丁香假单胞菌中的III型分泌系统及其调控机制研究进展

丁香假单胞菌(Pseudomonas syringae)是引起许多作物病害的一种革兰氏阴性病原细菌。该细菌入侵寄主植物细胞主要通过其III型分泌系统(type III secretion system,T3SS)将效应蛋白转入到寄主真核细胞内,抑制寄主免疫功能,以达到成功侵染和定殖的目的。III型分泌系统的主调控因子RhpR/S通过感受环境信号的变化直接调控hrpR/S及其他毒力相关通路。同时III型分泌系统基因的表达也受到其他调控因子的影响,包括σ因子HrpL、双组分系统GacA/S、Lon蛋白酶、第二信使分子和环境信号等。本文在简要介绍丁香假单胞菌III型分泌系统组成和功能的基础上,综述丁香假单胞菌III型分泌系统调控机制的最新研究进展,以期为深入探究病原菌的致病机制提供参考和思路。     

Pseudomonas syringae lytic transglycosylases coregulated with the type III secretion system contribute to the translocation of effector proteins into plant cells

Pseudomonas syringae translocates virulence effector proteins into plant cells via a type III secretion system (T3SS) encoded by hrp (for hypersensitive response and pathogenicity) genes. Three genes coregulated with the Hrp T3SS system in P. syringae pv. tomato DC3000 have predicted lytic transglycosylase domains: PSPTO1378 (here designated hrpH), PSPTO2678 (hopP1), and PSPTO852 (hopAJ1). hrpH is located between hrpR and avrE1 in the Hrp pathogenicity island and is carried in the functional cluster of P. syringae pv. syringae 61 hrp genes cloned in cosmid pHIR11. Strong expression of DC3000 hrpH in Escherichia coli inhibits bacterial growth unless the predicted catalytic glutamate at position 148 is mutated. Translocation tests involving C-terminal fusions with a Cya (Bordetella pertussis adenylate cyclase) reporter indicate that HrpH and HopP1, but not HopAJ1, are T3SS substrates. Pseudomonas fluorescens carrying a pHIR11 derivative lacking hrpH is poorly able to translocate effector HopA1, and this deficiency can be restored by HopP1 and HopAJ1, but not by HrpH(E148A) or HrpH_1-241. DC3000 mutants lacking hrpH or hrpH, hopP1, and hopAJ1 combined are variously reduced in effector translocation, elicitation of the hypersensitive response, and virulence. However, the mutants are not reduced in secretion of T3SS substrates in culture. When produced in wild-type DC3000, the HrpH(E148A) and HrpH_1-241 variants have a dominant-negative effect on the ability of DC3000 to elicit the hypersensitive response in nonhost tobacco and to grow and cause disease in host tomato. The three Hrp-associated lytic transglycosylases in DC3000 appear to have overlapping functions in contributing to T3SS functions during infection.     

Pseudomonas syringae pv. tomato DC3000 polymutants deploying coronatine and two type III effectors produce quantifiable chlorotic spots from individual bacterial colonies in Nicotiana benthamiana leaves

Primary virulence factors of Pseudomonas syringae pv. tomato DC3000 include the phytotoxin coronatine (COR) and a repertoire of 29 effector proteins injected into plant cells by the type III secretion system (T3SS). DC3000 derivatives differentially producing COR, the T3SS machinery and subsets of key effectors were constructed and assayed in leaves of Nicotiana benthamiana. Bacteria were inoculated by the dipping of whole plants and assayed for population growth and the production of chlorotic spots on leaves. The strains fell into three classes. Class I strains are T3SS⁺ but functionally effectorless, grow poorly in planta and produce faint chlorotic spots only if COR⁺. Class II strains are T3SS^– or, if T3SS⁺, also produce effectors AvrPtoB and HopM1. Class II strains grow better than class I strains in planta and, if COR⁺, produce robust chlorotic spots. Class III strains are T3SS⁺ and minimally produce AvrPtoB, HopM1 and three other effectors encoded in the P. syringae conserved effector locus. These strains differ from class II strains in growing better in planta, and produce chlorotic spots without COR if the precursor coronafacic acid is produced. Assays for chlorotic spot formation, in conjunction with pressure infiltration of low‐level inoculum and confocal microscopy of fluorescent protein‐labelled bacteria, revealed that single bacteria in the apoplast are capable of producing colonies and associated leaf spots in a 1 : 1 : 1 manner. However, COR makes no significant contribution to the bacterial colonization of the apoplast, but, instead, enables a gratuitous, semi‐quantitative, surface indicator of bacterial growth, which is determined by the strain's effector composition.     

