasma membrane was necessary for NbPtr1-dependent recognition. Furthermore, NbPtr1-dependent recognition of RipE1 natural variants is polymorphic, providing additional evidence for the indirect mode of activation of NbPtr1. Altogether, this work supports NbPtr1 relevance for resistance to bacterial wilt disease in Solanaceae.

A Fast and Easy Method to Study Ralstonia solanacearum Virulence upon Transient Gene Expression or Gene Silencing in Nicotiana benthamiana Leaves.

Ralstonia solanacearum is a devastating soil-borne bacterial pathogen that causes disease in multiple host plants worldwide. Typical assays to measure virulence of R. solanacearum in laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a novel inoculation protocol to analyze the replication of R. solanacearum upon infiltration into the leaves of Nicotiana benthamiana, in which gene expression has been altered using Agrobacterium tumefaciens. The protocol includes five major steps: 1) growth of N. benthamiana plants; 2) infiltration of A. tumefaciens; 3) R. solanacearum inoculation; 4) sample collection and bacterial quantitation; 5) data analysis and representation. The transient gene expression or gene silencing prior to R. solanacearum inoculation provides a straightforward way to perform genetic analysis of plant functions involved in the interaction between pathogen and host, using the appropriate combination of A. tumefaciens and R. solanacearum strains, with high sensitivity and accuracy provided by the quantitation of bacterial numbers in plant tissues.

Synaptotagmins at the endoplasmic reticulum-plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress.

Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

Split-luciferase Complementation Imaging Assay to Study Protein-protein Interactions in Nicotiana benthamiana.

The experimental identification of protein-protein interactions (PPIs) is critical to understand protein function. Thus, a plethora of sensitive and versatile approaches have been developed to detect PPIs in vitro or in vivo, such as protein pull-down, yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP), and bimolecular fluorescence complementation (BiFC) assays. The recently established split-luciferase complementation (Split-LUC) imaging assay has several advantages compared to other approaches to detect PPIs in planta: it is a relatively simple and fast method to detect PPIs in vivo; the results are quantitative, with high sensitivity and low background; it measures dynamic PPIs in real-time; and it requires limited experimental materials and instrumentation. In this assay, the amino-terminal and carboxyl-terminal halves of the luciferase enzyme are fused to two proteins of interest (POIs), respectively; the luciferase protein is reconstituted when two POIs interact with each other, giving rise to a measurable activity. Here, we describe a protocol for the Split-LUC imaging assay using a pair of modified gateway-compatible vectors upon Agrobacterium-mediated transient expression in Nicotiana benthamiana. With this setup, we have successfully confirmed a series of interactions among virus-plant proteins, virus-virus proteins, plant-plant proteins, or bacteria-plant proteins in N. benthamiana.

SGT1 is not required for plant LRR-RLK-mediated immunity.

Plant immune signalling activated by the perception of pathogen-associated molecular patterns (PAMPs) or effector proteins is mediated by pattern-recognition receptors (PRRs) and nucleotide-binding and leucine-rich repeat domain-containing receptors (NLRs), which often share cellular components and downstream responses. Many PRRs are leucine-rich repeat receptor-like kinases (LRR-RLKs), which mostly perceive proteinaceous PAMPs. The suppressor of the G2 allele of skp1 (SGT1) is a core immune regulator required for the activation of NLR-mediated immunity. In this work, we examined the requirement of SGT1 for immune responses mediated by several LRR-RLKs in both Nicotiana benthamiana and Arabidopsis. Using complementary genetic approaches, we found that SGT1 is not limiting for early PRR-dependent responses or antibacterial immunity. We therefore conclude that SGT1 does not play a significant role in bacterial PAMP-triggered immunity.

Intra-strain Elicitation and Suppression of Plant Immunity by Ralstonia solanacearum Type-III Effectors in Nicotiana benthamiana.

