Expression of avrPphB, an Avirulence Gene from Pseudomonas syringae pv. phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean

Abstract

Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola was examined. Incorporation of [35S]-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product. The 28-kDa native peptide was isolated from E. coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves. Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E. coli and P. syringae pv. phaseolicola. Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY- strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time. Expression of the hrp gene cluster of P. syringae pv. phaseolicola from plasmid pPPY430 in E. coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031). Despite constitutive expression of the hrp and avr genes in E. coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes. The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.

The Alternative Sigma Factor RpoN Is Required for hrp Activity in Pseudomonas syringae pv. Maculicola and Acts at the Level of hrpL Transcription

“…Despite some inconsistencies, these data shown that the loss of pathogenicity and HR phenotypes of the rpoN mutant is most easily explained by the failure to activate hrp and avr transcription in the rpoN mutant. In general, our data are consistent with previously proposed models of hrp regulation in P. syringae in which HrpS plus RpoN ( 54 ) activate hrpL and in which HrpL activates avr genes and the remaining genes in the hrp cluster (63,80,81). To further test this model, we placed hrpL expression under control of the E. coli lacZ promoter.…”

“…A conserved sequence, GGAACCNA-N14-CCAC NNA, which appears to be responsible for hrpL-dependent transcription, has been identified upstream of a variety of avr genes (81). In several cases, including avrRpt2 and avrRpm1, hrpL dependence has been confirmed experimentally (34,35,46,63,66,70,81). Thus, the loss of hrp by mutation of a putative hrp regulator such as rpoN should affect HR induction by blocking avr gene transcription and type III-mediated protein delivery.…”

Type III Protein Translocase

“…As expected, the cytoplasmic DnaK protein was almost exclusively detected in the soluble fraction. Similar results were obtained with E. coli MC4100 harboring the complete P. syringae phaseolicola TTS regulon on the low copy number cosmid pPPY430 and the hrpL factor under lac control on a separate plasmid (23,38). MC4100/ pPPY430/pHrpL grown in the presence (Fig.…”

Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

“…In the absence of a corresponding R gene product, the avr product enhances virulence; however, in hosts which have the corresponding R gene, recognition of the avr gene product enhances host resistance. Interestingly, most avr genes are also coordinately regulated with genes in the hrp cluster (27,29,46,67,71,77,97).The HR is accompanied by the induction of defense-related genes (7, 91) that are differentially expressed depending on the particular pair of avr and R gene products eliciting the HR (70, 72). Defense gene induction also occurs in the absence of the HR during compatible pathogen-host interactions, although usually later and at lower levels than those occurring during an HR (11,32,66).…”

“…In the absence of a corresponding R gene product, the avr product enhances virulence; however, in hosts which have the corresponding R gene, recognition of the avr gene product enhances host resistance. Interestingly, most avr genes are also coordinately regulated with genes in the hrp cluster (27,29,46,67,71,77,97).…”