Pathogenicity of and plant immunity to soft rot pectobacteria.
ABSTRACT: Soft rot pectobacteria are broad host range enterobacterial pathogens that cause disease on a variety of plant species including the major crop potato. Pectobacteria are aggressive necrotrophs that harbor a large arsenal of plant cell wall-degrading enzymes as their primary virulence determinants. These enzymes together with additional virulence factors are employed to macerate the host tissue and promote host cell death to provide nutrients for the pathogens. In contrast to (hemi)biotrophs such as Pseudomonas, type III secretion systems (T3SS) and T3 effectors do not appear central to pathogenesis of pectobacteria. Indeed, recent genomic analysis of several Pectobacterium species including the emerging pathogen Pectobacterium wasabiae has shown that many strains lack the entire T3SS as well as the T3 effectors. Instead, this analysis has indicated the presence of novel virulence determinants. Resistance to broad host range pectobacteria is complex and does not appear to involve single resistance genes. Instead, activation of plant innate immunity systems including both SA (salicylic acid) and JA (jasmonic acid)/ET (ethylene)-mediated defenses appears to play a central role in attenuation of Pectobacterium virulence. These defenses are triggered by detection of pathogen-associated molecular patterns (PAMPs) or recognition of modified-self such as damage-associated molecular patterns (DAMPs) and result in enhancement of basal immunity (PAMP/DAMP-triggered immunity or pattern-triggered immunity, PTI). In particular plant cell wall fragments released by the action of the degradative enzymes secreted by pectobacteria are major players in enhanced immunity toward these pathogens. Most notably bacterial pectin-degrading enzymes release oligogalacturonide (OG) fragments recognized as DAMPs activating innate immune responses. Recent progress in understanding OG recognition and signaling allows novel genetic screens for OG-insensitive mutants and will provide new insights into plant defense strategies against necrotrophs such as pectobacteria.
Project description:Plants possess an innate immune system capable of restricting invasion by most potential pathogens. At the cell surface, the recognition of microbe-associated molecular patterns (MAMPs) and/or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) represents the first event for the prompt mounting of an effective immune response. Pathogens have evolved effectors that block MAMP-triggered immunity. The Pseudomonas syringae effector AvrPto abolishes immunity triggered by the peptide MAMPs flg22 and elf18, derived from the bacterial flagellin and elongation factor Tu, respectively, by inhibiting the kinase function of the corresponding receptors FLS2 and EFR, as well as their co-receptors BAK1 and BKK1. Oligogalacturonides (OGs), a well-known class of DAMPs, are oligomers of ?-1,4-linked galacturonosyl residues, released on partial degradation of the plant cell wall homogalacturonan. We show here that AvrPto affects only a subset of the OG-triggered immune responses and that, among these responses, only a subset is affected by the concomitant loss of BAK1 and BKK1. However, the antagonistic effect on auxin-related responses is not affected by either AvrPto or the loss of BAK1/BKK1. These observations reveal an unprecedented complexity among the MAMP/DAMP response cascades. We also show that the signalling system mediated by Peps, another class of DAMPs, and their receptors PEPRs, contributes to OG-activated immunity. We hypothesize that OGs are sensed through multiple and partially redundant perception/transduction complexes, some targeted by AvrPto, but not necessarily comprising BAK1 and BKK1.
Project description:Damage-associated molecular patterns (DAMPs) are endogenous molecules that can activate the plant innate immunity. DAMPs can derive from the plant cell wall, which is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides. Fragments of pectin, called oligogalacturonides (OG), can be released after wounding or by pathogen-encoded cell wall degrading enzymes (CWDEs) such as polygalacturonases (PGs). OG are known to induce innate immune responses, including the activation of mitogen-activated protein kinases (MAPKs), production of H2O2, defense gene activation, and callose deposition. Thus, we hypothesized that xyloglucans (Xh), derived from the plant cell wall hemicellulose, could also act as an endogenous elicitor and trigger a signaling cascade similar to OG. Our results indicate that purified Xh elicit MAPK activation and immune gene expression in grapevine (Vitis vinifera) and Arabidopsis (Arabidopsis thaliana) to trigger induced resistance against necrotrophic (Botrytis cinerea) or biotrophic (Hyaloperonospora arabidopsidis) pathogens. Xh also induce resveratrol production in grapevine cell suspension and callose deposition in Arabidopsis which depends on the callose synthase PMR4. In addition, we characterized some signaling components of Xh-induced immunity using Arabidopsis mutants. Our data suggest that Xh-induced resistance against B. cinerea is dependent on the phytoalexin, salicylate, jasmonate, and ethylene pathways.
Project description:Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.
