Multiple loci of Pseudomonas syringae pv. syringae are involved in pathogenicity on bean: restoration of one lesion-deficient mutant requires two tRNA genes

Abstract

A mutational analysis of lesion-forming ability was undertaken in Pseudomonas syringae pv. syringae B728a, causal agent of bacterial brown spot disease of bean. Following a screen of 6,401 Tn5-containing derivatives of B728a on bean pods, 26 strains that did not form disease lesions were identified. Nine of the mutant strains were defective in the ability to elicit the hypersensitive reaction (HR) and were shown to contain Tn5 insertions within the P. syringae pv. syringae hrp region. Ten HR ؉ mutants were defective in the production of the toxin syringomycin, and a region of the chromosome implicated in the biosynthesis of syringomycin was deleted in a subset of these mutants. The remaining seven lesion-defective mutants retained the ability to produce protease and syringomycin. Marker exchange mutagenesis confirmed that the Tn5 insertion was causal to the mutant phenotype in several lesion-defective, HR ؉ strains. KW239, a lesion-and syringomycin-deficient mutant, was characterized at the molecular level. Sequence analysis of the chromosomal region flanking the Tn5 within KW239 revealed strong similarities to a number of known Escherichia coli gene products and DNA sequences: the nusA operon, including the complete initiator tRNA Met gene, metY; a tRNA Leu gene; the tpiA gene product; and the MrsA protein. Removal of sequences containing the two potential tRNA genes prevented restoration of mutant KW239 in trans. The Tn5 insertions within the lesion-deficient strains examined, including KW239, were not closely linked to each other or to the lemA or gacA genes previously identified as involved in lesion formation by P. syringae pv. syringae.The majority of Pseudomonas syringae genes known to be involved in plant disease also control plant interaction phenotypes not directly related to pathogenicity. Mutations in hrp genes, for example, disrupt symptom development, growth in association with host plants, and the hypersensitive reaction (HR) on nonhost plants (for a review, see reference 60). The discovery of the lemA gene of P. syringae pv. syringae B728a, a causal agent of bacterial brown spot disease of bean (Phaseolus vulgaris), demonstrated that lesion manifestation can be genetically separated from other interactions with plants. The lemA gene is required for disease lesion formation but not for nonhost hypersensitivity (59). Furthermore, the lemA gene is essential for production of the toxin syringomycin and an extracellular protease activity (21, 46). Genetic evidence and DNA sequence analysis of the lemA gene support a regulatory role for the predicted LemA protein, which is similar to a family of transmembrane histidine protein kinase sensors of two-component regulatory systems (4,20,46,61). More recently, a study of spontaneous mutants of B728a and other P. syringae pv. syringae strains led to the identification of the gacA gene as the second component, the response regulator (43, 47). Mutants of gacA have a phenotype similar to that of lemA mutants, being deficient in disease lesion formation and in ...

Role of the Hrp type III protein secretion system in growth of Pseudomonas syringae pv. syringae B728a on host plants in the field

“…Thus, we cannot rule out the possibility that the role of the type III secretion system in pathogenicity is completely mediated by its requirement for growth of Pss in the phyllosphere. Genes that play a more direct role in symptom development in the Pss-brown spot-snap bean system may reside in the gacS͞gacA regulon identified by Willis and colleagues (12)(13)(14)(15)39). The role of the Hrp type III secretion system in growth but not symptom development per se in Pss is consistent with the recent finding of Shea et al (40) on the role of the type III secretion system within Salmonella typhimurium pathogenicity island 2 (SPI2).…”

Global Regulation by gidA in Pseudomonas syringae

“…For example, extracellular protease production in B728a requires intact gac genes and has been shown to be separate from antibiotic production within the regulon (17,18). The production of this protease is unaffected in gidA mutants (32). In a similar fashion, loss of pyoverdin production is not a phenotype of gacS and gacA mutations in B728a.…”

“…The screening and isolation of KW2163 and KW2803 as random Tn5 transposon mutants affected for virulence and syringomycin production have been previously described (32). Sequence analysis of subclones made from cosmids containing the Tn5 insertions and flanking chromosomal DNA indicated that the two insertion sites mapped to closely placed but separate locations within the gidA gene ( Fig.…”

Phylogenetic Analysis of Pseudomonas syringae Pathovars Suggests the Horizontal Gene Transfer of argK and the Evolutionary Stability of hrp Gene Cluster

“…In general, plant pathogenic bacteria must have various factors that are related to host plant affinity and directly involved in destroying plant tissues to intrude into and infect plants and cause disease. Thus, the number of pathogenicity-related genes should be enormous (Baker et al 1997;Daniels et al 1988;Gopalan and He 1996;Rich and Willis 1997;Rudolph 1995). Moreover, there are data suggesting that dynamic evolutionary mechanisms such as horizontal gene transfer and genome rearrangement are involved in the diversification of pathogenicity-related genes existing on the P. syringae genome ("genome" refers to "bacterial chromosome" in this paper) and have accelerated and complicated the pathogenicity differentiation (Hatziloukas and Panopoulos 1992;Sawada et al 1995aSawada et al , 1996Sawada et al , 1997aSawada, unpublished data).…”