PIX is an N-terminal delivery domain that defines a class of polymorphic T6SS effectors in Enterobacterales.

The type VI secretion system (T6SS), a widespread protein delivery apparatus, plays a role in bacterial competition by delivering toxic effectors into neighboring cells. Identifying new T6SS effectors and deciphering the mechanism that governs their secretion remain major challenges. Here, we report two orphan antibacterial T6SS effectors in the pathogen Pantoea agglomerans (Pa). These effectors share an N-terminal domain, Pantoea type six (PIX), that defines a widespread class of polymorphic T6SS effectors in Enterobacterales. We show that the PIX domain is necessary and sufficient for T6SS-mediated effector secretion and that PIX binds to a specialized Pa VgrG protein outside its C-terminal toxic domain. Our findings underline the importance of identifying and characterizing delivery domains in polymorphic toxin classes as a tool to reveal effectors and shed light on effector delivery mechanisms.

Balancing growth amidst salt stress - lifestyle perspectives from the extremophyte model Schrenkiella parvula.

Schrenkiella parvula, a leading extremophyte model in Brassicaceae, can grow and complete its lifecycle under multiple environmental stresses, including high salinity. Yet, the key physiological and structural traits underlying its stress-adapted lifestyle are unknown along with trade-offs when surviving salt stress at the expense of growth and reproduction. We aimed to identify the influential adaptive trait responses that lead to stress-resilient and uncompromised growth across developmental stages when treated with salt at levels known to inhibit growth in Arabidopsis and most crops. Its resilient growth was promoted by traits that synergistically allowed primary root growth in seedlings, the expansion of xylem vessels across the root-shoot continuum, and a high capacity to maintain tissue water levels by developing thicker succulent leaves while enabling photosynthesis during salt stress. A successful transition from vegetative to reproductive phase was initiated by salt-induced early flowering, resulting in viable seeds. Self-fertilization in salt-induced early flowering was dependent upon filament elongation in flowers otherwise aborted in the absence of salt during comparable plant ages. The maintenance of leaf water status promoting growth, and early flowering to ensure reproductive success in a changing environment, were among the most influential traits that contributed to the extremophytic lifestyle of S. parvula.

Comparative sequence analysis of pPATH pathogenicity plasmids in Pantoea agglomerans gall-forming bacteria.

Acquisition of the pathogenicity plasmid pPATH that encodes a type III secretion system (T3SS) and effectors (T3Es) has likely led to the transition of a non-pathogenic bacterium into the tumorigenic pathogen Pantoea agglomerans. P. agglomerans pv. gypsophilae (Pag) forms galls on gypsophila (Gypsophila paniculata) and triggers immunity on sugar beet (Beta vulgaris), while P. agglomerans pv. betae (Pab) causes galls on both gypsophila and sugar beet. Draft sequences of the Pag and Pab genomes were previously generated using the MiSeq Illumina technology and used to determine partial T3E inventories of Pab and Pag. Here, we fully assembled the Pab and Pag genomes following sequencing with PacBio technology and carried out a comparative sequence analysis of the Pab and Pag pathogenicity plasmids pPATHpag and pPATHpab. Assembly of Pab and Pag genomes revealed a ~4 Mbp chromosome with a 55% GC content, and three and four plasmids in Pab and Pag, respectively. pPATHpag and pPATHpab share 97% identity within a 74% coverage, and a similar GC content (51%); they are ~156 kb and ~131 kb in size and consist of 198 and 155 coding sequences (CDSs), respectively. In both plasmids, we confirmed the presence of highly similar gene clusters encoding a T3SS, as well as auxin and cytokinins biosynthetic enzymes. Three putative novel T3Es were identified in Pab and one in Pag. Among T3SS-associated proteins encoded by Pag and Pab, we identified two novel chaperons of the ShcV and CesT families that are present in both pathovars with high similarity. We also identified insertion sequences (ISs) and transposons (Tns) that may have contributed to the evolution of the two pathovars. These include seven shared IS elements, and three ISs and two transposons unique to Pab. Finally, comparative sequence analysis revealed plasmid regions and CDSs that are present only in pPATHpab or in pPATHpag. The high similarity and common features of the pPATH plasmids support the hypothesis that the two strains recently evolved into host-specific pathogens.

Dynamic changes of the Prf/Pto tomato resistance complex following effector recognition.