Project description:BACKGROUND:Pectobacterium carotovorum subsp. brasiliense is a broad host range bacterial pathogen, which causes blackleg of potatoes and bacterial soft rot of vegetables worldwide. Production of plant cell wall degrading enzymes is usually critical for Pectobacterium infection. However, other virulence factors and the mechanisms of genetic adaptation still need to be studied in detail. RESULTS:In this study, the complete genome of P. carotovorum subsp. brasiliense strain SX309 isolated from cucumber was compared with eight other pathogenic bacteria belonging to the Pectobacterium genus, which were isolated from various host plants. Genome comparison revealed that most virulence genes are highly conserved in the Pectobacterium strains, especially for the key virulence determinants involved in the biosynthesis of extracellular enzymes and others including the type II and III secretion systems, quorum sensing system, flagellar and chemotactic genes. Nevertheless, some variable regions of the T6SS and the CRISP-Cas immune system are unique for P. carotovorum subsp. brasiliense. CONCLUSIONS:The extensive comparative genomics analysis revealed highly conserved virulence genes in the Pectobacterium strains. However, several variable regions of type VI secretion system and two subtype Cas mechanism-Cas immune systems possibly contribute to the process of Pectobacterium infection and adaptive immunity.
Project description:Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity.
Project description:Necrotrophic pathogens cause major pre- and post-harvest diseases in numerous agronomic and horticultural crops inflicting significant economic losses. In contrast to biotrophs, obligate plant parasites that infect and feed on living cells, necrotrophs promote the destruction of host cells to feed on their contents. This difference underpins the divergent pathogenesis strategies and plant immune responses to biotrophic and necrotrophic infections. This chapter focuses on Arabidopsis immunity to necrotrophic pathogens. The strategies of infection, virulence and suppression of host defenses recruited by necrotrophs and the variation in host resistance mechanisms are highlighted. The multiplicity of intraspecific virulence factors and species diversity in necrotrophic organisms corresponds to variations in host resistance strategies. Resistance to host-specific necrotophs is monogenic whereas defense against broad host necrotrophs is complex, requiring the involvement of many genes and pathways for full resistance. Mechanisms and components of immunity such as the role of plant hormones, secondary metabolites, and pathogenesis proteins are presented. We will discuss the current state of knowledge of Arabidopsis immune responses to necrotrophic pathogens, the interactions of these responses with other defense pathways, and contemplate on the directions of future research.
Project description:The plant pathogen Pseudomonas syringae pv.tomato (DC3000) causes virulence by delivering effector proteins into host plant cells through its type three secretion system (T3SS). In response to the plant environment DC3000 expresses hypersensitive response and pathogenicity genes (hrp). Pathogenesis depends on the ability of the pathogen to manipulate the plant metabolism and to inhibit plant immunity, which depends to a large degree on the plant's capacity to recognize both pathogen and microbial determinants (PAMP/MAMP-triggered immunity). We have developed and employed MS-based shotgun and targeted proteomics to (i) elucidate the extracellular and secretome composition of DC3000 and (ii) evaluate temporal features of the assembly of the T3SS and the secretion process together with its dependence of pH. The proteomic screen, under hrp inducing in vitro conditions, of extracellular and cytoplasmatic fractions indicated the segregated presence of not only T3SS implicated proteins such as HopP1, HrpK1, HrpA1 and AvrPto1, but also of proteins not usually associated with the T3SS or with pathogenicity. Using multiple reaction monitoring MS (MRM-MS) to quantify HrpA1 and AvrPto1, we found that HrpA1 is rapidly expressed, at a strict pH-dependent rate and is post-translationally processed extracellularly. These features appear to not interfere with rapid AvrPto1 expression and secretion but may suggest some temporal post-translational regulatory mechanism of the T3SS assembly. The high specificity and sensitivity of the MRM-MS approach should provide a powerful tool to measure secretion and translocation in infected tissues.
Project description:Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen Acidovorax avenae causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of A. avenae K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein (Lrp) gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 Lrp gene. An Lrp-deleted K1 strain (K?Lrp) did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 (N?Lrp) did not interfere with infection of finger millet. In addition, N?Lrp retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain ?, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain ? was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation.
Project description:Pectobacterium species are enterobacterial plant-pathogens that cause soft rot disease in diverse plant species. Unlike hemi-biotrophic plant pathogenic bacteria, the type III secretion system (T3SS) of Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) appears to secrete only one effector protein, DspE. Previously, we found that the T3SS regulator HrpL and the effector DspE are required for P. carotovorum pathogenesis on leaves. Here, we identified genes up-regulated by HrpL, visualized expression of dspE in leaves, and established that DspE causes host cell death. DspE required its full length and WxxxE-like motifs, which are characteristic of the AvrE-family effectors, for host cell death. We also examined expression in plant leaves and showed that hrpL is required for the expression of dspE and hrpN, and that the loss of a functional T3SS had unexpected effects on expression of other genes during leaf infection. These data support a model where P. carotovorum uses the T3SS early in leaf infection to initiate pathogenesis through elicitation of DspE-mediated host cell death.
Project description:Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host-pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis.