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

Abstract

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.

Identification of Harpins in Pseudomonas syringae pv. tomato DC3000, Which Are Functionally Similar to HrpK1 in Promoting Translocation of Type III Secretion System Effectors

“…However, this same analysis found only three effectors in the medium, i.e., AvrPto1, HopM1, and HopAM1. It is noteworthy that all three of these effectors are strongly expressed (21) and hopAM1 is duplicated in the DC3000 genome (43). On the basis of all of these observations, we conclude that the repertoire of active harpins in DC3000 is complete with HrpZ1, HrpW1, HopAK1, and HopP1.…”

“…tomato DC3000. The genome of this strain had previously been sequenced and analyzed by microarray and bioinformatic analyses for promoters regulated by the HrpL alternative sigma factor and for downstream open reading frames with N-terminal amino acid patterns predictive of T3SS substrates (8,21,38,63). We screened the open reading frames predicted to encode HrpL-regulated T3SS substrates for those lacking cysteine and found several harpin candidates.…”

“…Exogenous HR elicitation is associated only with the N-terminal harpin domain (13), but HrpW1 homology is much more strongly conserved in its pectate lyase domain than in its harpin domain (11). HrpZ1 and HrpW1 are both conserved among various P. syringae pathovars and are rapidly expressed at high levels when hrp-hrc genes are induced (21,43). However, a double knockout of both genes in P. syringae pv.…”

Pseudomonas syringae Lytic Transglycosylases Coregulated with the Type III Secretion System Contribute to the Translocation of Effector Proteins into Plant Cells

“…Microarray analysis of the kinetics of gene expression in P. syringae pv. tomato DC3000 after bacte- rial transfer into Hrp-inducing medium reveals three putative LT genes that are preceded by Hrp promoter sequences and are activated by the HrpL alternative sigma factor in coordination with genes encoding the T3SS and major effectors (21). These are PSPTO2678, PSPTO852, and PSPTO1378.…”

“…The avrPto1 promoter was chosen because microarray analysis revealed it to be typical of promoters that are strongly upregulated by HrpL, whereas the hopA1 promoter is weaker and the hrpZ1 is located downstream of hrpA1 in a polycistronic operon (21). For each test protein, the level of translocation was substantially reduced in the mutants (Fig.…”