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U Bonas

Publications and source records attributed to U Bonas.

At least 19 recordsLinked to original sources
PubMed2001

Chromosome landing at the tomato Bs4 locus.

The tomato (Lycopersicon esculentum) Bs4 gene confers resistance to strains of Xanthomonas campestris pathovar vesicatoria that express the avirulence protein AvrBs4. As part of a map-based cloning strategy for the isolation of Bs4, we converted Bs4-linked amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers into locus-specific sequence-tagged-site (STS) markers. The use of these markers for the analysis of 1972 meiotic events allowed high-resolution genetic mapping within a 1.2-cM interval containing the target gene. Two tomato yeast artificial chromosome (YAC) clones, each harboring inserts of approximately 250 kb, were identified using the marker most closely linked to Bs4. YAC end-specific markers were established and employed to construct a local YAC contig. The ratio of physical to genetic distance at Bs4 was calculated to be 280 kb/cM, revealing that recombination rates in this region are about three times higher than the genome-wide average. Mapping of YAC end-derived markers demonstrated that the Bs4 locus maps within a region of 250 kb, corresponding to a genetic interval of 0.9 cM.

Chromosome Mapping↗
PubMed2001

Molecular secrets of bacterial type III effector proteins.

Most Gram-negative phytopathogenic bacteria are thought to inject effector proteins into the plant cell via a type III secretion system that is essential for pathogenicity. Plant targets and the mode of action of type III effector proteins, which include avirulence (Avr) proteins, are largely unknown. However, recent findings have shed light on the molecular mechanisms of Avr action. Here, we focus on two classes of Avr proteins (the AvrBs3 and AvrRxv/YopJ families) that have been suggested to act as transcription factors and proteases, respectively.

Apoptosis↗
PubMed2001

Eukaryotic features of the Xanthomonas type III effector AvrBs3: protein domains involved in transcriptional activation and the interaction with nuclear import receptors from pepper.

The AvrBs3 protein of the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria is targeted to host-plant cells by the bacterial Hrp type III secretion system. In pepper plants containing the Bs3 resistance gene, AvrBs3 induces the hypersensitive response (HR). AvrBs3 recognition is thought to occur in the plant cell nucleus as HR induction is dependent on nuclear localization signals (NLSs) and an acidic transcription activation domain (AAD). In a search for AvrBs3-interacting pepper proteins using the yeast two-hybrid system, we have isolated eight different classes of cDNA inserts including two genes for importin alpha proteins. Importin alpha is part of the nuclear import machinery and interacts with AvrBs3 through an NLS in the carboxy-terminus of the protein, both in yeast and in vitro. The mechanism of AvrBs3 recognition was further studied by analysis of the C-terminal AAD. This putative transcription-activation domain was shown to be required for AvrBs3 HR-inducing activity, and could be functionally replaced with the VP16 AAD from the Herpes simplex virus. Our data support the model in which the AvrBs3 effector localizes to the nucleus, where the Bs3-mediated surveillance system of resistant plants detects AvrBs3 through its interference with host gene transcription.

Active Transport, Cell Nucleus↗
PubMed2001

cDNA-AFLP analysis unravels a genome-wide hrpG-regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria.

The Hrp type III protein secretion system is essential for pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria. Expression of the hrp gene cluster is controlled by HrpG, a two-component response regulator, and HrpX, an AraC-type transcriptional activator. Using the cDNA-AFLP technique, 30 hrpG-induced (hgi) and five hrpG-repressed (hgr) cDNA fragments were identified, defining a large hrpG-regulon in X. campestris pv. vesicatoria. Expression of most genes in the hrpG-regulon was dependent on hrpX. Seven cDNA fragments map to the known hrp gene cluster and flanking regions. All other genes appear to be scattered over the chromosome and endogenous plasmids. Sequence analysis identified genes encoding putative extracellular proteases, a putative transcriptional regulator and XopJ and XopB (Xanthomonas outer proteins), homologues of YopJ from Yersinia spp. and the avirulence protein AvrPphD of Pseudomonas syringae respectively. XopB is secreted by the Hrp type III secretion system. Analysis of deletion mutants in several hgi genes revealed a new virulence locus. This study demonstrates that cDNA-AFLP is a powerful tool to study prokaryotic transcriptomes and to identify genes contributing to Xanthomonas virulence and putative effector proteins.

Bacterial Proteins↗
PubMed2001

Genetic mapping and functional analysis of the tomato Bs4 locus governing recognition of the Xanthomonas campestris pv. vesicatoria AvrBs4 protein.

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.). Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X. campestris pv. vesicatoria strains identified 15 resistant and two susceptible tomato genotypes. Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no. 4). Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus. A standard tomato mapping population was employed to localize Bs4-linked markers on the short arm of chromosome 5. Investigation of X. campestris pv. vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent. Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X. campestris pv. vesicatoria strains, suggesting symplastic perception of the avirulence protein. Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition. Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X. campestris pv. vesicatoria avirulence proteins AvrBs4 and AvrBs3.