Effector proteins delivered inside plant cells are powerful weapons for bacterial pathogens, but this exposes the pathogen to potential recognition by the plant immune system. Therefore, the effector repertoire of a given pathogen must be balanced for a successful infection. Ralstonia solanacearum is an aggressive pathogen with a large repertoire of secreted effectors. One of these effectors, RipE1, is conserved in most R. solanacearum strains sequenced to date. In this work, we found that RipE1 triggers immunity in N. benthamiana, which requires the immune regulator SGT1, but not EDS1 or NRCs. Interestingly, RipE1-triggered immunity induces the accumulation of salicylic acid (SA) and the overexpression of several genes encoding phenylalanine-ammonia lyases (PALs), suggesting that the unconventional PAL-mediated pathway is responsible for the observed SA biosynthesis. Surprisingly, RipE1 recognition also induces the expression of jasmonic acid (JA)-responsive genes and JA biosynthesis, suggesting that both SA and JA may act cooperatively in response to RipE1. We further found that RipE1 expression leads to the accumulation of glutathione in plant cells, which precedes the activation of immune responses. R. solanacearum secretes another effector, RipAY, which is known to inhibit immune responses by degrading cellular glutathione. Accordingly, RipAY inhibits RipE1-triggered immune responses. This work shows a strategy employed by R. solanacearum to counteract the perception of its effector proteins by plant immune system.

A bacterial effector protein prevents MAPK-mediated phosphorylation of SGT1 to suppress plant immunity.

Nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as sensors that perceive pathogen molecules and activate immunity. In plants, the accumulation and activation of NLRs is regulated by SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1). In this work, we found that an effector protein named RipAC, secreted by the plant pathogen Ralstonia solanacearum, associates with SGT1 to suppress NLR-mediated SGT1-dependent immune responses, including those triggered by another R. solanacearum effector, RipE1. RipAC does not affect the accumulation of SGT1 or NLRs, or their interaction. However, RipAC inhibits the interaction between SGT1 and MAP kinases, and the phosphorylation of a MAPK target motif in the C-terminal domain of SGT1. Such phosphorylation is enhanced upon activation of immune signaling and contributes to the activation of immune responses mediated by the NLR RPS2. Additionally, SGT1 phosphorylation contributes to resistance against R. solanacearum. Our results shed light onto the mechanism of activation of NLR-mediated immunity, and suggest a positive feedback loop between MAPK activation and SGT1-dependent NLR activation.

A Bacterial Effector Protein Hijacks Plant Metabolism to Support Pathogen Nutrition.

Many bacterial plant pathogens employ a type III secretion system to inject effector proteins within plant cells to suppress plant immunity. Whether and how effector proteins also co-opt plant metabolism to support extensive bacterial replication remains an open question. Here, we show that Ralstonia solanacearum, the causal agent of bacterial wilt disease, secretes the effector protein RipI, which interacts with plant glutamate decarboxylases (GADs) to alter plant metabolism and support bacterial growth. GADs are activated by calmodulin and catalyze the biosynthesis of gamma-aminobutyric acid (GABA), an important signaling molecule in plants and animals. RipI promotes the interaction of GADs with calmodulin, enhancing the production of GABA. R. solanacearum is able to replicate efficiently using GABA as a nutrient, and both RipI and plant GABA contribute to a successful infection. This work reveals a pathogenic strategy to hijack plant metabolism for the biosynthesis of nutrients that support microbial growth during plant colonization.

Molecular Characterization of ZosmaNRT2, the Putative Sodium Dependent High-Affinity Nitrate Transporter of Zostera marina L.

One of the most important adaptations of seagrasses during sea colonization was the capacity to grow at the low micromolar nitrate concentrations present in the sea. In contrast to terrestrial plants that use H+ symporters for high-affinity NO3- uptake, seagrasses such as Zostera marina L. use a Na+-dependent high-affinity nitrate transporter. Interestingly, in the Z. marina genome, only one gene (Zosma70g00300.1; NRT2.1) is annotated to this function. Analysis of this sequence predicts the presence of 12 transmembrane domains, including the MFS domains of the NNP transporter family and the "nitrate signature" that appears in all members of the NNP family. Phylogenetic analysis shows that this sequence is more related to NRT2.5 than to NRT2.1, sharing a common ancestor with both monocot and dicot plants. Heterologous expression of ZosmaNRT2-GFP together with the high-affinity nitrate transporter accessory protein ZosmaNAR2 (Zosma63g00220.1) in Nicotiana benthamiana leaves displayed four-fold higher fluorescence intensity than single expression of ZosmaNRT2-GFP suggesting the stabilization of NRT2 by NAR2. ZosmaNRT2-GFP signal was present on the Hechtian-strands in the plasmolyzed cells, pointing that ZosmaNRT2 is localized on the plasma membrane and that would be stabilized by ZosmaNAR2. Taken together, these results suggest that Zosma70g00300.1 would encode a high-affinity nitrate transporter located at the plasma membrane, equivalent to NRT2.5 transporters. These molecular data, together with our previous electrophysiological results support that ZosmaNRT2 would have evolved to use Na+ as a driving ion, which might be an essential adaptation of seagrasses to colonize marine environments.

