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), N(epsilon)-(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.
Fouts
Derrick E
DE
Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203, USA.
Abramovitch
Robert B
RB
Alfano
James R
JR
Baldo
Angela M
AM
Buell
C Robin
CR
Cartinhour
Samuel
S
Chatterjee
Arun K
AK
D'Ascenzo
Mark
M
Gwinn
Michelle L
ML
Lazarowitz
Sondra G
SG
Lin
Nai-Chun
NC
Martin
Gregory B
GB
Rehm
Amos H
AH
Schneider
David J
DJ
van Dijk
Karin
K
Tang
Xiaoyan
X
Collmer
Alan
A
D001426:Bacterial Proteins / Q000235:genetics*; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D017930:Genes, Reporter; D016680:Genome, Bacterial; D018551:Solanum lycopersicum / Q000382:microbiology; D008390:Markov Chains; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000235:genetics* / Q000472:pathogenicity*; D012313:RNA / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics*; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C085855:HrpL protein, Pseudomonas syringae; D012808:Sigma Factor; D012313:RNA