Bacterial Proteins↗
PubMed2000

Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants.

Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Agrobacterium tumefaciens↗
PubMed2000

HrpB2 and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant.

The interaction between the plant pathogen Xanthomonas campestris pv. vesicatoria and its host plants is controlled by hrp genes (hypersensitive reaction and pathogenicity), which encode a type III protein secretion system. Among type III-secreted proteins are avirulence proteins, effectors involved in the induction of plant defence reactions. Using non-polar mutants, we investigated the role of 12 hrp genes in the secretion of the avirulence protein AvrBs3 from X. c. pv. vesicatoria and a heterologous protein, YopE, from Yersinia pseudotuberculosis. Genes conserved among type III secretion systems (hrcQ, hrcR, hrcS and hrcT) as well as non-conserved genes (hrpB1, hrpB2, hrpB4, hrpB5, hrpD5 and hrpD6) were shown to be required for secretion. Protein localization studies using specific antibodies showed that HrpB1 and HrpB4, as well as the putative ATPase HrcN, were mainly found in the soluble fraction of the bacterial cell. In contrast, HrpB2 and HrpF, which is related to NolX of Rhizobium fredii, are secreted into the culture medium in an hrp-dependent manner. As HrpB2, but not HrpF, is essential for type III protein secretion, there might be a hierarchy in the secretion process. We propose that HrpF, which is dispensable for protein secretion but required for AvrBs3 recognition in planta, functions as a translocator of effector proteins into the host cell.

Bacterial Proteins↗
PubMed2000

Resistance of tomato and pepper to T3 strains of Xanthomonas campestris pv. vesicatoria is specified by a plant-inducible avirulence gene.

Tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria (Xcv) elicits a hypersensitive response (HR) in leaves of Lycopersicon esculentum near-isogenic line (NIL) 216 and pepper genotypes. One cosmid clone (35 kb) selected from a genomic library of a T3 strain induced an HR in all resistant plants. A 1.5-kb active subclone containing the putative avirulence (avr) gene, designated avrXv3, was sequenced. The avrXv3 gene encodes a 654-bp open reading frame (ORF) with no homology to any known gene. Expression studies with a fusion of this gene and uidA indicated that avrXv3 is plant inducible and controlled by the hypersensitivity and pathogenicity (hrp) regulatory system. Mutational analysis and transcription activation assays revealed that AvrXv3 has transcription activation activity in yeast, and that the putative domain responsible for that activity is located at the C terminus of the AvrXv3 protein. Agrobacterium tumefaciens-mediated transient expression confirmed the direct role of AvrXv3 in eliciting the HR in tomato NIL 216 and supported the hypothesis that Avr proteins must be present inside the plant host cell to trigger the HR.

Base Sequence↗
PubMed1999

The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens.

Studies of essential pathogenicity determinants in Gram-negative bacteria have revealed the conservation of type III protein secretion systems that allow delivery of virulence factors into host cells from plant and animal pathogens. Ten of 21 Hrp proteins of the plant pathogen Xanthomonas campestris pv. vesicatoria have been suggested to be part of a type III machinery. Here, we report the hrp-dependent secretion of two avirulence proteins, AvrBs3 and AvrRxv, by X. campestris pv. vesicatoria strains that constitutively express hrp genes. Secretion occurred without leakage of a cytoplasmic marker in minimal medium containing BSA, at pH 5.4. Secretion was strictly hrp-dependent because a mutant carrying a deletion in hrcV, a conserved hrp gene, did not secrete AvrBs3 and AvrRxv. Moreover, the Hrp system of X. campestris pv. vesicatoria was able to secrete proteins from two other plant pathogens: PopA, a protein secreted via the Hrp system in Ralstonia solanacearum, and AvrB, an avirulence protein from Pseudomonas syringae pv. glycinea. Interestingly, X. campestris pv. vesicatoria also secreted YopE, a type III-secreted cytotoxin of the mammalian pathogen Yersinia pseudotuberculosis in a hrp-dependent manner. YerA, a YopE-specific chaperone, was required for YopE stability but not for secretion in X. campestris pv. vesicatoria. Our results demonstrate the functional conservation of the type III system of X. campestris for secretion of proteins from both plant and mammalian pathogens and imply recognition of their respective secretion signals.

Animals↗
PubMed1999

Gene-for-gene interactions: bacterial avirulence proteins specify plant disease resistance.

Resistance of plants to bacterial pathogens is often controlled by corresponding genes for resistance and avirulence in host and pathogen, respectively. Fifty years after discovery of the genetic basis of gene-for-gene interactions, several avirulence and plant resistance genes have been isolated and are being studied on the molecular level. Tremendous progress has been made due to a better understanding of type III secretion systems that are required for bacterial pathogenicity. We are beginning to grasp how the plant actually recognizes bacterial avirulence determinants. The current view is that the bacterium translocates avirulence proteins into the host cell by the Hrp type III secretion system and that recognition occurs in the plant cell.

