Discovery of the Hrp Type III Secretion System in Phytopathogenic Bacteria: How Investigation of Hypersensitive Cell Death in Plants Led to a Novel Protein Injector System and a World of Inter-Organismal Molecular Interactions Within Plant Cells.

In the early 1960s, Pseudomonas syringae and other host-specific phytopathogenic proteobacteria were discovered to elicit a rapid, resistance-associated death when infiltrated at high inoculum levels into nonhost tobacco leaves. This hypersensitive reaction (or response; HR) was a useful indicator of basic pathogenic ability. Research over the next 20 years failed to identify an elicitor of the HR but revealed that its elicitation required contact between metabolically active bacterial and plant cells. Beginning in the early 1980s, molecular genetic tools were applied to the HR puzzle, revealing the presence in P. syringae of clusters of hrp genes, so named because they are required for the HR and pathogenicity, and of avr genes, so named because their presence confers HR-associated avirulence in resistant cultivars of a host plant species. A series of breakthroughs over the next two decades revealed that (i) hrp gene clusters encode a type III secretion system (T3SS), which injects Avr (now "effector") proteins into plant cells, where their recognition triggers the HR; (ii) T3SSs, which are typically present in pathogenicity islands acquired by horizontal gene transfers, are found in many bacterial pathogens of plants and animals and inject many effector proteins, which are collectively essential for pathogenicity; and (iii) a primary function of phytopathogen effectors is to subvert non-HR defenses resulting from recognition of conserved microbial features presented outside of plant cells. In the 2000s, Hrp system research shifted to extracellular components enabling effector delivery across plant cell walls and plasma membranes, regulation, and tools for studying effectors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Natural variation for unusual host responses and flagellin-mediated immunity against Pseudomonas syringae in genetically diverse tomato accessions.

The interaction between tomato and Pseudomonas syringae pv tomato (Pst) is a well-developed model for investigating the molecular basis of the plant immune system. There is extensive natural variation in Solanum lycopersicum (tomato) but it has not been fully leveraged to enhance our understanding of the tomato-Pst pathosystem. We screened 216 genetically diverse accessions of cultivated tomato and a wild tomato species for natural variation in their response to three strains of Pst. The host response to Pst was investigated using multiple Pst strains, tomato accessions with available genome sequences, reactive oxygen species (ROS) assays, reporter genes and bacterial population measurements. The screen uncovered a broad range of previously unseen host symptoms in response to Pst, and one of these, stem galls, was found to be simply inherited. The screen also identified tomato accessions that showed enhanced responses to flagellin in bacterial population assays and in ROS assays upon exposure to flagellin-derived peptides, flg22 and flgII-28. Reporter genes confirmed that the host responses were due primarily to pattern recognition receptor-triggered immunity. This study revealed extensive natural variation in tomato for susceptibility and resistance to Pst and will enable elucidation of the molecular mechanisms underlying these host responses.

Modular Study of the Type III Effector Repertoire in Pseudomonas syringae pv. tomato DC3000 Reveals a Matrix of Effector Interplay in Pathogenesis.

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of Nicotiana benthamiana and other plants by injecting a complex repertoire of type III secretion effector (T3E) proteins. Effectorless polymutant DC3000D36E was used with a modularized system for native delivery of the 29 DC3000 T3Es singly and in pairs. Assays of the performance of this T3E library in N. benthamiana leaves revealed a matrix of T3E interplay, with six T3Es eliciting death and eight others variously suppressing the death activity of the six. The T3E library was also interrogated for effects on DC3000D36E elicitation of a reactive oxygen species burst, for growth in planta, and for T3Es that reversed these effects. Pseudomonas fluorescens and Agrobacterium tumefaciens heterologous delivery systems yielded notably different sets of death-T3Es. The DC3000D36E T3E library system highlights the importance of 13 T3Es and their interplay in interactions with N. benthamiana.

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.

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors.

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors.

Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Polymutants Reveal an Interplay between HopAD1 and AvrPtoB.

