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

Abstract

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 HrpLresponsive 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 virulenceimplicated 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 HrpLdependent 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), N -(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. P seudomonas syringae pv. tomato DC3000 has emerged as an important model organism in molecular plant pathology because of its genetic tractability, its pathogenicity on both tomato and Arabidopsis thaliana, and its ability to deliver virulence effector proteins into host cells via a type III protein secretion system. To support worldwide functional genomic investigations of P. s. tomato DC3000, we are determining the complete sequence of the DC3000 genome. A draft sequence is now available (http:͞͞www.tigr.org͞tdb͞mdb͞ mdbinprogress.html), and we have initiated a genomewide investigation of potential virulence factors with a search for virulencerelated genes in the same regulon as the type III secretion system.Virulence effector proteins delivered to or into host cells by type III secretion systems are key factors in the pathogenicity of many bacteria, including animal pathogens in the genera Salmonella, Yersinia, Shigella, and Escherichia, and plant pathogens in the genera Pseudomonas, Erwinia, Xanthomonas, Ralstonia, and Pantoea (1). In plant pathogens, the type III secretion machinery is referred to as the hypersensitive response and pathogenicity (Hrp) system ...

Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000

“…Two of them (Psyr2576 and Psyr2577) have orthologs in Pst DC3000 that appear to be controlled by the sigma factor HrpL (45). Another (Psyr3722) has an ortholog in Pst DC3000 that is truncated and therefore likely to be nonfunctional or to produce a different peptide.…”

The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000

“…Hrp effectors are primarily known as Hop (Hrp outer protein) or Avr (avirulence) proteins, based on the phenotype by which they were discovered (11). Functional analysis of the DC3000 genome has yielded genes for 31 effectors and 7 additional proteins secreted by the Hrp system that likely have a role in translocating effectors across the plant cell wall and plasma membrane (8)(9)(10)(11)(12)36). The associated hop, avr, and hol (Hop-like candidate) genes may provide markers to regions of the genome that are enriched in other virulence genes (Fig.…”

“…Importantly, the pathogenicity of DC3000 resembles that of most animal and plant pathogens in the gamma Proteobacteria, which rely on a type III secretion system (TTSS) to inject virulence effector proteins into host cells (6). Highlighting the role of DC3000 as a model for plant-pathogen interactions, Ͼ35 genes encoding TTSS substrates have been experimentally identified in this strain (7)(8)(9)(10)(11)(12), which exceeds the number demonstrated for any other bacterial pathogen to date. Furthermore, the pathogenicity of DC3000 resembles that of many important bacterial and fungal pathogens of plants where host specificity is controlled by ''gene-for-gene'' interactions in which a dominant allele in the host and a dominant allele in the pathogen condition the outcome of the plant-pathogen interaction (13).…”

Comparative Genomics of Host-Specific Virulence in Pseudomonas syringae

“…syringae as a species has the largest known arsenal of T3SEs of any pathogen (Fouts et al 2002;Guttman et al 2002;Petnicki-Ocwieja et al 2002;ZwieslerVollick et al 2002;Greenberg and Vinatzer 2003;Chang et al 2005). Functional studies have shown that while the T3SS as a whole is required for pathogenesis, many T3SEs have redundant or subtle roles (Lorang and Keen 1995;Charkowski et al 1998;Guttman and Greenberg 2001;Badel et al 2002Badel et al , 2003Losada et al 2004;Lim and Kunkel 2005;Lin and Martin 2005).…”