Global transcriptional responses of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> DC3000 to changes in iron bioavailability <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Pseudomonas syringae pv tomato DC3000 (DC3000) is a Gram-negative model plant pathogen that is found in a wide variety of environments. To survive in these diverse conditions it must sense and respond to various environmental cues. One micronutrient required for most forms of life is iron. Bioavailable iron has been shown to be an important global regulator for many bacteria where it not only regulates a wide variety of genes involved in general cell physiology but also virulence determinants. In this study we used microarrays to study differential gene regulation in DC3000 in response to changes in levels of cell-associated iron.     

Global Analysis of the HrpL Regulon in the Plant Pathogen Pseudomonas syringae pv. tomato DC3000 Reveals New Regulon Members with Diverse Functions

The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen Pseudomonas syringae pv. tomato DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a promoter sequence, often designated as the “hrp promoter.” Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely hrp promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new hrp promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. PSPTO_5633 is the only new HrpL regulon member that is potentially an effector and is now designated HopBM1. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or ΔhopQ1-1 backgrounds, do not affect the hypersensitive response or in planta growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new hrp promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors.     

The c‐di‐GMP phosphodiesterase BifA is involved in the virulence of bacteria from the Pseudomonas syringae complex

In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c‐di‐GMP) phosphodiesterase‐encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free‐living and virulence‐related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour‐inducing pathogen of woody hosts, P. savastanoi pv. savastanoi NCPPB 3335, and the pathogen of tomato and Arabidopsis, P. syringae pv. tomato DC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P. savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real‐time monitoring of olive plants infected with green fluorescent protein (GFP)‐tagged P. savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild‐type strain. All free‐living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P. aeruginosa BifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P. syringae and P. savastanoi BifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P. syringae complex provides confirmation of the role of c‐di‐GMP signalling in the virulence of this group of plant pathogens.     

GacA directly regulates expression of several virulence genes in Pseudomonas syringae pv. tabaci 11528

A two-component system comprising GacS and GacA affects a large number of traits in many Gram-negative bacteria. However, the signals to which GacS responds, the regulation mechanism for GacA expression, and the genes GacA controls are not yet clear. In this study, several phenotypic tests and tobacco-leaf pathogenicity assays were conducted using a gacA deletion mutant strain (BL473) of Pseudomonas syringae pv. tabaci 11528. To determine the regulation mechanism for gacA gene expression and to identify GacA-regulated genes, we conducted quantitative RT-PCR and electrophoretic mobility shift assay (EMSA) experiments. The results indicated that virulence traits related to the pathogenesis of P. syringae pv. tabaci 11528 are regulated coordinately by GacA and iron availability. They also revealed that several systems coordinately regulate gacA gene expression in response to iron concentration and bacterial cell density and that GacA and iron together control the expression of several virulence genes. EMSA results provided genetic and molecular evidence for direct control of virulence genes by GacA.     

Consequences of flagellin export through the type III secretion system of Pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant Nicotiana benthamiana

Bacterial flagellin is perceived as a microbe (or pathogen)‐associated molecular pattern (MAMP or PAMP) by the extracellular pattern recognition receptors, FLS2 and TLR5, of plants and mammals respectively. Flagellin accidently translocated into mammalian cells by pathogen type III secretion systems (T3SSs) is recognized by nucleotide‐binding leucine‐rich repeat receptor NLRC4 as a pattern of pathogenesis and induces a death‐associated immune response. The non‐pathogen Pseudomonas fluorescens Pf0‐1, expressing a Pseudomonas syringae T3SS, and the plant pathogen P. syringae pv. tomato DC3000 were used to seek evidence of an analogous cytoplasmic recognition system for flagellin in the model plant Nicotiana benthamiana. Flagellin (FliC) was secreted in culture and translocated into plant cells by the T3SS expressed in Pf0‐1 and DC3000 and in their ΔflgGHI flagellar pathway mutants. ΔfliC and ΔflgGHI mutants of Pf0‐1 and DC3000 were strongly reduced in elicitation of reactive oxygen species production and in immunity induction as indicated by the ability of challenge bacteria inoculated 6 h later to translocate a type III effector–reporter and to elicit effector‐triggered cell death. Agrobacterium‐mediated transient expression in N. benthamiana of FliC with or without a eukaryotic export signal peptide, coupled with virus‐induced gene silencing of FLS2, revealed no immune response that was not FLS2 dependent. Transiently expressed FliC from DC3000 and Pectobacterium carotovorum did notinduce cell death in N. benthamiana, tobacco or tomato leaves. Flagellin is the major Pseudomonas MAMP perceived by N. benthamiana, and although flagellin secretion through the plant cell wall by the T3SS may partially contribute to FLS2‐dependent immunity, flagellin in the cytosol does not elicit immune‐associated cell death. We postulate that a death response to translocated MAMPs would produce vulnerability to the many necrotrophic pathogens of plants, such as P. carotovorum, which differ from P. syringae and other (hemi)biotrophic pathogens in benefitting from death‐associated immune responses.     

