Genetic and transcriptional organization of the hrp cluster of Pseudomonas syringae pv. phaseolicola

Abstract

The hrp cluster of Pseudomonas syringae pv. phaseolicola encodes functions that are essential for pathogenicity on bean plants and for the elicitation of the hypersensitive response on resistant plants. The cluster was saturated with insertions of transposon Tn3-spice that served both as a mutagen and as a sensitive reporter of the expression of the target regions. The mutations covered a 17.5-kb segment in strain NPS3121, in which seven hrp::Tn5 insertions had been previously mapped, and regions outside this segment. The cluster is organized into seven distinct complementation groups (hrpL, hrpAB, hrpC, hrpD, hrpE, hrpF, and hrpSR) on the basis of the analysis of over 100 Tn3-spice insertions in plasmids and 43 similar insertions in the chromosome; it spans nearly 22 kb and is chromosomally located. The transcriptional orientation of all genes in the cluster was established by measuring the level of ice nucleation activity of complemented merodiploids carrying chromosomal hrp::inaZ fusions after inoculation in Red Kidney bean leaves. Although all seven loci were actively expressed in Red Kidney bean leaves, none of them was substantially expressed when the bacteria were grown in King B broth medium. Mutations in all loci, except those in hrpC, greatly reduced the ability of the bacteria to multiply in bean leaves. Mutations in the hrpC locus, although preventing the bacteria from eliciting a hypersensitive reaction on tobacco, allowed the bacteria to produce delayed and attenuated symptoms in Red Kidney bean leaves and to multiply to a level 102. to 103-fold lower than that of the wild-type strain. This is the first comprehensive report of the genetic and transcriptional organization of the hrp gene cluster in a phytopathogenic bacterium.The bean halo blight pathogen, Pseudomonas syringae pv. phaseolicola, possesses a group of genes, designated hrp ("harp"), that are essential for the development of disease symptoms and for the elicitation of the hypersensitive reaction on resistant plants. The majority of known hrp genes in this bacterium are clustered in a large genomic region that has been designated the hrp cluster, whereas one locus (hrpM) is located elsewhere in the genome (24,29,31,36). Besides P. syringae pv. phaseolicola, hrp genes and/or gene clusters from several other members of the P. syringae taxon have been described and cloned (5, 13-16, 24, 30, 33, 37-39), as have those from phylogenetically distinct plant pathogens, such as Pseudomonas solanacearum (2, 14), Erwinia amylovora (44, 45), and some pathovars of Xanthomonas campestris (2,6,42). Homology between hrp genes has been reported among members of the P. syringae taxon (8a, 24) as well as between P. solanacearum and members of the X. campestris group (2).The nature and precise role of most hrp gene products in the plant-bacterium interaction are unknown. Three hrp loci have been analyzed in some detail in P. syringae pv. phaseolicola. A locus designated hrpS encodes a protein that shares sequence similarity to NtrC-like regulatory proteins (...

Organization and environmental regulation of the Pseudomonas syringae pv. syringae 61 hrp cluster

“…Several P. syningae pv. phaseolicola hrp genes are rapidly induced in leaves of susceptible bean plants (27); this has been attributed to an unidentified plant effector (9,38). Our results, which were obtained with a different host range variant of P. syningae, focused on the early phases of the interaction with a nonhost plant species, and utilized a different reporter gene system (GUS versus ice nucleation), are inconsistent with the involvement of a plant factor in the regulation of P. syningae pv.…”

“…The designations chosen by Huang et al (15) were retained to avoid confusion with the designations used for the partially homologous P. syringae pv. phaseolicola hrp cluster (38) transcriptional unit failed to complement any TnphoA insertions within that unit; Tn5-gusAl insertions in downstream complementation groups retained the ability to complement insertions in upstream complementation groups. This suggests that hrpIII-IV, hrpV-VII, and hrpXI-XII represent multicistronic transcriptional units.…”

“…syringae 61 hrp/hrm genes are thus likely genes to be induced during the induction stage of the HR. Enhanced expression of several hrp genes has been reported during pathogenesis of P. syringae pv. phaseolicola in its susceptible host, bean plants (9,27,38).…”

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

“…It is not surprising that we identified a large number of fusions to the hrp/hrc region, as these genes are known to be induced upon inoculation of bacteria into plant tissue (Rahme et al, 1991). Likewise, we anticipated identifying genes that encode known or predicted effectors of the type III secretion system, such as avr and vir genes and members of the conserved effector locus (CEL), as these genes are known or expected to be co-regulated with the hrp/hrc locus He, 1998;Alfano et al, 2000).…”

“…Many known P. syringae pathogenicity and virulence determinants, including hrp/hrc and avr genes, are induced upon inoculation into plant tissue (for examples, see Rahme et al, 1991;Innes et al, 1993;Salmeron and Staskawicz, 1993). Thus, we hypothesized that we could identify additional P. syringae genes important for colonization, growth in planta and disease production by screening for bacterial genes that are expressed after inoculation into plant tissue.…”