Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria.
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Conserved Sequence↗
Biomedical subjects
U Bonas
Publications and source records attributed to U Bonas.
HrpG, a key hrp regulatory protein of Xanthomonas campestris pv. vesicatoria is homologous to two-component response regulators.
Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease of pepper and tomato plants. Expression of its basic pathogenicity genes, the hrp genes, is induced in planta and in XVM2 medium and is dependent on the hrp regulatory gene hrpXv for five out of six loci in the 23-kb hrp cluster. Here we describe the isolation of a novel hrp gene, hrpG, that was identified after chemical mutagenesis and that is located next to the hrpXv gene. In a hrpG mutant induction of expression of the seven loci hrpA to hrpF, and hrpXv is abolished, suggesting that hrpG functions at the top of the hrp gene regulatory cascade. hrpG is the only gene in the locus and encodes a putative protein of 263 amino acids with a molecular mass of 28.9 kDa. The HrpG amino acid sequence shows similarity to response regulator proteins of the OmpR subclass of two-component systems, being mostly related to the ChvI proteins of Agrobacterium tumefaciens and Rhizobium spp., and TctD of Salmonella typhimurium. Expression of hrpG is low in complex medium, is increased in XVM2 by a factor of four, and is independent of other hrp loci. A model on hrp gene regulation in Xanthomonas campestris pv. vesicatoria is discussed.
Amino Acid Sequence↗
The rpoN gene of Xanthomonas campestris pv. vesicatoria is not required for pathogenicity.
We have cloned the rpoN region of the pepper and tomato pathogen Xanthomonas campestris pv. vesicatoria to analyze its role in pathogenicity and hrp gene activation. Open reading frame 3 (ORF3) from this region is predicted to encode a protein that is up to 45% identical and 65% similar to previously described RpoN proteins and was shown to be essential for the utilization of nitrate as sole nitrogen source. The X. campestris pv. vesicatoria rpoN gene is, however, not required for pathogenicity and hrp gene expression.
Amino Acid Sequence↗
HrpXv, an AraC-type regulator, activates expression of five of the six loci in the hrp cluster of Xanthomonas campestris pv. vesicatoria.
hrp genes, basic pathogenicity genes of the pepper and tomato pathogen Xanthomonas campestris pv. vesicatoria, are regulated dependent on environmental conditions. We isolated the hrpXv gene, which was found to be outside the large hrp cluster comprising the six loci hrpA to hrpF. The predicted HrpXv protein is 476 amino acids long and has a molecular mass of 52.5 kDa. HrpX is highly conserved among xanthomonads and is a member of the AraC family of regulatory proteins. An hrpXv insertion mutant has a typical hrp phenotype and no longer allows induction of the five hrp loci hrpB to hrpF in the new hrp induction medium XVM2, indicating that HrpXv is the positive regulator of these loci. An hrpXv mutant could be partially complemented by the related hrpB gene of Burkholderia solanacearum, the protein product of which shows 40 and 58% amino acid identity and similarity, respectively, to HrpXv. The hrpXv gene itself has a low basal level of expression that is enhanced in XVM2. Expression of hrpXv as well as that of the hrpA locus is independent of the hrpXv gene. The transcription start site of hrpXv was mapped. Comparison between the hrpXv promoter and the corresponding region of the hrpXc gene from X. campestris pv. campestris revealed sequence conservation up to position -84. A putative helix-turn-helix motif in the C-terminal region of HrpXv and its possible interaction with a conserved hrp promoter element, the plant-inducible promoter box, are discussed.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
hrp genes of phytopathogenic bacteria.
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Bacterial Outer Membrane Proteins↗
Bacterial home goal by harpins.
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Animals↗
Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria.