A Boolean Model of the Pseudomonas syringae hrp Regulon Predicts a Tightly Regulated System

The Type III secretion system (TTSS) is a protein secretion machinery used by certain gram-negative bacterial pathogens of plants and animals to deliver effector molecules to the host and is at the core of the ability to cause disease. Extensive molecular and biochemical study has revealed the components and their interactions within this system but reductive approaches do not consider the dynamical properties of the system as a whole. In order to gain a better understanding of these dynamical behaviours and to create a basis for the refinement of the experimentally derived knowledge we created a Boolean model of the regulatory interactions within the hrp regulon of Pseudomonas syringae pathovar tomato strain DC3000 Pseudomonas syringae. We compared simulations of the model with experimental data and found them to be largely in accordance, though the hrpV node shows some differences in state changes to that expected. Our simulations also revealed interesting dynamical properties not previously predicted. The model predicts that the hrp regulon is a biologically stable two-state system, with each of the stable states being strongly attractive, a feature indicative of selection for a tightly regulated and responsive system. The model predicts that the state of the GacS/GacA node confers control, a prediction that is consistent with experimental observations that the protein has a role as master regulator. Simulated gene “knock out” experiments with the model predict that HrpL is a central information processing point within the network.     

Pseudomonas syringae colonizes distant tissues in Nicotiana benthamiana through xylem vessels

The ability to move from the primary infection site and colonize distant tissue in the leaf is an important property of bacterial plant pathogens, yet this aspect has hardly been investigated for model pathogens. Here we show that GFP‐expressing Pseudomonas syringae pv. syringae DC3000 that lacks the HopQ1‐1 effector (PtoDC3000ΔhQ) has a strong capacity to colonize distant leaf tissue from wound‐inoculated sites in N. benthamiana. Distant colonization occurs within 1 week after toothpick inoculation and is characterized by distant colonies in the apoplast along the vasculature. Distant colonization is blocked by the non‐host resistance response triggered by HopQ1‐1 in an SGT1‐dependent manner and is associated with an explosive growth of the bacterial population, and displays robust growth differences between compatible and incompatible interactions. Scanning electron microscopy revealed that PtoDC3000ΔhQ bacteria are present in xylem vessels, indicating that they use the xylem to move through the leaf blade. Distant colonization does not require flagellin‐mediated motility, and is common for P. syringae pathovars that represent different phylogroups.     

FleQ Coordinates Flagellum-Dependent and -Independent Motilities in Pseudomonas syringae pv. tomato DC3000

Motility plays an essential role in bacterial fitness and colonization in the plant environment, since it favors nutrient acquisition and avoidance of toxic substances, successful competition with other microorganisms, the ability to locate the preferred hosts, access to optimal sites within them, and dispersal in the environment during the course of transmission. In this work, we have observed that the mutation of the flagellar master regulatory gene, fleQ, alters bacterial surface motility and biosurfactant production, uncovering a new type of motility for Pseudomonas syringae pv. tomato DC3000 on semisolid surfaces. We present evidence that P. syringae pv. tomato DC3000 moves over semisolid surfaces by using at least two different types of motility, namely, swarming, which depends on the presence of flagella and syringafactin, a biosurfactant produced by this strain, and a flagellum-independent surface spreading or sliding, which also requires syringafactin. We also show that FleQ activates flagellum synthesis and negatively regulates syringafactin production in P. syringae pv. tomato DC3000. Finally, it was surprising to observe that mutants lacking flagella or syringafactin were as virulent as the wild type, and only the simultaneous loss of both flagella and syringafactin impairs the ability of P. syringae pv. tomato DC3000 to colonize tomato host plants and cause disease.     

