Plant and environmental sensory signals control the expression of hrp genes in Pseudomonas syringae pv. phaseolicola

Abstract

The hrp genes of Pseudomonas syringae pv. phaseolicola control the development of primary disease symptoms in bean plants and the elicitation of the hypersensitive response in resistant plants. We examined the expression of the seven operons located in the 22-kb hrp cluster (L. G. Rahme, M. N. Mindrinos, and N. J. Panopoulos, J. Bacteriol. 173:575-586, 1991) A set of genes called hrp ("harp") (27) controls the ability of phytopathogenic bacteria to cause disease on susceptible plants and to elicit the hypersensitive response on resistant plants (46). Previous studies established the pathological significance of these genes in Pseudomonas syringae pv. phaseolicola, the casual agent of halo blight disease in bean plants (27). Most of the hrp genes in this bacterium lie in a -22-kb region (the hrp cluster), which is chromosomally located and genetically composed of seven complementation groups, hrpL, hrpAB, hrpC, hrpD, hrpE, hrpF, and hrpRS (39) (see Fig. 1). An additional hrp gene, hrpM, is unlinked to this cluster (12). Several investigators have described genes with similar or analogous functions in phytopathogenic gram-negative bacteria that represent four major taxa: P. syringae, Pseudomonas solanacearum, Erwinia spp., and Xanthomonas campestris (6,7,26,35,44) In this study, we investigated the nature and role of physiological factors involved in the transcriptional regulation of the hip genes of P. syringae pv. phaseolicola by using chromosomally located hrp: :inaZ fusions in complemented and noncomplemented strains grown in planta and in vitro. Our studies show that expression of the hrp genes is controlled by a specific plant signal(s), pH, osmolarity, and the nature of the carbon source. The biological significance and the mechanistic implications of these diverse physicochemical inputs in hip gene expression are critically discussed, and a hypothesis concerning the sequence of events which take place early in the plant-bacterium interaction is proposed. MATERIALS AND METHODSMedia, reagents, and growth conditions. P. syringae pv. phaseolicola NPS3121 and all its derivatives were routinely grown at 24°C in King's B medium (21) or in modified King's B medium lacking glycerol as specified in the text and legends. Defined mineral salts media were made by adding various carbohydrates, amino acids, and organic acids from concentrated, filter-sterilized stocks to M9-salts medium (28). Except as otherwise stated in the text and legends, (i) the carbon source used in M9 medium was sucrose and (ii) the final concentrations of carbon sources or other individual supplements were 5 mM and the pH was adjusted to 5.5. Final concentrations of antibiotics in the media were 15, 20, 50, and 100 ,ug/ml for tetracycline, spectinomycin, nalidixic acid, and rifamycin, respectively.Transfer from nutritionally complex to defined M9 medium caused a variable growth lag in the cultures depending on the carbon source present in the M9 medium. To minimize this lag, the carbon substrate present in the M9 medium 3499 JOURNAL

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

“…In planta, hrpS-uidA expression per cell was about 10-fold higher than in vitro, a finding similar to what has been reported previously for P. syringae pv. phaseolicola (65). 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.…”

“…Strains were grown overnight in KB media which suppresses hrp gene expression. The bacteria were then pelleted, washed, and resuspended in minimal M9 medium, a medium that derepresses hrp and avr gene expression (65). At various times, samples were taken and ␤-glucuronidase activity was determined, with zero time points taken in KB media before resuspension.…”

Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana

“…In P. syringae, expression of many genes involved in pathogenesis, such as hrp/hrc and avr genes, is governed by the alternative RNA polymerase sigma factor HrpL (Rahme et al, 1992;Innes et al, 1993;Salmeron and Staskawicz, 1993;, and it is likely that HrpL also regulates the expression of additional genes involved in virulence. One way to identify ipx genes potentially regulated by HrpL is to examine the promoter regions of these genes for the presence of an 'hrp box', a regulatory element believed to be recognized by HrpL .…”

“…Twenty strains were found to carry more than one pIPET vector and were not analysed further. The resulting DNA blots were also hybridized with probes from the hrp/hrc region to identify strains carrying ipx fusions with promoters from the hrp/hrc cluster, which have previously been demonstrated to be induced in planta (Rahme et al, 1992). Thirty-six independent hrp/hrc fusions were identified in this manner and were not characterized further.…”

“…As a first step towards analysing the ipx promoters, the GUS expression patterns of the ipx fusions were qualitatively assessed on rich, minimal and hrp-inducing medium, a minimal medium often used to induce the expression of P. syringae hrp/hrc and avr genes in culture (Huynh et al, 1989;Rahme et al, 1992;Salmeron and Staskawicz, 1993). The majority of the ipx fusions exhibited GUS activity on hrp-inducing medium and/or on minimal medium containing succinate (Table 1; data not shown).…”