Bacteria↗
PubMed1999

Mutations in the regulatory gene hrpG of Xanthomonas campestris pv. vesicatoria result in constitutive expression of all hrp genes.

hrpG is a key regulatory gene for transcriptional activation of pathogenicity genes (hrp) of Xanthomonas campestris pv. vesicatoria. We identified three mutations in hrpG which render hrp gene expression constitutive in normally suppressing medium. The mutations in hrpG result in novel amino acid substitutions compared to mutations in related proteins, such as OmpR. In addition, mutated hrpG enhances the timing and intensity of plant reactions in infection assays.

Amino Acid Sequence↗
PubMed1998

hpaA mutants of Xanthomonas campestris pv. vesicatoria are affected in pathogenicity but retain the ability to induce host-specific hypersensitive reaction.

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants. We reported previously that the main hrp (hypersensitive reaction and pathogenicity) gene cluster in X. c. pv. vesicatoria contains six transcription units, designated hrpA to hrpF. We present here the sequence of the hrpD operon and an analysis of non-polar mutants in each of the six genes. Three genes, hrcQ, hrcR and hrcS, are predicted to encode conserved components of type III protein secretion systems in plant and mammalian pathogenic bacteria. For hrpD5 and hrpD6, homologues have only been found in Ralstonia solanacearum. Interestingly, the hrpD operon contains one gene, hpaA (for hrp-associated), which is specifically required for disease development. hpaA mutants are affected in pathogenicity, but retain in part the ability to induce avirulence gene-mediated, host-specific hypersensitive reaction (HR). In addition, HpaA was found to contain two functional nuclear localization signals, which are important for the interaction with the plant. We propose that HpaA is an effector protein that may be translocated into the host cell via the Hrp secretion pathway.

Amino Acid Sequence↗
PubMed1997

Bacterial avirulence proteins as triggers of plant disease resistance.

Disease resistance in plants is often characterized by matching resistance and avirulence genes in host and pathogen, respectively. It has recently been shown that expression of bacterial avirulence genes in plants induces resistance gene-dependent defense reactions. The finding that avirulence proteins act inside plant cells represents a major advance in our understanding of host-pathogen specificity.

Bacteria↗
PubMed1997

Recognition of bacterial avirulence proteins occurs inside the plant cell: a general phenomenon in resistance to bacterial diseases?

One of the recent exciting developments in the research area of plant-microbe interactions is a breakthrough in understanding part of the initial signalling between avirulent Gram-negative bacteria and resistant plants. For resistance to occur, both interacting organisms need to express matching genes, the plant resistance gene and the bacterial avirulence gene. The biochemical function of bacterial avirulence genes and the nature of the signal molecules recognized by the plant have been a mystery for a long time. Recently, several laboratories have shown that bacterial avirulence proteins function as elicitors that are perceived within the plant cell.

Bacterial Proteins↗
PubMed1997

hrpf of Xanthomonas campestris pv. vesicatoria encodes an 87-kDa protein with homology to NoIX of Rhizobium fredii.

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants. The main hrp (hypersensitive reaction and pathogenicity) gene cluster in X. campestris pv. vesicatoria spans a 23-kb chromosomal region, comprising six complementation groups designated hrpA to hrpF. Analysis of the hrpF locus revealed a single open reading frame encoding HrpF (86.4 kDa). HrpF is predominantly hydrophilic, and contains two hydrophobic domains in the C terminus. An interesting feature is the presence of two imperfect direct repeats in the N-terminal region. Deletion studies showed that one repeat is sufficient for function. Epitope tagging of HrpF allowed detection of the protein in X. campestris pv. vesicatoria. Subcellular localization studies suggest that HrpF is both in the soluble fraction and in the inner membrane. Interestingly, HrpF is 48% identical (67% similar) to the Rhizobium fredii NoIX protein that is part of the host specificity locus. Since several Hrp proteins are believed to be components of the types of III hrp protein secretion apparatus, allowing export of proteins essential for the interaction with the plant, the possible role of hrpF and NoIX in secretion is discussed.

Amino Acid Sequence↗
PubMed1997

Characterization of the fimA gene encoding bundle-forming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria.

The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5' end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::omega mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.

Amino Acid Sequence↗
PubMed1996

Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell.

The molecular mechanism by which bacterial avirulence genes mediate recognition by resistant host plants has been enigmatic for more than a decade. In this paper we provide evidence that the Xanthomonas campestris pv. vesicatoria avirulence protein AvrBs3 is recognized inside the plant cell. Transient expression of avrBs3 in pepper leaves, using Agrobacterium tumefaciens for gene delivery, results in hypersensitive cell death, specifically on plants carrying the resistance gene Bs3. In addition, for its intracellular recognition, AvrBs3 requires nuclear localization signals that are present in the C-terminal region of the protein. We propose that AvrBs3 is translocated into plant cells via the Xanthomonas Hrp type III secretion system and that nuclear factors are involved in AvrBs3 perception.

Amino Acid Sequence↗