Three pairs of oligonucleotide primers specific for different regions of the hrp gene (hypersensitive reaction and pathogenicity) cluster of Xanthomonas campestris pv. vesicatoria were designed and tested for amplification of DNA isolated from a large number of different bacteria. DNA sequences related to the hrp genes were successfully amplified from X. fragariae and from 28 pathovars of X. campestris. No DNA amplification occurred with genomic DNA from phytopathogenic strains of X. campestris pv. secalis, X. campestris pv. translucens, and X. albilineans or from nonpathogenic opportunistic xanthomonads and phytopathogenic strains of the genera Acidovorax, Agrobacterium, Clavibacter, Erwinia, Pseudomonas, and Xylella. The DNA from those bacteria also failed to hybridize to hrp-specific fragments in Southern blot analysis. DNA fragments amplified with a particular primer pair were of identical size from each of the different phytopathogenic xanthomonads. However, restriction analysis of these fragments by using frequently cutting endonucleases revealed variation in the pattern for these hrp-related fragments amplified from the different Xanthomonas strains. The restriction patterns generated for the different fragments allowed distinction of the strains representing a pathovar or species of phytopathogenic xanthomonads. We believe that DNA amplification with hrp-specific oligonucleotide primers is a highly sensitive and specific method that can be applied for detection and identification of phytopathogenic xanthomonads.
Base Sequence↗
Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3.
Bacterial spot disease of tomato and pepper caused by Xanthomonas campestris pv vesicatoria is prevented by resistance genes in the plant that match genes for avirulence in the bacterium. Based on DNA homology to the avirulence gene avrBs3, which induces the resistance response on pepper, we have isolated another avirulence gene from X. c. vesicatoria, designated avrBs3-2. This gene differs in specificity from avrBs3 in inducing the hypersensitive response on tomato but not on pepper. Sequence analysis of the avrBs3-2 gene revealed a high degree of conservation: the 3480 bp open reading frame contains an internal region of 17.5 nearly identical 102 bp repeat units that differ in their order from those present in the avrBs3 gene. The coding region is 97% identical to avrBs3 and expresses constitutively a 122 kDa protein, thus representing a natural allele of this gene. The previously isolated 1.7 kb avrBsP gene from X. c. vesicatoria is 100% identical to the corresponding avrBs3-2 sequence, indicating that these genes might be identical. Interestingly, derivatives of avrBs3-2 lacking the C-terminal region and part of the repetitive region are still able to confer incompatibility in tomato. The avrBs3-2 gene is compared with the sequence of avrBs3 derivatives generated by deletion of repeat units that also have avirulence activity on tomato. Both genes, avrBs3 and avrBs3-2, are flanked by a 62 bp long inverted repeat, which prompts speculations about the origin of the members of the avrBs3 gene family.
Alleles↗
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.
Capsicum↗
Expression of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria is not under the control of hrp genes and is independent of plant factors.
The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria pepper race 1 is responsible for the induction of a race-specific hypersensitive reaction in resistant pepper cultivars. A DNA region of 3.7 kb, containing several open reading frames and an internal repetitive region, was shown previously to be necessary for avirulence activity (U. Bonas, R. E. Stall, and B. Staskawicz, Mol. Gen. Genet. 218:127-136, 1989). The promoter of avrBs3 was identified by using gene fusions to beta-glucuronidase. Also, we mapped the transcription start site and showed that the avrBs3 gene is expressed constitutively in cells grown in minimal or complex medium and in planta. Polyclonal antibodies raised against a fusion protein produced in Escherichia coli allowed the identification of a 122-kDa protein in X. campestris pv. vesicatoria cells expressing the avrBs3 gene. The antibody is specific for AvrBs3 in X. campestris pv. vesicatoria cells but also recognizes homologous proteins in other pathovars of X. campestris. We found that AvrBs3 is localized intracellularly in X. campestris pv. vesicatoria and is mainly in the soluble fraction. The effect of mutations in the hrp gene cluster on the function of AvrBs3 was examined. Expression of AvrBs3 in X. campestris pv. vesicatoria grown in minimal or complex medium is independent of the hrp gene cluster that determines pathogenicity and hypersensitivity to X. campestris pv. vesicatoria. In the plant, however, the hrp genes are required for elicitation of a race-specific resistance response.
Amino Acid Sequence↗
Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria.
The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6-3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125,000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%-100% homology with each other.
Amino Acid Sequence↗
Transposon-induced alterations in the promoter region affect transcription of the chalcone synthase gene of Antirrhinum majus.