Distinct Pseudomonas type‐III effectors use a cleavable transit peptide to target chloroplasts

The pathogen Pseudomonas syringae requires a type‐III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type‐III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type‐III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector‐triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP‐triggered immune responses compared with wild‐type plants. An N‐terminal region of HopK1 shares similarity with the corresponding region in the well‐studied type‐III effector AvrRps4; however, their C‐terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast localized, indicating that the shared N‐terminal regions of these type‐III effectors represent a chloroplast transit peptide. The HopK1 contribution to virulence and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4‐induced HR did not. Our results suggest that a primary virulence target of these type‐III effectors resides in chloroplasts, and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type‐III effectors use a cleavable transit peptide to localize to chloroplasts, and that targets within this organelle are important for immunity.     

Pseudomonas syringae naturally lacking the canonical type III secretion system are ubiquitous in nonagricultural habitats,are phylogenetically diverse and can be pathogenic

The type III secretion system (T3SS) is an important virulence factor of pathogenic bacteria, but the natural occurrence of variants of bacterial plant pathogens with deficiencies in their T3SS raises questions about the significance of the T3SS for fitness. Previous work on T3SS-deficient plant pathogenic bacteria has focused on strains from plants or plant debris. Here we have characterized T3SS-deficient strains of Pseudomonas syringae from plant and nonplant substrates in pristine nonagricultural contexts, many of which represent recently described clades not yet found associated with crop plants. Strains incapable of inducing a hypersensitive reaction (HR⁻) in tobacco were detected in 65% of 126 samples from headwaters of rivers (mountain creeks and lakes), snowpack, epilithic biofilms, wild plants and leaf litter and constituted 2 to 100% of the P. syringae population associated with each sample. All HR⁻ strains lacked at least one gene in the canonical hrp/hrc locus or the associated conserved effector locus, but most lacked all six of the genes tested (hrcC, hrpL, hrpK1, avrE1 and hrpW1) and represented several disparate phylogenetic clades. Although most HR⁻ strains were incapable of causing symptoms on cantaloupe seedlings as expected, strains in the recently described TA-002 clade caused severe symptoms in spite of the absence of any of the six conserved genes of the canonical T3SS according to PCR and Southern blot assays. The phylogenetic context of the T3SS variants we observed provides insight into the evolutionary history of P. syringae as a pathogen and as an environmental saprophyte.     

Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.Many proteobacterial pathogens use a type III secretion system (T3SS) as their primary mechanism to overcome and infect eukaryotic hosts. T3SSs are complex macromolecular machines that span both the bacterial cell envelope and host cell barriers to deliver proteins, commonly termed effectors, from the bacterial cytoplasm into the host cytoplasm (13, 19). After delivery into the host, effector proteins manipulate host cell function and suppress host defenses, allowing bacterial proliferation and disease development (6, 20). Bacteria that rely on T3SS to cause disease include plant pathogens such as Pseudomonas syringae, Ralstonia solanacearum, Erwinia and Xanthomonas species and animal pathogens in the genera Yersinia, Salmonella, Shigella, Escherichia, and Pseudomonas. While the repertoire of effectors delivered by a given T3SS is unique, the T3SS machinery is more universal (13). T3SS includes a core set of eight conserved proteins. These proteins, which are also conserved in bacterial flagellar biogenesis machines, make up the multiringed base structure, or basal body, that spans the bacterial membranes and cell wall. T3SS machines are also comprised of less-conserved and unique proteins that vary between systems. These include regulatory proteins that orchestrate construction of the machine and the extracellular components that function to translocate effectors across host barriers.The extracellular portion of the T3SS is comprised of the pilus or needle appendage (in plant or animal pathogens, respectively), which acts as a conduit for effector delivery, and the translocon complex, which creates the pore in the host cell membrane. These substructures vary between different T3SSs; presumably these external structures have adapted to allow different bacteria to infect different types of host cells. For Yersinia enterocolitica to infect macrophage cells, the T3SS needle must be a particular length (∼58 nm) to bridge the lipopolysaccharides extending from the bacterial outer membrane and reach the host cell membrane (35). Several other animal pathogens have T3SS needles of a defined length (48). Enteropathogenic Escherichia coli also has an additional extension beyond the needle called the EspA filament that functions to span the mucous layer found outside enterocyte cells (13). In plant pathogens, however, the extracellular gap between a bacterium and a plant cell includes a thick plant cell wall that is variable in width between plant species. Consequently, plant pathogenic Pseudomonas syringae has a pilus that can measure over 1 μm in vitro (25).Another major difference between the T3SS machineries of animal and plant pathogens is their translocon complexes. In animal pathogens, these are typically comprised of three essential proteins, but there is growing evidence that plant pathogen translocons employ diverse, functionally redundant components (28). There is growing interest in understanding the regulatory players that orchestrate the construction of diverse machinery. It is hypothesized that the assembly of the T3SS must involve several tightly regulated steps that allow secretion of the required components, followed by that of effectors upon completion. Of particular interest here is the control of pilus/needle subunit secretion, which is necessary when the pilus/needle is being constructed but would presumably compete with translocon and effector secretion after the T3SS is complete.We study the model plant pathogen P. syringae pv. tomato (Pto) DC3000, the causal agent of bacterial speck of tomato and Arabidopsis thaliana (8). DC3000 has a T3SS that delivers ca. 28 effectors and is essential for pathogenesis (11, 12, 30, 43). The P. syringae T3SS is encoded by hrp and hrc genes (hypersensitive response and pathogenicity/conserved), which are located in a pathogenicity island on the chromosome (4). hrc genes encode the conserved core components present in every T3SS. hrp genes encode T3SS components that are divergent or unique to P. syringae and enterobacterial plant pathogens, which also possess Hrp1 class T3SS (13). In contrast, plant pathogenic Ralstonia and Xanthomonas spp. have Hrp2 class T3SS, as indicated by several different Hrp proteins and distinct regulatory systems.To better understand the T3SS machinery, we previously conducted a survey of the hrp genes of P. syringae pv. syringae (Psy) 61 to complete the inventory of all those encoding proteins capable of traveling the T3SS into plant cells when expressed from a constitutive promoter (39). We hypothesized that these proteins might aid in pilus or translocon construction or regulate the construction process. HrpP was one protein found to be a T3SS substrate and important for secretion and translocation of the model effector AvrPto. Importantly, HrpP is related to a well-studied protein from Yersinia enterocolitica, YscP, which is a T3SS-secreted protein and a regulator responsible for switching the T3SS from secreting needle subunits to secreting effector proteins (15, 38, 47). It has also been shown that secretion of YscP into the culture medium is not essential for the switch function and that there may be two type III secretion signals embedded in YscP (2).The phenotype of a yscP mutant is unregulated secretion of the needle subunit, no secretion of effectors, and production of needles of indeterminate length. The switching phenotype requires a domain at the C terminus of YscP called the type III secretion substrate specificity switch (T3S4) domain, which is a conserved feature unifying its homologs (1). YscP has been proposed to act as a molecular ruler because the length of the YscP protein is directly correlated with the length of the Ysc needle (26). According to this model, when the needle has reached its proper length, YscP signals to the T3SS machinery to stop secreting needle subunits and begin secreting effector proteins. However, other functional models have been hypothesized for homologs of YscP. A recent study of the Salmonella enterica serovar Typhimurium YscP homolog InvJ showed that an invJ mutant lacked an inner rod. When the inner rod protein PrgJ was overexpressed, the length of the needle decreased relative to that of the wild type, leading the researchers to conclude that InvJ controls the inner rod, which in turn controls needle length (33). Recent evidence in Yersinia has lent more support to this model. YscP was found to negatively control secretion of YscI, the inner rod protein (51). Also, certain YscI mutations affected needle assembly but not effector secretion, implying that YscI may be a key player in substrate switching. Little is known about HrpB, the inner rod homolog in P. syringae (22), other than that the protein can be translocated into plant cells and is essential for T3SS function (39).Other models for length control/substrate switching have been proposed, such as the “C-ring cup model” in flagella, which was based on the observation that certain mutations in proteins that make up the inner membrane C ring of the basal body lead to shorter hooks (the flagellar equivalent of the needle), thus suggesting that C-ring capacity controls hook length (32). A more recent, flagellar “molecular-clock” model suggests that because overexpression of hook subunits leads to longer hooks and hook polymerization-defective mutants make shorter hooks, hook polymerization initiates a countdown, and the timing, in cooperation with the YscP homolog FliK, determines final hook length (34).HrpP is considered a member of the YscP/FliK family due mostly to the presence of a T3S4 domain at its C terminus. HrpP is also proline rich (10.6%), which is considered a characteristic of the family. The most striking feature of HrpP is its small size; the protein is 189 amino acids, compared with YscP from Y. enterocolitica, which is 453 amino acids and 8.4% proline. We were intrigued by how HrpP functions in P. syringae to regulate a pilus that can measure several hundred nanometers in length. Also, unlike animal pathogen needles and flagellar hooks, the pilus of P. syringae is predicted to be indeterminate in length, based on the fact that plant cell walls vary in width between species (40).We hypothesized that HrpP would be a main player in regulating pilus construction in P. syringae by allowing the system to make the transition between secretion of pilus subunits and secretion of translocon or effector proteins, though perhaps by a novel mechanism. In this study, we more precisely define the role of HrpP in the P. syringae T3SS. We show that HrpP is a T3SS substrate in DC3000, is translocated into plant cells at levels equivalent to those of effectors, and is essential for the function of the T3SS. Though it is highly translocated and variable, we found that HrpP from different P. syringae pathovars could complement the DC3000 hrpP mutant. Analysis of truncations of HrpP and an impassible HrpP-green fluorescent protein (GFP) fusion suggests that it has structural similarities to YscP, but surprisingly, HrpP was found to be required for full secretion of the pilus subunit HrpA as well as for translocation of HrpB.     

