Hrp Loci Research Articles

Distribution of avirulence genes avrA and popP1 in 22 Japanese phylotype I strains of Ralstonia solanacearum

Sequence analysis of hrp loci and effector genes in the flanking regions showed significantly high similarities between two phylotype I strains of Ralstonia solanacearum, GMI1000 and Japanese strain OE1-1. Further sequence analysis of the distribution of avrA and popP1, known as determinants of a hypersensitive response (HR) induction on Nicotiana tabacum (tobacco), in 22 Japanese phylotype I strains revealed that all strains had one of the two distinct avrA alleles and that 10 strains had an identical popP1 but the other 12 did not. After infiltration of tobacco leaves, more than half of these 22 strains elicited HR. In combination with the ability to induce HR, avrA and popP1 are thus not likely to be the sole determinants of HR in Japanese phylotype I strains.

Relative effects on virulence of mutations in the sap, pel, and hrp loci of Erwinia chrysanthemi.

We constructed strains of Erwinia chrysanthemi EC16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the pel (CUCPB5006) and hrp (CUCPB5039) mutations. This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037). The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel-Hrp-Sap (BT108) suggested that the effects of these mutations are additive. In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers. The competitive indices of strains BT105, UM1005 (Pel-), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro. These results indicate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in pel and sap cannot.

Expression and localization of HrpA1, a protein of Xanthomonas campestris pv. vesicatoria essential for pathogenicity and induction ofthe hypersensitive reaction.

The hrp cluster of the pepper and tomato pathogen Xanthomonas campestris pv. vesicatoria is required for both pathogenicity on susceptible host plants and induction of the hypersensitive reaction on resistant plants. The hrpA locus is located at the left end of the 25-kb hrp region and encodes a single 64-kDa Hrp protein, HrpA1, which belongs to the PulD superfamily of proteins involved in type II and type III protein secretion. In this study, we developed a defined medium without any plant-derived molecules that induces expression of hrpA in vitro. The hrpA transcription start site was mapped in the coding region of the hrpB8 gene, which is the last gene of the hrpB operon. The inducible hrpA promoter shows no homology to known promoter elements or other hrp loci of X. campestris pv. vesicatoria. hrpA expression was shown to be independent of the hrp regulatory gene hrpX. The amino acid sequence of the HrpA1 protein is predicted to contain an N-terminal signal sequence and no further transmembrane domains and to be rich in beta-sheet stretches. Expression of HrpA1 in Escherichia coli cells causes induction of the psp operon like some of its counterparts, suggesting some commonality of function and that HrpA1 forms multimers. The protein product of hrpA1 was identified by using a specific polyclonal antibody. Cell fractionation studies demonstrated that the HrpA1 protein is localized in the outer membrane of X. campestris pv. vesicatoria. HrpA1 is the first component of the Hrp secretion system whose localization has been determined in the original organism.

HrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of sigma factors

hrpL of Erwinia amylovora Ea321 encodes a 21.7-kDa regulatory protein, similar to members of the ECF (extra cytoplasmic functions) subfamily of eubacterial RNA polymerase sigma factors. hrpL is a single-gene operon in complementation group VI of the E. amylovora hrp gene cluster. Its product is required by Ea321 to elicit the hypersensitive response (HR) and to cause disease. HrpL controls the expression of five independent hrp loci, including hrpN, which encodes harpin, a proteinaceous elicitor of the HR. hrpL is environmentally regulated, and its expression is affected by hrpS, another regulatory gene of the hrp gene cluster of E. amylovora. pCPP1078, a multicopy plasmid carrying hrpL, is able to restore HR-eliciting ability to hrpS mutants. A conserved motif was identified upstream of the hrpI and hrpN operons, which are transcriptionally regulated by hrpL. This conserved motif shares a high degree of similarity with other biochemically defined or putative ECF-dependent promoter sequences, including sequences upstream of Streptomyces coelicolor dagA P2, Pseudomonas aeruginosa algD, Pseudomonas syringae pv. syringae 61 hrpZ, and P. syringae pv. tomato avrD. In spite of the similarity between the hrpL genes of E. amylovora and P. syringae 61, no functional cross-complementation was observed.

Altered Expression of Erwinia amylovora HRP Genes in Tobacco Leaves Pretreated with Bacterial Protein‐Lipopolysaccharides

AbstractInfiltration of protein‐lipopolysaccharide complexes (pr LPS) (250 μg/ml) of Pseudomonas syringae pv. aptata into tobacco leaves protected against subsequent elicitation of the hypersensitive response (HR) by Erwinia amylovora. The effect of prLPS on the expression of E. amylovora hrp (hypersensitive response and pathogenicity) gencs was tested in protected tobacco leaves and in a defined medium in which hrp genes were highly expressed. Two,E. amylovora hrp loci transcriptionally fused with the Escherichia coliβ‐glucuronidase (Gus) coding sequence were used as chromosomal reporter genes. The prLPS treatment did not affect hrp gene expression of Hrp‐ mutants both in planta and in vitro, so the effect of prLPS treatment on hrp gene expression of two E. amylovora Hrp+ transformants was assayed during HR development in unprotected tissue and during the same time in prLPS protected tissue. A plasmid containing the same Gus fusions previously located in the chromosome was introduced into a wild‐type strain of E. amylovora. Gus activity was significantly lower in protected tissue. We suggest that HR inhibition by prLPS treatment requires the entire hrp gene cluster in order for the bacteria to send a signal to the plant. which, in turn, inhibits the expression of hrp genes.

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

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) in planta and in vitro under different physiological and nutritional conditions by using chromosomally located hrp::inaZ reporter fusions. We show that (i) a plant signal(s) is specifically required for the induction of the seven hrp operons, during both compatible and incompatible interactions; (ii) hrpL and hrpRS are regulated by different mechanisms in planta and in vitro; and (iii) expression of individual hrp loci is differentially affected by pH, osmotic strength, and type of carbon source: hrpAB, hrpC, and hrpD were downregulated similarly by osmolarity, pH, and certain carbon sources; hrpE expression was affected strongly by pH and carbon substrate and slightly by osmolarity; and hrpF was not substantially affected by any of these factors. These findings suggest complex signaling mechanisms taking place during plant-pathogen interactions.

Expression of Erwinia amylovora hrp genes in response to environmental stimuli.

Seven hrp loci that are essential for the hypersensitive reaction elicited by Erwinia amylovora were transcriptionally fused with a derivative of transposon Tn5, containing the promoterless Escherichia coli beta-glucuronidase reporter gene. The seven hrp fusions were used to monitor expression of the hrp loci in vitro and in planta. No significant expression was detected in rich medium for any of the fusions. However, five of them were expressed highly in planta and in inducing medium that contains mannitol, salts, and 5 mM (NH4)2SO4. Expression of these five hrp loci is regulated by ammonium, nicotinic acid, complex-nitrogen sources, certain carbon sources, temperature, and pH. Under well-defined conditions, i.e., in inducing medium, no specific plant components were required for transcriptional activation of the hrp loci. The high levels of expression detected in vitro were comparable to those determined during the development of the hypersensitive reaction in tobacco. Differential levels of expression of the hrp loci occurred in host and nonhost plants. In pear, a host plant, expression of the hrp loci was delayed and greatly reduced compared with expression in tobacco leaves, a nonhost.

Expression of the Xanthomonas campestris pv. vesicatoria hrp gene cluster, which determines pathogenicity and hypersensitivity on pepper and tomato, is plant inducible.

The hrp gene cluster from Xanthomonas campestris pv. vesicatoria determines functions necessary not only for pathogenicity on the host plants pepper and tomato but also for the elicitation of the hypersensitive reaction on resistant host and nonhost plants. Transcriptional orientation and expression of the hrp loci were determined with hrp::Tn3-gus fusions. In addition, expression of the hrp loci was studied by RNA hybridization experiments. Expression of the hrp genes was not detectable after growth of the bacteria in complex medium or in minimal medium. However, high levels of induction of hrp gene expression were measured during growth of the bacteria in the plant. To search for a plant molecule responsible for this induction, we examined a variety of materials of plant origin for their ability to induce hrp gene expression. Filtrates from plant suspension cultures induced hrp genes to levels comparable to those induced in the plant. The inducing molecule(s) was found to be heat stable and hydrophilic and to have a molecular mass of less than 1,000 daltons.

A Xanthomonas Pathogenicity Locus Is Induced by Sucrose and Sulfur-Containing Amino Acids.

Expression of hrp (hypersensitive reaction and pathogenicity) genes from Xanthomonas campestris pv vesicatoria is suppressed in complex media but induced in the plant. We examined the effects of macronutrients on transcription of hrp-gusA ([beta]-glucuronidase) fusions by growth of the bacteria in defined medium. Modified MM1 minimal medium, supplemented with casamino acids, was able to induce hrpF strongly when sucrose or fructose was added as a carbon source. However, high concentrations of casamino acids suppressed hrpF induction. Sulfur-containing amino acids were required for induction, with methionine induction being comparable to induction in plants. Both sucrose and methionine were required for induction. Induction in medium optimal for hrpF induction, designated XVM1, occurred at pH 5.5 to pH 7.5. High concentrations of phosphate or sodium chloride suppressed gene activation. Gene induction was inhibited by succinate, citrate, pyruvate, and glutamine. Expression levels of different hrp loci from X. c. vesicatoria in XVM1 varied, dependent on the genetic background of the Xanthomonas strain used. The results suggest that several control mechanisms might be involved in the expression of hrp genes.