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen recognition and activation of innate immunity. In plants, NLRs recognise pathogen-derived effector proteins and initiate effector-triggered immunity (ETI). However, the molecular mechanisms that link NLR-mediated effector recognition and downstream signalling are not fully understood. By exploiting the well-characterised tomato Prf/Pto NLR resistance complex, we identified the 14-3-3 proteins TFT1 and TFT3 as interacting partners of both the NLR complex and the protein kinase MAPKKKα. Moreover, we identified the helper NRC proteins (NLR-required for cell death) as integral components of the Prf /Pto NLR recognition complex. Notably our studies revealed that TFTs and NRCs interact with distinct modules of the NLR complex and, following effector recognition, dissociate facilitating downstream signalling. Thus, our data provide a mechanistic link between activation of immune receptors and initiation of downstream signalling cascades.

Tomato receptor-like cytoplasmic kinase Fir1 interacts with a negative regulator of jasmonic acid signaling.

Plant cells detect potential pathogens through plasma membrane-localized pattern recognition receptors (PRRs) that recognize microbe-associated molecular patterns (MAMPs) and activate pattern-triggered immunity (PTI). PRR-mediated MAMP perception is linked to PTI signaling by receptor-like cytoplasmic kinases (RLCKs). In tomato, Flagellin-sensing 2 (Fls2)/Fls3 interacting RLCK 1 (Fir1) is involved in PTI triggered by flagellin perception. Fir1 is necessary for regulation of jasmonic acid (JA) signaling and is involved in pre-invasion immunity. We show that Fir1 physically interacts with JASMONATE-ZIM-DOMAIN PROTEIN 3 (JAZ3), a negative regulator of JA signaling. This finding suggests that Fir1 modulates JA signaling by regulating JAZ3.

Tomato receptor-like cytoplasmic kinase Fir1 is involved in flagellin signaling and preinvasion immunity.

Detection of bacterial flagellin by the tomato (Solanum lycopersicum) receptors Flagellin sensing 2 (Fls2) and Fls3 triggers activation of pattern-triggered immunity (PTI). We identified the tomato Fls2/Fls3-interacting receptor-like cytoplasmic kinase 1 (Fir1) protein that is involved in PTI triggered by flagellin perception. Fir1 localized to the plasma membrane and interacted with Fls2 and Fls3 in yeast (Saccharomyces cerevisiae) and in planta. CRISPR/Cas9-generated tomato fir1 mutants were impaired in several immune responses induced by the flagellin-derived peptides flg22 and flgII-28, including resistance to Pseudomonas syringae pv. tomato (Pst) DC3000, production of reactive oxygen species, and enhanced PATHOGENESIS-RELATED 1b (PR1b) gene expression, but not MAP kinase phosphorylation. Remarkably, fir1 mutants developed larger Pst DC3000 populations than wild-type plants, whereas no differences were observed in wild-type and fir1 mutant plants infected with the flagellin deficient Pst DC3000ΔfliC. fir1 mutants failed to close stomata when infected with Pst DC3000 and Pseudomonas fluorescens and were more susceptible to Pst DC3000 than wild-type plants when inoculated by dipping, but not by vacuum-infiltration, indicating involvement of Fir1 in preinvasion immunity. RNA-seq analysis detected fewer differentially expressed genes in fir1 mutants and altered expression of jasmonic acid (JA)-related genes. In support of JA response deregulation in fir1 mutants, these plants were similarly susceptible to Pst DC3000 and to the coronatine-deficient Pst DC3118 strain, and more resistant to the necrotrophic fungus Botrytis cinerea following PTI activation. These results indicate that tomato Fir1 is required for a subset of flagellin-triggered PTI responses and support a model in which Fir1 negatively regulates JA signaling during PTI activation.

The Emerging Role of PP2C Phosphatases in Tomato Immunity.

The antagonistic effect of plant immunity on growth likely drove evolution of molecular mechanisms that prevent accidental initiation and prolonged activation of plant immune responses. Signaling networks of pattern-triggered and effector-triggered immunity, the two main layers of plant immunity, are tightly regulated by the activity of protein phosphatases that dephosphorylate their protein substrates and reverse the action of protein kinases. Members of the PP2C class of protein phosphatases have emerged as key negative regulators of plant immunity, primarily from research in the model plant Arabidopsis thaliana, revealing the potential to employ PP2C proteins to enhance plant disease resistance. As a first step towards focusing on the PP2C family for both basic and translational research, we analyzed the tomato genome sequence to ascertain the complement of the tomato PP2C family, identify conserved protein domains and signals in PP2C amino acid sequences, and examine domain combinations in individual proteins. We then identified tomato PP2Cs that are candidate regulators of single or multiple layers of the immune signaling network by in-depth analysis of publicly available RNA-seq datasets. These included expression profiles of plants treated with fungal or bacterial pathogen-associated molecular patterns, with pathogenic, nonpathogenic, and disarmed bacteria, as well as pathogenic fungi and oomycetes. Finally, we discuss the possible use of immunity-associated PP2Cs to better understand the signaling networks of plant immunity and to engineer durable and broad disease resistance in crop plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

An antibacterial T6SS in Pantoea agglomerans pv. betae delivers a lysozyme-like effector to antagonize competitors.