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered plant innate immune system by injecting a complex repertoire of type III secretion effector (T3E) proteins. Beyond redundancy and interplay, individual T3Es may interact with multiple immunity-associated proteins, rendering their analysis challenging. We constructed a Pst DC3000 polymutant lacking all 36 T3Es and restored individual T3Es or their mutants to explore the interplay among T3Es. The weakly expressed T3E HopAD1 was sufficient to elicit immunity-associated cell death in Nicotiana benthamiana. HopAD1-induced cell death was suppressed partially by native AvrPtoB and completely by AvrPtoBM3, which has mutations disrupting its E3 ubiquitin ligase domain and two known domains for interacting with immunity-associated kinases. AvrPtoBM3 also gained the ability to interact with the immunity-kinase MKK2, which is required for HopAD1-dependent cell death. Thus, AvrPtoB has alternative, competing mechanisms for suppressing effector-triggered plant immunity. This approach allows the deconvolution of individual T3E activities.

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.

Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 family member, is needed to produce L-allo-isoleucine, a precursor for the phytotoxin coronatine.

Pseudomonas syringae pv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plant Nicotiana benthamiana in a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed in N. benthamiana with cfa, cma, and cmaL mutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either the cma operon or cmaL accumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered in cmaL mutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaL mutant when it is supplemented with 50 µg/ml l-allo-isoleucine, the starting unit for CMA production. cmaL is found in all other sequenced P. syringae strains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family.

Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae.

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant pathogen Pseudomonas syringae pathovar tomato strain DC3000 possess characteristic patterns, including (i) greater than 10% serine within the first 50 amino acids, (ii) an aliphatic residue or proline at position 3 or 4, and (iii) a lack of acidic amino acids within the first 12 residues. Here, the functional significance of the P. syringae T3SS substrate compositional patterns was tested. A mutant AvrPto effector protein lacking all three patterns was secreted into culture and translocated into plant cells, suggesting that the compositional characteristics are not absolutely required for T3SS targeting and that other recognition mechanisms exist. To further analyze the unique properties of T3SS targeting signals, we developed a computational algorithm called TEREE (Type III Effector Relative Entropy Evaluation) that distinguishes DC3000 T3SS substrates from other proteins with a high sensitivity and specificity. Although TEREE did not efficiently identify T3SS substrates in Salmonella enterica, it was effective in another P. syringae strain and Ralstonia solanacearum. Thus, the TEREE algorithm may be a useful tool for identifying new effector genes in plant pathogens. The nature of T3SS targeting signals was additionally investigated by analyzing the N-terminus of FtsX, a putative membrane protein that was classified as a T3SS substrate by TEREE. Although the first 50 amino acids of FtsX were unable to target a reporter protein to the T3SS, an AvrPto protein substituted with the first 12 amino acids of FtsX was translocated into plant cells. These results show that the T3SS targeting signals are highly mutable and that secretion may be directed by multiple features of substrates.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression.

Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae.

The virulence of Pseudomonas syringae and many other proteobacterial pathogens is dependent on complex repertoires of effector proteins injected into host cells by type III secretion systems. The 28 well-expressed effector genes in the repertoire of the model pathogen P. syringae pv. tomato DC3000 were deleted to produce polymutant DC3000D28E. Growth of DC3000D28E in Nicotiana benthamiana was symptomless and 4 logs lower than that of DC3000ΔhopQ1-1, which causes disease in this model plant. DC3000D28E seemed functionally effectorless but otherwise WT in diagnostic phenotypes relevant to plant interactions (for example, ability to inject the AvrPto-Cya reporter into N. benthamiana). Various effector genes were integrated by homologous recombination into native loci or by a programmable or random in vivo assembly shuttle (PRIVAS) system into the exchangeable effector locus in the Hrp pathogenicity island of DC3000D28E. The latter method exploited dual adapters and recombination in yeast for efficient assembly of PCR products into programmed or random combinations of multiple effector genes. Native and PRIVAS-mediated integrations were combined to identify a minimal functional repertoire of eight effector genes that restored much of the virulence of DC3000ΔhopQ1-1 in N. benthamiana, revealing a hierarchy in effector function: AvrPtoB acts with priority in suppressing immunity, enabling other effectors to promote further growth (HopM1 and HopE1), chlorosis (HopG1), lesion formation (HopAM1-1), and near full growth and symptom production (AvrE, HopAA1-1, and/or HopN1 functioning synergistically with the previous effectors). DC3000D28E, the PRIVAS method, and minimal functional repertoires provide new resources for probing the plant immune system.

Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

Methods to study PAMP-triggered immunity using tomato and Nicotiana benthamiana.

Understanding the molecular basis of plant responses to pathogen-associated molecular patterns (PAMPs) is an active area of research in the field of plant-microbe interactions. A growing number of plant genes involved in various steps of PAMP-triggered immunity (PTI) pathways and microbial factors involved in the elicitation or suppression of PTI have been identified. These studies have largely relied on Arabidopsis thaliana and, therefore, most of the PTI assays have been developed and optimized for that model plant system. Although PTI is a conserved feature among plants, the response spectra vary across different species. Thus, there is a need for robust PTI assays in other pathosystems, such as those involving Solanaceae plant-pathogen interactions, which include many economically important plants and their diseases. We have optimized molecular, cellular, and whole-plant methods to measure PTI responses in two widely studied solanaceous species, tomato (Solanum lycopersicum) and Nicotiana benthamiana. Here, we provide detailed protocols for measuring various PTI-associated phenotypes, including bacterial populations after pretreatment of leaves with PAMPs, induction of reporter genes, callose deposition, activation of mitogen-activated protein kinases, and a luciferase-based reporter system. These methods will facilitate limited genetic screens and detailed characterization of potential PTI-related genes in model and economically important Solanaceae spp.-pathogen interactions.

Identification of Nicotiana benthamiana genes involved in pathogen-associated molecular pattern-triggered immunity.

In order to identify components of pathogen-associated molecular pattern-triggered immunity (PTI) pathways in Nicotiana benthamiana, we conducted a large-scale forward-genetics screen using virus-induced gene silencing and a cell-death-based assay for assessing PTI. The assay relied on four combinations of PTI-inducing nonpathogens and cell-death-causing challenger pathogens and was first validated in plants silenced for FLS2 or BAK1. Over 3,200 genes were screened and 14 genes were identified that, when silenced, compromised PTI as judged by the cell-death-based assay. Further analysis indicated that the 14 genes were not involved in a general cell death response. A subset of the genes was found to act downstream of FLS2-mediated PTI induction, and silencing of three genes compromised production of reactive oxygen species in leaves exposed to flg22. The 14 genes encode proteins with potential functions in defense and hormone signaling, protein stability and degradation, energy and secondary metabolism, and cell wall biosynthesis and provide a new resource to explore the molecular basis for the involvement of these processes in PTI.

Components of the Pseudomonas syringae type III secretion system can suppress and may elicit plant innate immunity.

The type III secretion system (T3SS) of Pseudomonas syringae translocates into plant cells multiple effectors that suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). P. syringae pv. tomato DC3000 no longer delivers the T3SS translocation reporter AvrPto-Cya in Nicotiana benthamiana leaf tissue in which PTI was induced by prior inoculation with P. fluorescens(pLN18). Cosmid pLN18 expresses the T3SS system of P. syringae pv. syringae 61 but lacks the hopA1(Psy61) effector gene. P. fluorescens(pLN18) expressing HrpH(PtoDC3000) or HopP1(PtoDC3000), two T3SS-associated putative lytic transglycosylases, suppresses PTI, based on multiple assays involving DC3000 challenge inoculum (AvrPto-Cya translocation, hypersensitive response elicitation, and colony development in planta) or on plant responses (vascular dye uptake or callose deposition). Analysis of additional mutations in pHIR11 derivatives revealed that the pLN18-encoded T3SS elicits a higher level of reactive oxygen species (ROS) than does P. fluorescens without a T3SS, that enhanced ROS production is dependent on the HrpK1 translocator, and that HopA1(Psy61) suppresses ROS elicitation attributable to both the P. fluorescens PAMPs and the presence of a functional T3SS.

Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors.