A systematic screen of conserved Ralstonia solanacearum effectors reveals the role of RipAB, a nuclear-localized effector that suppresses immune responses in potato.

Both Solanum tuberosum and Ralstonia solanacearum phylotype IIB originated in South America and share a long-term co-evolutionary history. However, our knowledge of potato bacterial wilt pathogenesis is scarce as a result of the technical difficulties of potato plant manipulation. Thus, we established a multiple screening system (virulence screen of effector mutants in potato, growth inhibition of yeast and transient expression in Nicotiana benthamiana) of core type III effectors (T3Es) of a major potato pathovar of phylotype IIB, to provide more research perspectives and biological tools. Using this system, we identified four effectors contributing to virulence during potato infection, with two exhibiting multiple phenotypes in two other systems, including RipAB. Further study showed that RipAB is an unknown protein with a nuclear localization signal (NLS). Furthermore, we generated a ripAB complementation strain and transgenic ripAB-expressing potato plants, and subsequent virulence assays confirmed that R. solanacearum requires RipAB for full virulence. Compared with wild-type potato, transcriptomic analysis of transgenic ripAB-expressing potato plants showed a significant down-regulation of Ca2+ signalling-related genes in the enriched Plant-Pathogen Interaction (PPI) gene ontology (GO) term. We further verified that, during infection, RipAB is required for the down-regulation of four Ca2+ sensors, Stcml5, Stcml23, Stcml-cast and Stcdpk2, and a Ca2+ transporter, Stcngc1. Further evidence showed that the immune-associated reactive oxygen species (ROS) burst is attenuated in ripAB transgenic potato plants. In conclusion, a systematic screen of conserved R. solanacearum effectors revealed an important role for RipAB, which interferes with Ca2+ -dependent gene expression to promote disease development in potato.

The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses.

The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen-induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ-glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.

The Ralstonia solanacearum csp22 peptide, but not flagellin-derived peptides, is perceived by plants from the Solanaceae family.

Ralstonia solanacearum, the causal agent of bacterial wilt disease, is considered one of the most destructive bacterial pathogens due to its lethality, unusually wide host range, persistence and broad geographical distribution. In spite of the extensive research on plant immunity over the last years, the perception of molecular patterns from R. solanacearum that activate immunity in plants is still poorly understood, which hinders the development of strategies to generate resistance against bacterial wilt disease. The perception of a conserved peptide of bacterial flagellin, flg22, is regarded as paradigm of plant perception of invading bacteria; however, no elicitor activity has been detected for R. solanacearum flg22. Recent reports have shown that other epitopes from flagellin are able to elicit immune responses in specific species from the Solanaceae family, yet our results show that these plants do not perceive any epitope from R. solanacearum flagellin. Searching for elicitor peptides from R. solanacearum, we found several protein sequences similar to the consensus of the elicitor peptide csp22, reported to elicit immunity in specific Solanaceae plants. A R. solanacearum csp22 peptide (csp22Rsol ) was indeed able to trigger immune responses in Nicotiana benthamiana and tomato, but not in Arabidopsis thaliana. Additionally, csp22Rsol treatment conferred increased resistance to R. solanacearum in tomato. Transgenic A. thaliana plants expressing the tomato csp22 receptor (SlCORE) gained the ability to respond to csp22Rsol and became more resistant to R. solanacearum infection. Our results shed light on the mechanisms for perception of R. solanacearum by plants, paving the way for improving current approaches to generate resistance against R. solanacearum.

