Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species.
High humidity has a profound influence on the development of numerous phyllosphere diseases in crop fields and natural ecosystems, but the molecular basis of this humidity effect is not understood. Previous studies emphasize immune suppression as a key step in bacterial pathogenesis. Here we show that humidity-dependent, pathogen-driven establishment of an aqueous intercellular space (apoplast) is another crucial step in bacterial infection of the phyllosphere. Bacterial effectors, such as Pseudomonas syringae HopM1, induce establishment of the aqueous apoplast and are sufficient to transform non-pathogenic P. syringae strains into virulent pathogens in immune-deficient Arabidopsis under high humidity. Arabidopsis quadruple mutants simultaneously defective in a host target (MIN7) of HopM1 and in pattern-triggered immunity could not only recapitulate the basic features of bacterial infection, but also exhibit humidity-dependent dyshomeostasis of the endophytic commensal bacterial community in the phyllosphere. These results highlight a new conceptual framework for understanding diverse phyllosphere-bacterial interactions.
AvrPto from the Pseudomonas cell. To examine this, avrPto deletions C⌬25, C⌬41, and C⌬74 were placed into pBI121 and tested with the Agrobacterium transient assay. Agrobacterium EHA105 containing avrPto induced an HR in 2 days, whereas EHA105 containing the avrPto deletion C⌬25 induced an HR after 4 days; the other deletions did not elicit an HR ( X. Tang and G. Martin, unpublished results). This suggests that the carboxyl terminal 25 amino acids of AvrPto are not required for secretion from the bacterial cell; this portion of AvrPto may serve as an activation domain, interact with other components in the signaling pathway, or have a role in AvrPto stability.
Diverse gram-negative bacteria deliver effector proteins into the cells of their eukaryotic hosts using the type III secretion system. Collectively, these type III effector proteins function to optimize the host cell environment for bacterial growth. Type III effector proteins are essential for the virulence of Pseudomonas syringae, Xanthomonas spp., Ralstonia solanacearum and Erwinia species. Type III secretion systems are also found in nonpathogenic pseudomonads and in species of symbiotic nitrogen-fixing Rhizobium. We discuss the functions of type III effector proteins of plant-associated bacteria, with an emphasis on pathogens. Plant pathogens tend to carry diverse collections of type III effectors that likely share overlapping functions. Several effectors inhibit host defense responses. The eukaryotic host targets of only a few type III effector proteins are currently known. We also discuss possible mechanisms for diversification of the suite of type III effector proteins carried by a given bacterial strain.
Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.
Pseudomonas syringae strains deliver variable numbers of type III effector proteins into plant cells during infection. These proteins are required for virulence, because strains incapable of delivering them are nonpathogenic. We implemented a whole-genome, highthroughput screen for identifying P. syringae type III effector genes. The screen relied on FACS and an arabinose-inducible hrpL factor to automate the identification and cloning of HrpLregulated genes. We determined whether candidate genes encode type III effector proteins by creating and testing full-length protein fusions to a reporter called ⌬79AvrRpt2 that, when fused to known type III effector proteins, is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 plant disease resistance protein. ⌬79AvrRpt2 is thus a marker for type III secretion system-dependent translocation, the most critical criterion for defining type III effector proteins. We describe our screen and the collection of type III effector proteins from two pathovars of P. syringae. This stringent functional criteria defined 29 type III proteins from P. syringae pv. tomato, and 19 from P. syringae pv. phaseolicola race 6. Our data provide full functional annotation of the hrpL-dependent type III effector suites from two sequenced P. syringae pathovars and show that type III effector protein suites are highly variable in this pathogen, presumably reflecting the evolutionary selection imposed by the various host plants.host-microbe interaction ͉ plant pathogenesis ͉ Arabidopsis ͉ FACS
We propose a new statistical test for assessing differential gene expression using RNA sequencing (RNA-Seq) data. Commonly used probability distributions, such as binomial or Poisson, cannot appropriately model the count variability in RNA-Seq data due to overdispersion. The small sample size that is typical in this type of data also prevents the uncritical use of tools derived from large-sample asymptotic theory. The test we propose is based on the NBP parameterization of the negative binomial distribution. It extends an exact test proposed by Robinson and Smyth (2007, 2008). In one version of Robinson and Smyth’s test, a constant dispersion parameter is used to model the count variability between biological replicates. We introduce an additional parameter to allow the dispersion parameter to depend on the mean. Our parametric method complements nonparametric regression approaches for modeling the dispersion parameter. We apply the test we propose to an Arabidopsis data set and a range of simulated data sets. The results show that the test is simple, powerful and reasonably robust against departures from model assumptions.
Innate immunity in plants can be triggered by microbe- and pathogen-associated molecular patterns. The pathogen-associated molecular pattern-triggered immunity (PTI) is often suppressed by pathogen effectors delivered into the host cell. Plants can overcome pathogen suppression of PTI and reestablish pathogen resistance through effector-triggered immunity (ETI). An unanswered question is how plants might overcome pathogen-suppression of PTI during ETI. Findings described in this paper suggest a possible mechanism. During Pseudomonas syringae pathovar tomato ( Pst ) DC3000 infection of Arabidopsis , a host ADP ribosylation factor guanine nucleotide exchange factor, AtMIN7, is destabilized by the pathogen effector HopM1 through the host 26S proteasome. In this study, we discovered that AtMIN7 is required for not only PTI, consistent with the notion that Pst DC3000 degrades AtMIN7 to suppress PTI, but also ETI. The AtMIN7 level in healthy plants is low, but increases posttranscriptionally in response to activation of PTI. Whereas DC3000 infection led to degradation of AtMIN7, activation of ETI by three different effectors, AvrRpt2, AvrPphB, and HopA1, in Col-0 plants blocks the ability of Pst DC3000 to destabilize AtMIN7. Further analyses of bacterial translocation of HopM1 and AtMIN7 stability in HopM1 transgenic plants show that ETI prevents HopM1-mediated degradation of AtMIN7 inside the plant cell. Both AtMIN7 and HopM1 are localized to the trans -Golgi network/early endosome, a subcellular compartment that is not previously known to be associated with bacterial pathogenesis in plants. Thus, blocking pathogen degradation of trans -Golgi network/early endosome-associated AtMIN7 is a critical part of the ETI mechanism to counter bacterial suppression of PTI.
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