The gamma-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III secretion system to inject ca. 28 Avr/Hop effector proteins into plants, which enables the bacterium to grow from low inoculum levels to produce bacterial speck symptoms in tomato, Arabidopsis thaliana, and (when lacking hopQ1-1) Nicotiana benthamiana. The effectors are collectively essential but individually dispensable for the ability of the bacteria to defeat defenses, grow, and produce symptoms in plants. Eighteen of the effector genes are clustered in six genomic islands/islets. Combinatorial deletions involving these clusters and two of the remaining effector genes revealed a redundancy-based structure in the effector repertoire, such that some deletions diminished growth in N. benthamiana only in combination with other deletions. Much of the ability of DC3000 to grow in N. benthamiana was found to be due to five effectors in two redundant-effector groups (REGs), which appear to separately target two high-level processes in plant defense: perception of external pathogen signals (AvrPto and AvrPtoB) and deployment of antimicrobial factors (AvrE, HopM1, HopR1). Further support for the membership of HopR1 in the same REG as AvrE was gained through bioinformatic analysis, revealing the existence of an AvrE/DspA/E/HopR effector superfamily, which has representatives in virtually all groups of proteobacterial plant pathogens that deploy type III effectors.

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 DeltahrpP 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 DeltahrpP. 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 DeltahrpP 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 DeltahrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 DeltahrpP 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.

A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae.

The injection of nearly 30 effector proteins by the type III secretion system underlies the ability of Pseudomonas syringae pv. tomato DC3000 to cause disease in tomato and other host plants. The search for effector functions is complicated by redundancy within the repertoire and by plant resistance (R)-gene sentinels, which may convert effector virulence activities into a monolithic defense response. On the premise that some effectors target universal eukaryotic processes and that yeast (Saccharomyces cerevisiae) lacks R genes, the DC3000 effector repertoire was expressed in yeast. Of 27 effectors tested, HopAD1, HopAO1, HopD1, HopN1, and HopU1 were found to inhibit growth when expressed from a galactose-inducible GAL1 promoter, and HopAA1-1 and HopAM1 were found to cause cell death. Catalytic site mutations affecting the tyrosine phosphatase activity of HopAO1 and the cysteine protease activity of HopN1 prevented these effectors from inhibiting yeast growth. Expression of HopAA1-1, HopAM1, HopAD1, and HopAO1 impaired respiration in yeast, as indicated by tests with ethanol glycerol selective media. HopAA1-1 colocalized with porin to yeast mitochondria and was shown to cause cell death in yeast and plants in a domain-dependent manner. These results support the use of yeast for the study of plant-pathogen effector repertoires.

Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type III secretion system effectors.

Harpins are a subset of type III secretion system (T3SS) substrates found in all phytopathogenic bacteria that utilize a T3SS. Pseudomonas syringae pv. tomato DC3000 was previously reported to produce two harpins, HrpZ1 and HrpW1. DC3000 was shown here to deploy two additional proteins, HopAK1 and HopP1, which have the harpin-like properties of lacking cysteine, eliciting the hypersensitive response (HR) when partially purified and infiltrated into tobacco leaves, and possessing a two-domain structure similar to that of the HrpW1 class of harpins. Unlike the single-domain harpin HrpZ1, the two-domain harpins have C-terminal enzyme-like domains: pectate lyase for HopAK1 and lytic transglycosylase for HopP1. Genetic techniques to recycle antibiotic markers were applied to DC3000 to generate a quadruple harpin gene polymutant. The polymutant was moderately reduced in the elicitation of the HR and translocation of the T3SS effector AvrPto1 fused to a Cya translocation reporter, but the mutant was unaffected in the secretion of AvrPto1-Cya. The DC3000 hrpK1 gene encodes a putative translocator in the HrpF/NopX family and was deleted in combination with the four harpin genes. The hrpK1 quadruple harpin gene polymutant was strongly reduced in HR elicitation, virulence, and translocation of AvrPto1-Cya into plant cells but not in the secretion of representative T3SS substrates in culture. HrpK1, HrpZ1, HrpW1, and HopAK1, but not HopP1, were independently capable of restoring some HR elicitation to the hrpK1 quadruple harpin gene polymutant, which suggests that a consortium of semiredundant translocators from three protein classes cooperate to form the P. syringae T3SS translocon.

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.