Four solid-colour revertants were isolated from the highly variegated niv-53::Tam1 mutant, in which the transposable element Tam1 is integrated in the promoter region of the chalcone synthase (chs) gene. DNA sequence analysis revealed that in all four lines the Tam1 element was deleted together with flanking nucleotides of the chs promoter. In one case the TATA box of the chs gene was removed resulting in extremely low expression of the gene, and initiation of transcription occurring at a new position. The other three deletions defined a sequence motif (TAC-CAT) which is apparently required for maximal gene expression. Thus transposable elements seem to be useful for probing gene structure, in this case the signal structure in the promoter region, by virtue of imprecise excision.
Acyltransferases↗
Developmental regulation of embryonic genes in plants.
Somatic embryogenesis from cultured carrot cells progresses through successive morphogenetic stages termed globular, heart, and torpedo. To understand the molecular mechanisms underlying plant embryogenesis, we isolated two genes differentially expressed during embryo development. The expression of these two genes is associated with heart-stage embryogenesis. By altering the culture conditions and examining their expressions in a developmental variant cell line, we found that these genes were controlled by the developmental program of embryogenesis and were not directly regulated by 2,4-dichlorophenoxyacetic acid, the growth regulator that promotes unorganized growth of cultured cells and suppresses embryo morphogenesis. These genes are also expressed in carrot zygotic embryos but not in seedlings or mature plants.
Journal Article↗
The 17-kb Tam1 element of Antirrhinum majus induces a 3-bp duplication upon integration into the chalcone synthase gene.
The DNA sequence of the termini and the flanking regions of the 17-kb transposable element Tam1 was determined. Tam1 is integrated in the chalcone synthase gene of the niv-53 mutant of Antirrhinum majus. The element has a 13-bp perfect inverted repeat at its termini and appears to induce a 3-bp duplication of the target site upon integration. The DNA sequence of a niv revertant was analyzed and found to differ from the wild-type sequence by an additional 2 bp that seem to derive from the target site duplication. Stretches of homologous sequences have been found between the ends of Tam1, within each terminus of the element, and between the termini and target site sequences. Structural similarities between the ends of Tam1 and the Spm-18 element of Zea mays reflect a possible horizontal spread of a common progenitor.
Journal Article↗
Transposable elements in Antirrhinum majus and Zea mays.
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Base Sequence↗
A gene from Xanthomonas campestris pv. vesicatoria that determines avirulence in tomato is related to avrBs3.
Strains of Xanthomonas campestris pv. vesicatoria that were avirulent in tomato leaves but virulent in pepper leaves were identified. A cloned gene, avrBsP, from one of the strains, Xv 87-7, converted a virulent strain in tomato to avirulent in tomato. A 1.7-kb subclone containing the avirulence gene cross-hybridized with the avirulence gene, which determines race 1 within the pepper group of strains (avrBs3). However, the two avirulence genes differ in their biological activity. The base sequences of the two avirulence genes were almost identical through the 1.7-kb segment of avrBsP, with significant differences only in some bases in the repeat region.
Amino Acid Sequence↗
Determinants of pathogenicity in Xanthomonas campestris pv. vesicatoria are related to proteins involved in secretion in bacterial pathogens of animals.
One of the model systems investigated for studying plant bacterial pathogenesis is Xanthomonas campestris pv vesicatoria, the causal agent of bacterial spot disease of pepper and tomato. Genes necessary for both basic pathogenicity and the induction of the hypersensitive response in resistant plants (hrp genes) were previously isolated from X. c. pv. vesicatoria and characterized genetically. As a first step toward functional analysis, part of the hrp gene cluster, making up several loci, was sequenced. Here, we report the first indications of the function of hrp genes. Striking similarities to proteins from the mammalian pathogens Shigella flexneri, Yersinia enterocolitica, Y. pestis, and other bacteria were discovered. Proteins encoded by genes within the X. c. pv. vesicatoria loci hrpA, hrpB, and hrpC are similar to ATPases and to Yersinia Ysc and LcrD proteins, which are involved in secretion of Yop proteins, a particular class of essential pathogenicity factors produced by Yersinia species. This finding indicates, for the first time, that the fundamental determinants of pathogenicity may be conserved among bacterial pathogens of plants and animals. We hypothesize that hrp genes are involved in the secretion of molecules essential for the interaction of X. c. pv. vesicatoria with the plant.
Amino Acid Sequence↗