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

doi:10.1073/pnas.032514099

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

^*Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203; ^†Boyce Thompson Institute for Plant Research, Ithaca, NY 14853-1801; ^‡Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, NE 68588-0660; ^§United States Department of Agriculture–Agricultural Research Service Center for Agricultural Bioinformatics, Cornell Theory Center, Ithaca, NY 14853-2901; ^¶Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850; ^‖Department of Plant Microbiology and Pathology, University of Missouri, Columbia, MO 65211; ^**Cornell Theory Center, Cornell University, Ithaca, NY 14853; and ^††Department of Plant Pathology, Kansas State University, Manhattan, KS 66506-5502

Edited by Eugene W. Nester, University of Washington, Seattle, WA, and approved December 11, 2001 (received for review October 1, 2001)

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 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), N ^ɛ-(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.

Footnotes

↵ ‡‡ Present address: United States Department of Agriculture–Agricultural Research Service Center for Agricultural Bioinformatics, Ithaca, NY 14853-2901.
↵ §§ To whom reprint requests should be addressed. E-mail: arc2{at}cornell.edu.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. J03681, J03682, L11336, M15194, M21965, L20425, Z21715, X84843, L14926, I16119, AF232006, M22219, AJ251482, X67808, AJ22647, M86401, AJ277494–AJ277496, AF231453, PSJ224433, L41863, and AF268940).
Abbreviations:

Hrp,

hypersensitive response and pathogenicity;

Avr,

avirulence;

Hop,

Hrp-dependent outer protein;

CEL,

conserved effector locus;

HMM,

hidden Markov model;

E,

expectation (value);

EEL,

exchangeable effector locus