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 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.

Figure 1

Physical map of miniTn5gus insertions in the P. s. tomato CUCPB5114 Hrp pathogenicity island EEL and CEL regions and in avrPto_DC3000 and AvrPtoB. Transposon insertions were identified on the basis of the higher β-glucuronidase activity in CUCPB5114∷miniTn5gus(pCPP5032) mutants grown in media containing tetracycline, which prevents loss of the P_nptII-hrpL plasmid. Arrowheads upstream of ORFs denote Hrp promoters. Independent Tn5gus insertions are indicated by triangles.

Figure 2

A sequence logo representing the Hrp promoter HMM used for searching the P. s. tomato DC3000 genome (51). The vertical axis is information content in bits. The height of a nucleotide reflects its representation in the sequence at that position. Nucleotides are displayed upside-down in positions where they occur less frequently than 25%. The expected frequency of nucleotides is listed below each position. This figure represents only the expected nucleotide frequencies at each position, but not the underlying transition and insertion/deletion probabilities. Positions of relaxed insertion probabilities are indicated with an “i.”

Figure 3

RNA blot analysis of HrpL-dependent expression of representative virulence-implicated genes. Each well was loaded with 25 μg of total RNA isolated from CUCPB5114 cultures carrying either vector control pCPP5031 or P_nptII-hrpL plasmid pCPP5032 (lanes 2 and 3, respectively). PCR-amplified internal fragments were used as probes; lane 1 in each case contains PCR product of the corresponding probe. AvrPpiB11_Pto and AvrPpiB12_Pto are 100% identical, therefore their signals cannot be distinguished.