Analysis of PAMP-Triggered ROS Burst in Plant Immunity.

The plant perception of pathogen-associated molecular patterns triggers a plethora of cellular immune responses. One of these responses is a rapid and transient burst of reactive oxygen species (ROS) mediated by plasma membrane-localized NADPH oxidases. The ROS burst requires a functional receptor complex and the contribution of several additional regulatory components. In laboratory conditions, the ROS burst can be detected a few minutes after the treatment with an immunogenic microbial elicitor. For these reasons, the elicitor-triggered ROS burst has been often exploited as readout to probe the contribution of plant components to early immune responses. Here, we describe a detailed protocol for the measurement of elicitor-triggered ROS burst in a simple, fast, and easy manner.

A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

Genetic analysis of the individual contribution to virulence of the type III effector inventory of Pseudomonas syringae pv. phaseolicola.

Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1(Pto) (DC3000) to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of analysing their functions within the context of the infection.

The Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity.

*The Pseudomonas syringae pv syringae type III effector HopZ1a is a member of the HopZ effector family of cysteine-proteases that triggers immunity in Arabidopsis. This immunity is dependent on HopZ1a cysteine-protease activity, and independent of known resistance genes. We have previously shown that HopZ1a-triggered immunity is partially additive to that triggered by AvrRpt2. These partially additive effects could be caused by at least two mechanisms: their signalling pathways share a common element(s), or one effector interferes with the response triggered by the other. *Here, we investigate the molecular basis for the partially additive effect displayed by AvrRpt2- and HopZ1a-triggered immunities, by analysing competitive indices, hypersensitive response and symptom induction, PR-1 accumulation, expression of PR genes, and systemic acquired resistance (SAR) induction. *Partially additive effects between these defence responses require HopZ1a cysteine-protease activity, and also take place between HopZ1a and AvrRps4 or AvrRpm1-triggered responses. We establish that HopZ1a-triggered immunity is independent of salicylic acid (SA), EDS1, jasmonic acid (JA) and ethylene (ET)-dependent pathways, and show that HopZ1a suppresses the induction of PR-1 and PR-5 associated with P. syringae pv tomato (Pto)-triggered effector-triggered immunity (ETI)-like defences, AvrRpt2-triggered immunity, and Pto or Pto (avrRpt2) activation of SAR, and that suppression requires HopZ1a cysteine-protease activity. *Our results indicate that HopZ1a triggers an unusual resistance independent of known pathways and suppresses SA and EDS1-dependent resistance.

Positive regulation of the Hrp type III secretion system in Pseudomonas syringae pv. phaseolicola.

Disease in compatible hosts and induction of the hypersensitive response in resistant plants by most plant-pathogenic bacteria require a functional type III secretion system (T3SS). Expression of T3SS genes responds to host and environmental factors and is induced within the plant. In Pseudomonas syringae, expression of the T3SS requires HrpL, which is transcriptionally upregulated by HrpR and HrpS. In some pathovars, expression of the hrpRS genes is upregulated by the GacA/S two-component system. Additionally, HrpA, the major component of the T3SS pilus, has also been linked to the regulation of the hrpRS gene expression. Previous studies concerning regulation of hypersensitive response and pathogenesis/hypersensitive response conserved (hrp/hrc) gene expression have used mostly in vitro inducing conditions, different pathovars, and methodology. Here, we analyze the roles of HrpL, GacA, and HrpA in the bean pathogen, using single, double, and triple mutants as well as strains ectopically expressing the regulators. We use real-time polymerase chain reaction analysis in vitro and in planta to quantify gene expression and competitive indices and other assays to assess bacterial fitness. Our results indicate that i) HrpL acts as a general virulence regulator that upregulates non-T3SS virulence determinants and downregulates flagellar function; ii) GacA modulates the expression of hrpL, and its contribution to virulence is entirely HrpL dependent; iii) there is a basal HrpL-independent expression of the T3SS genes in rich medium that is important for full activation of the system, maybe by keeping the system primed for rapid activation upon contact with the plant; and iv) HrpA upregulates expression of the T3SS genes and is essential to activate expression of the hrpZ operon upon contact with the plant.