The type VI secretion system (T6SS) is deployed by numerous Gram-negative bacteria to deliver toxic effectors into neighbouring cells. The genome of Pantoea agglomerans pv. betae (Pab) phytopathogenic bacteria contains a gene cluster (T6SS1) predicted to encode a complete T6SS. Using secretion and competition assays, we found that T6SS1 in Pab is a functional antibacterial system that allows this pathogen to outcompete rival plant-associated bacteria found in its natural environment. Computational analysis of the T6SS1 gene cluster revealed that antibacterial effector and immunity proteins are encoded within three genomic islands that also harbour arrays of orphan immunity genes or toxin and immunity cassettes. Functional analyses indicated that VgrG, a specialized antibacterial effector, contains a C-terminal catalytically active glucosaminidase domain that is used to degrade prey peptidoglycan. Moreover, we confirmed that a bicistronic unit at the end of the T6SS1 cluster encodes a novel antibacterial T6SS effector and immunity pair. Together, these results demonstrate that Pab T6SS1 is an antibacterial system delivering a lysozyme-like effector to eliminate competitors, and indicate that this bacterium contains additional novel T6SS effectors.

BRASSINOSTEROID-SIGNALLING KINASES 7 and 8 associate with the FLS2 immune receptor and are required for flg22-induced PTI responses.

Pattern-triggered immunity (PTI) is typically initiated in plants by recognition of pathogen- or damage-associated molecular patterns (PAMP/DAMPs) by cell surface-localized pattern recognition receptors (PRRs). Here, we investigated the role in PTI of Arabidopsis thaliana brassinosteroid-signalling kinases 7 and 8 (BSK7 and BSK8), which are members of the receptor-like cytoplasmic kinase subfamily XII. BSK7 and BSK8 localized to the plant cell periphery and interacted in yeast and in planta with FLS2, but not with other PRRs. Consistent with a role in FLS2 signalling, bsk7 and bsk8 single and bsk7,8 double mutant plants were impaired in several immune responses induced by flg22, but not by other PAMP/DAMPs. These included resistance to Pseudomonas syringae and Botrytis cinerea, reactive oxygen species accumulation, callose deposition at the cell wall, and expression of the defence-related gene PR1, but not activation of MAP kinases and expression of the FRK1 and WRKY29 genes. bsk7, bsk8, and bsk7,8 plants also displayed enhanced susceptibility to P. syringae and B. cinerea. Finally, BSK7 and BSK8 variants mutated in their myristoylation site or in the ATP-binding site failed to complement defective phenotypes of the corresponding mutants, suggesting that localization to the cell periphery and kinase activity are critical for BSK7 and BSK8 functions. Together, these findings demonstrate that BSK7 and BSK8 play a role in PTI initiated by recognition of flg22 by interacting with the FLS2 immune receptor.

Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III-secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus.

The cucurbit pathogenic bacterium Acidovorax citrulli requires a functional type III secretion system (T3SS) for pathogenicity. In this bacterium, as with Xanthomonas and Ralstonia spp., an AraC-type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III-secreted effectors (T3Es). The annotation of a sequenced A. citrulli strain revealed 11 T3E genes. Assuming that this could be an underestimation, we aimed to uncover the T3E arsenal of the A. citrulli model strain, M6. Thorough sequence analysis revealed 51 M6 genes whose products are similar to known T3Es. Furthermore, we combined machine learning and transcriptomics to identify novel T3Es. The machine-learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA-Seq revealed differential gene expression between wild-type M6 and a mutant defective in HrpX: 159 and 28 genes showed significantly reduced and increased expression in the mutant relative to wild-type M6, respectively. Data combined from these approaches led to the identification of seven novel T3E candidates that were further validated using a T3SS-dependent translocation assay. These T3E genes encode hypothetical proteins that seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study places A. citrulli among the 'richest' bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.

