David J. Schneider

Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor.

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), Nɛ-(indole-3-acetyl)-l-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.

Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors

Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete. A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain. These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P. syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.

The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from todays Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

A Draft Genome Sequence of Pseudomonas syringae pv. tomato T1 Reveals a Type III Effector Repertoire Significantly Divergent from That of Pseudomonas syringae pv. tomato DC3000

Diverse gene products including phytotoxins, pathogen-associated molecular patterns, and type III secreted effectors influence interactions between Pseudomonas syringae strains and plants, with additional yet uncharacterized factors likely contributing as well. Of particular interest are those interactions governing pathogen-host specificity. Comparative genomics of closely related pathogens with different host specificity represents an excellent approach for identification of genes contributing to host-range determination. A draft genome sequence of Pseudomonas syringae pv. tomato T1, which is pathogenic on tomato but nonpathogenic on Arabidopsis thaliana, was obtained for this purpose and compared with the genome of the closely related A. thaliana and tomato model pathogen P. syringae pv. tomato DC3000. Although the overall genetic content of each of the two genomes appears to be highly similar, the repertoire of effectors was found to diverge significantly. Several P. syringae pv. tomato T1 effectors absent from strain DC3000 were confirmed to be translocated into plants, with the well-studied effector AvrRpt2 representing a likely candidate for host-range determination. However, the presence of avrRpt2 was not found sufficient to explain A. thaliana resistance to P. syringae pv. tomato T1, suggesting that other effectors and possibly type III secretion system-independent factors also play a role in this interaction.

Whole-Genome Expression Profiling Defines the HrpL Regulon of Pseudomonas syringae pv. tomato DC3000, Allows de novo Reconstruction of the Hrp cis Clement, and Identifies Novel Coregulated Genes

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and deltahrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the deltahrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.

Bioinformatics-Enabled Identification of the HrpL Regulon and Type III Secretion System Effector Proteins of Pseudomonas syringae pv. phaseolicola 1448A

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.

Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads

Bacteria that survive under variable conditions possess an assortment of genetic regulators to meet these challenges. The group IV or extracytoplasmic function (ECF) sigma factors regulate gene expression in response to specific environmental signals by altering the promoter specificity of RNA polymerase. We have undertaken a study of PvdS, a group IV sigma factor encoded by Pseudomonas syringae pv. tomato DC3000 (DC3000), a plant pathogen that is likely to encounter variations in nutrient availability as well as plant host defences. The gene encoding PvdS was previously identified by sequence similarity to the Pseudomonas aeruginosa orthologue, which directs transcription of genes encoding the biosynthesis of pyoverdine, a siderophore involved in iron acquisition, and is responsible for the characteristic fluorescence of the pseudomonads. We identified 15 promoters regulated by PvdS in DC3000 and characterized the promoter motif using computational analysis. Mutagenesis of conserved nucleotides within the motif interfered with promoter function and the degree of the effect was different depending on which region of the motif was mutated. Hidden Markov models constructed from alignments of sequence motifs extracted from DC3000 and PAO1 were used to query genomes of DC3000 and other fluorescent pseudomonads for similar motifs. We conclude that the role of PvdS as a regulator of pyoverdine synthesis is conserved among the fluorescent pseudomonads, but the promoters recognized by PvdS orthologues may differ subtly from species to species.

The Effect of Target Vector Selection on the Invariance of Classifier Performance Measures

In this paper, the multiclass supervised training problem is considered when a discrete set of classes is assumed. Upon generating affine models for finite data sets, we have observed the invariance of certain measures of performance after a trained classifier has been presented with test data of unknown classification. Specifically, after constructing mappings between training vectors and their desired targets, the class membership and ranking of test data has been found to remain either invariant or close to invariant under a transformation of the set of target vectors. Therefore, we derive conditions explaining how this type of invariance can arise when the multiclass problem is phrased in the context of linear networks. A bioinformatics example is then presented in order to demonstrate various principles outlined in this work.