Recombineering and stable integration of the Pseudomonas syringae pv. syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0‐1

Many Gram‐negative bacteria use a type III secretion system (T3SS) to establish associations with their hosts. The T3SS is a conduit for direct injection of type‐III effector proteins into host cells, where they manipulate the host for the benefit of the infecting bacterium. For plant‐associated pathogens, the variations in number and amino acid sequences of type‐III effectors, as well as their functional redundancy, make studying type‐III effectors challenging. To mitigate this challenge, we developed a stable delivery system for individual or defined sets of type‐III effectors into plant cells. We used recombineering and Tn5‐mediated transposition to clone and stably integrate, respectively, the complete hrp/hrc region from Pseudomonas syringae pv. syringae 61 into the genome of the soil bacterium Pseudomonas fluorescens Pf0‐1. We describe our development of Effector‐to‐Host Analyzer (EtHAn), and demonstrate its utility for studying effectors for their in planta functions.     

IDL6‐HAE/HSL2 impacts pectin degradation and resistance to Pseudomonas syringae pv tomato DC3000 in Arabidopsis leaves

Plant cell walls undergo dynamic structural and chemical changes during plant development and growth. Floral organ abscission and lateral root emergence are both accompanied by cell‐wall remodeling, which involves the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)‐derived peptide and its receptors, HAESA (HAE) and HAESA‐LIKE2 (HSL2). Plant cell walls also act as barriers against pathogenic invaders. Thus, the cell‐wall remodeling during plant development could have an influence on plant resistance to phytopathogens. Here, we identified IDA‐like 6 (IDL6), a gene that is prominently expressed in Arabidopsis leaves. IDL6 expression in Arabidopsis leaves is significantly upregulated when the plant is suffering from attacks of the bacterial Pseudomonas syringae pv. tomato (Pst) DC3000. IDL6 overexpression and knockdown lines respectively decrease and increase the Arabidopsis resistance to Pst DC3000, indicating that the gene promotes the Arabidopsis susceptibility to Pst DC3000. Moreover, IDL6 promotes the expression of a polygalacturonase (PG) gene, ADPG2, and increases PG activity in Arabidopsis leaves, which in turn reduces leaf pectin content and leaf robustness. ADPG2 overexpression restrains Arabidopsis resistance to Pst DC3000, whereas ADPG2 loss‐of‐function mutants increase the resistance to the bacterium. Pst DC3000 infection elevates the ADPG2 expression partially through HAE and HSL2. Taken together, our results suggest that IDL6‐HAE/HSL2 facilitates the ingress of Pst DC3000 by promoting pectin degradation in Arabidopsis leaves, and Pst DC3000 might enhance its infection by manipulating the IDL6‐HAE/HSL2‐ADPG2 signaling pathway.