Two Pantoea agglomerans type III effectors can transform nonpathogenic and phytopathogenic bacteria into host-specific gall-forming pathogens.

Pantoea agglomerans (Pa), a widespread commensal bacterium, has evolved into a host-specific gall-forming pathogen on gypsophila and beet by acquiring a plasmid harbouring a type III secretion system (T3SS) and effectors (T3Es). Pantoea agglomerans pv. gypsophilae (Pag) elicits galls on gypsophila and a hypersensitive response on beet, whereas P. agglomerans pv. betae (Pab) elicits galls on beet and gypsophila. HsvG and HsvB are two paralogous T3Es present in both pathovars and act as host-specific transcription activators on gypsophila and beet, respectively. PthG and PseB are major T3Es that contribute to gall development of Pag and Pab, respectively. To establish the minimal combinations of T3Es that are sufficient to elicit gall symptoms, strains of the nonpathogenic bacteria Pseudomonas fluorescens 55, Pa 3-1, Pa 98 and Escherichia coli, transformed with pHIR11 harbouring a T3SS, and the phytopathogenic bacteria Erwinia amylovora, Dickeya solani and Xanthomonas campestris pv. campestris were transformed with the T3Es hsvG, hsvB, pthG and pseB, either individually or in pairs, and used to infect gypsophila and beet. Strikingly, all the tested nonpathogenic and phytopathogenic bacterial strains harbouring hsvG and pthG incited galls on gypsophila, whereas strains harbouring hsvB and pseB, with the exception of E. coli, incited galls on beet.

Overexpression of BSK5 in Arabidopsis thaliana Provides Enhanced Disease Resistance.

Plant surface-localized pattern recognition receptors (PRRs) recognize pathogen- or damage-associated molecular patterns (PAMP/DAMPs) and activate pattern-triggered immunity (PTI). PRRs recruit receptor-like cytoplasmic kinases (RLCKs) to transduce the perceived signal to downstream signaling components. Brassinosteroid-signaling kinase 5 (BSK5) is a member of the RLCK XII subfamily and mutational analysis revealed its involvement in plant immunity. Here, we provide evidence that overexpression of BSK5 in transgenic Arabidopsis plants enhanced disease resistance to the bacterial pathogen Pseudomonas syringae and to the fungus Botrytis cinerea. Remarkably, upon treatment with the flg22, elf18 and pep1 PAMP/DAMPs, BSK5-overexpressing plants displayed higher levels of immune responses, including production of reactive oxygen species, callose deposition at the cell wall, and PATHOGENESIS-RELATED1 (PR1) gene expression. Together, these findings further substantiate the role of BSK5 in plant immunity and illustrate its potential use for improving plant disease resistance.

Mai1 Protein Acts Between Host Recognition of Pathogen Effectors and Mitogen-Activated Protein Kinase Signaling.

The molecular mechanisms acting between host recognition of pathogen effectors by nucleotide-binding leucine-rich repeat receptor (NLR) proteins and mitogen-activated protein kinase (MAPK) signaling cascades are unknown. MAPKKKα (M3Kα) activates MAPK signaling leading to programmed cell death (PCD) associated with NLR-triggered immunity. We identified a tomato M3Kα-interacting protein, SlMai1, that has 80% amino acid identity with Arabidopsis brassinosteroid kinase 1 (AtBsk1). SlMai1 has a protein kinase domain and a C-terminal tetratricopeptide repeat domain that interacts with the kinase domain of M3Kα. Virus-induced gene silencing of Mai1 homologs in Nicotiana benthamiana increased susceptibility to Pseudomonas syringae and compromised PCD induced by four NLR proteins. PCD was restored by expression of a synthetic SlMai1 gene that resists silencing. Expression of AtBsk1 did not restore PCD in Mai1-silenced plants, suggesting SlMai1 is functionally divergent from AtBsk1. PCD caused by overexpression of M3Kα or MKK2 was unaffected by Mai1 silencing, suggesting Mai1 acts upstream of these proteins. Coexpression of Mai1 with M3Kα in leaves enhanced MAPK phosphorylation and accelerated PCD. These findings suggest Mai1 is a molecular link acting between host recognition of pathogens and MAPK signaling.

BRASSINOSTEROID-SIGNALING KINASE5 Associates with Immune Receptors and Is Required for Immune Responses.

Plants utilize cell surface-localized pattern recognition receptors (PRRs) to detect pathogen- or damage-associated molecular patterns (PAMP/DAMPs) and initiate pattern-triggered immunity (PTI). Here, we investigated the role of Arabidopsis (Arabidopsis thaliana) BRASSINOSTEROID-SIGNALING KINASE5 (BSK5), a member of the receptor-like cytoplasmic kinase subfamily XII, in PRR-initiated immunity. BSK5 localized to the plant cell periphery, interacted in yeast and in planta with multiple receptor-like kinases, including the ELONGATION FACTOR-TU RECEPTOR (EFR) and PEP1 RECEPTOR1 (PEPR1) PRRs, and was phosphorylated in vitro by PEPR1 and EFR in the kinase activation loop. Consistent with a role in PTI, bsk5 mutant plants displayed enhanced susceptibility to the bacterial pathogen Pseudomonas syringae and to the fungus Botrytis cinerea Furthermore, bsk5 mutant plants were impaired in several immune responses induced by the elf18, pep1, and flg22 PAMP/DAMPs, including resistance to P. syringae and B. cinerea, production of reactive oxygen species, callose deposition at the cell wall, and enhanced PATHOGENESIS-RELATED1 gene expression. However, bsk5 plants were not affected in PAMP/DAMP activation of mitogen-activated protein kinases and expression of the FLG22-INDUCED RECEPTOR-LIKE KINASE1 or the WRKY domain-containing gene WRKY29 BSK5 variants mutated in the BSK5 myristoylation site, ATP-binding site, and kinase activation loop failed to complement defective PTI phenotypes of bsk5 mutant plants, suggesting that localization to the cell periphery, kinase activity, and phosphorylation by PRRs are critical for the function of BSK5 in PTI. These findings demonstrate that BSK5 plays a role in PTI by interacting with multiple immune receptors.

Tomato 14-3-3 Proteins Are Required for Xv3 Disease Resistance and Interact with a Subset of Xanthomonas euvesicatoria Effectors.

The 14-3-3 phospho-binding proteins with scaffolding activity play central roles in the regulation of enzymes and signaling complexes in eukaryotes. In plants, 14-3-3 isoforms are required for disease resistance and key targets of pathogen effectors. Here, we examined the requirement of the tomato (Solanum lycopersicum) 14-3-3 isoform (TFT) protein family for Xv3 disease resistance in response to the bacterial pathogen Xanthomonas euvesicatoria. In addition, we determined whether TFT proteins interact with the repertoire of X. euvesicatoria type III secretion effector proteins, including AvrXv3, the elicitor of Xv3 resistance. We show that multiple TFT contribute to Xv3 resistance. We also show that one or more TFT proteins physically interact with multiple effectors (AvrXv3, XopE1, XopE2, XopN, XopO, XopQ, and XopAU). Genetic analyses indicate that none of the identified effectors interfere with AvrXv3-elicited resistance into Xv3 tomato leaves; however, XopE1, XopE2, and XopO are required to suppress symptom development in susceptible tomato leaves. Phospho-peptide mapping revealed that XopE2 is phosphorylated at multiple residues in planta and residues T66, T131, and S334 are required for maximal binding to TFT10. Together, our data support the hypothesis that multiple TFT proteins are involved in immune signaling during X. euvesicatoria infection.

Effector Gene xopAE of Xanthomonas euvesicatoria 85-10 Is Part of an Operon and Encodes an E3 Ubiquitin Ligase.

The type III effector XopAE from the Xanthomonas euvesicatoria strain 85-10 was previously shown to inhibit plant immunity and enhance pathogen-induced disease symptoms. Evolutionary analysis of 60 xopAE alleles (AEal) revealed that the xopAE locus is conserved in multiple Xanthomonas species. The majority of xopAE alleles (55 out of 60) comprise a single open reading frame (ORF) (xopAE), while in 5 alleles, including AEal 37 of the X. euvesicatoria 85-10 strain, a frameshift splits the locus into two ORFs (hpaF and a truncated xopAE). To test whether the second ORF of AEal 37 (xopAE85-10 ) is translated, we examined expression of yellow fluorescent protein (YFP) fused downstream to truncated or mutant forms of the locus in Xanthomonas bacteria. YFP fluorescence was detected at maximal levels when the reporter was in proximity to an internal ribosome binding site upstream of a rare ATT start codon in the xopAE85-10 ORF but was severely reduced when these elements were abolished. In agreement with the notion that xopAE85-10 is a functional gene, its protein product was translocated into plant cells by the type III secretion system, and translocation was dependent on its upstream ORF, hpaF Homology modeling predicted that XopAE85-10 contains an E3 ligase XL box domain at the C terminus, and in vitro assays demonstrated that this domain displays monoubiquitination activity. Remarkably, the XL box was essential for XopAE85-10 to inhibit pathogen-associated molecular pattern (PAMP)-induced gene expression in Arabidopsis protoplasts. Together, these results indicate that the xopAE85-10 gene resides in a functional operon, which utilizes the alternative start codon ATT and encodes a novel XL box E3 ligase.IMPORTANCEXanthomonas bacteria utilize a type III secretion system to cause disease in many crops. This study provides insights into the evolution, translocation, and biochemical function of the XopAE type III secreted effector, contributing to the understanding of Xanthomonas-host interactions. We establish XopAE as a core effector of seven Xanthomonas species and elucidate the evolution of the Xanthomonas euvesicatoriaxopAE locus, which contains an operon encoding a truncated effector. Our findings indicate that this operon evolved from the split of a multidomain gene into two ORFs that conserved the original domain function. Analysis of xopAE85-10 translation provides the first evidence for translation initiation from an ATT codon in Xanthomonas Our data demonstrate that XopAE85-10 is an XL box E3 ubiquitin ligase and provide insights into the structure and function of this effector family.

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.

Revealing the inventory of type III effectors in Pantoea agglomerans gall-forming pathovars using draft genome sequences and a machine-learning approach.

Pantoea agglomerans, a widespread epiphytic bacterium, has evolved into a hypersensitive response and pathogenicity (hrp)-dependent and host-specific gall-forming pathogen by the acquisition of a pathogenicity plasmid containing a type III secretion system (T3SS) and its effectors (T3Es). Pantoea agglomerans pv. betae (Pab) elicits galls on beet (Beta vulgaris) and gypsophila (Gypsophila paniculata), whereas P. agglomerans pv. gypsophilae (Pag) incites galls on gypsophila and a hypersensitive response (HR) on beet. Draft genome sequences were generated and employed in combination with a machine-learning approach and a translocation assay into beet roots to identify the pools of T3Es in the two pathovars. The genomes of the sequenced Pab4188 and Pag824-1 strains have a similar size (∼5 MB) and GC content (∼55%). Mutational analysis revealed that, in Pab4188, eight T3Es (HsvB, HsvG, PseB, DspA/E, HopAY1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on beet and gypsophila. In Pag824-1, nine T3Es (HsvG, HsvB, PthG, DspA/E, HopAY1, HopD1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on gypsophila, whereas the PthG effector triggers HR on beet. HsvB, HsvG, PthG and PseB appear to endow pathovar specificities to Pab and Pag, and no homologous T3Es were identified for these proteins in other phytopathogenic bacteria. Conversely, the remaining T3Es contribute to the virulence of both pathovars, and homologous T3Es were found in other phytopathogenic bacteria. Remarkably, HsvG and HsvB, which act as host-specific transcription factors, displayed the largest contribution to disease development.

Assessing the Ability of Salmonella enterica to Translocate Type III Effectors Into Plant Cells.

Salmonella enterica serovar Typhimurium, a human enteric pathogen, has the ability to multiply and survive endophytically in plants. Genes encoding the type III secretion system (T3SS) or its effectors (T3Es) may contribute to its colonization. Two reporter plasmids for T3E translocation into plant cells that are based on hypersensitive response domains of avirulence proteins from the Pantoea agglomerans-beet and Xanthomonas euvesicatoria-pepper pathosystems were employed in this study to investigate the role of T3Es in the interaction of Salmonella ser. Typhimurium 14028 with plants. The T3Es of Salmonella ser. Typhimurium, SipB and SifA, which are translocated into animal cells, could not be delivered by Salmonella ser. Typhimurium into cells of beet roots or pepper leaves. In contrast, these effectors were translocated into plant cells by the phytopathogenic bacteria P. agglomerans pv. betae, Erwinia amylovora, and X. euvesicatoria. Similarly, HsvG, a T3E of P. agglomerans pv. gypsophilae, and XopAU of X. euvesicatoria could be translocated into beet roots and pepper leaves, respectively, by the plant pathogens but not by Salmonella ser. Typhimurium. Mutations in Salmonella ser. Typhimurium T3SS genes invA, ssaV, sipB, or sifA, did not affect its endophytic colonization of lettuce leaves, supporting the notion that S. enterica cannot translocate T3Es into plant cells.