We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G؉C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads.In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 M in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.Many gram-negative plant-pathogenic bacteria possess two sets of genes that modulate their interactions with plants. The avirulence gene determines host specificity based on gene-forgene interactions, and the hrp (hypersensitive reaction and pathogenicity) genes are involved in pathogenicity and the induction of hypersensitive responses (HRs) in nonhost plants (6). The nine hrp genes, which are highly conserved in plants and bacterial pathogens of animals, are known as the hrc (hrp conserved) genes (5). The hpa (hrp-associated) genes contribute to pathogenicity and to the induction of HR in nonhost plants but are not essential for the pathogenic interactions of bacteria with plants (19). These genes are generally clustered in a chromosomal region that spans 20 to 30 kb, and most of the Hrp and Hrc proteins function as type III protein secretion systems (16).Type III secretion systems mediate the translocation of effector proteins across the bacterial membrane and into the host and are often important for virulence and in the modulation of host defense responses (16). The hrp-hrc regions are now designated pathogenicity islands (PAIs) in various plantpathogenic bacteria (2). PAIs contain many virulence genes, are present only in pathogenic bacteria, have different GϩC contents compared with the host bac...

Harpins are heat-stable, glycine-rich type III-secreted proteins produced by plant pathogenic bacteria, which cause a hypersensitive response (HR) when infiltrated into the intercellular space of tobacco leaves; however, the biochemical mechanisms by which harpins cause plant cell death remain unclear. In this study, we determined the biochemical characteristics of HpaG, the first harpin identified from a Xanthomonas species, under plant apoplast-like conditions using electron microscopy and circular dichroism spectroscopy. We found that His 6 -HpaG formed biologically active spherical oligomers, protofibrils, and ␤-sheet-rich fibrils, whereas the null HR mutant In many Gram-negative plant pathogenic bacteria, the hrp (hypersensitive response and pathogenicity) genes, which mostly encode proteins necessary for type III protein secretion systems, are involved in the secretion of harpins (1, 2). Harpins are heat-stable, glycine-rich type III-secreted proteins that cause a hypersensitive response (HR) 2 when filtrated into the intercellular space of tobacco leaves (1, 3, 4). The HR in plants is an early defense response that restricts the growth of plant pathogens by causing cell death. The plant HR is similar to programmed cell death, or apoptosis, in animal cells, and the biochemical changes that occur during the HR in plant cells have been well documented (5-8). Two categories of HR exist in plants, pathogen-driven and harpin protein-dependent. A typical pathogen-driven HR occurs when a pathogen carrying an avirulence gene enters the intracellular environment of a plant carrying its cognate resistance gene. The second type of HR results from the activity of bacterial harpins outside of plant cells, including those found in Pseudomonas syringae pv. syringae, Erwinia amylovora, Erwinia chrysanthemi, and Xanthomonas axonopodis pv. glycines (1, 3, 9 -12). In Ralstonia solanacearum, a harpin-like protein, PopA, has been shown to induce an HR in nonhost tobacco plants (13).Among the harpins, HrpN (3), HrpZ (9), and HrpW (11) are well characterized HR elicitors. We previously reported the identification of a harpin, HpaG, which is produced by X. axonopodis pv. glycines 8ra (1), and we discovered that an hpaG mutant lacking the ability to induce an HR was much less virulent in susceptible soybean cultivars. HpaG induces an HR in various nonhost plants, but it exhibits specificity for those plants; HpaG induces an HR in tobacco and pepper plants but not in tomato or Chinese cabbage (1). In a previous study (4), we reported that HpaG has two predicted ␣-helices (motifs 1 and 3) in the N-and C-terminal regions, and that a Gln and Gly repeated sequence (motif 2; QGQGQGQGG) is located between the two ␣-helices (Fig. 1, A and B). The motif 2 region is homologous to the prion-forming domain (PrD) of a yeast prion protein, Rnq1p (14). Using mutagenesis and deletion analysis, we deduced that 23 amino acids in motif 1 are essential for inducing the HR. This was confirmed by showing that a synthetic peptide comprising these 23 ami...

Effector proteins play crucial roles in plant-parasite interactions by suppressing plant defenses and hijacking plant physiological responses to facilitate parasite invasion and propagation. Although effector proteins have been characterized in many microbial plant pathogens, their nature and role in adaptation to host plants are largely unknown in insect herbivores. Aphids rely on salivary effector proteins injected into the host plants to promote phloem sap uptake. Therefore, gaining insight into the repertoire and evolution of aphid effectors is key to unveiling the mechanisms responsible for aphid virulence and host plant specialization. With this aim in mind, we assembled catalogues of putative effectors in the legume specialist aphid, Acyrthosiphon pisum, using transcriptomics and proteomics approaches. We identified 3,603 candidate effector genes predicted to be expressed in A. pisum salivary glands (SGs), and 740 of which displayed up-regulated expression in SGs in comparison to the alimentary tract. A search for orthologs in 17 arthropod genomes revealed that SG-up-regulated effector candidates of A. pisum are enriched in aphid-specific genes and tend to evolve faster compared with the whole gene set. We also found that a large fraction of proteins detected in the A. pisum saliva belonged to three gene families, of which certain members show evidence consistent with positive selection. Overall, this comprehensive analysis suggests that the large repertoire of effector candidates in A. pisum constitutes a source of novelties promoting plant adaptation to legumes.

HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and sitedirected mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were In many interactions between gram-negative plant-pathogenic bacteria and plants, hrp (for hypersensitive reaction and pathogenicity) genes are required for pathogenicity in the host plant and induction of the hypersensitive response (HR) in nonhost plants (17). Regions that contain a cluster of hrp genes and other components required for pathogenicity are designated pathogenicity islands (PAIs) (3, 15). Most hrp genes that encode components of the type III protein secretion system mediate the translocation of effector proteins, such as Avr (avirulence) proteins, across the bacterial membrane and the walls and plasma membranes of plant cells (10).HR is a highly localized plant cell death that occurs when nonhost plants or resistant cultivars of host plants are infiltrated with the plant pathogen or HR elicitor molecules, such as Avr proteins and harpins. HR is thought be a resistance reaction of plants to microbial pathogens (11). Harpins are a group of HR elicitors that are secreted by the type III secretion pathway and elicit HR when infiltrated into the apoplast of leaves of nonhost plants. Unlike Avr proteins, which must be delivered inside the cell to exert their functions, harpins can elicit HR when delivered to the intercellular space of plant cells (10). Since the first known harpin, HrpN, was identified from Erwinia amylovora, many harpins have been reported from Pseudomonas, Ralstonia, and Xanthomonas species (4,8,12,14,15,27). Harpins are glycine-rich, heat stable, and lack cysteine, but the biochemical mechanisms of HR elicitation in nonhost plants are unclear. One reason for this is that the amino acid sequences of harpins do not share significant homology with other known proteins or among themselves.The mode of action of harpins is still controversial. HrpZ of Pseudomonas syringae pv. syringae associates with the walls rather than the membranes of plant cells, and the protein elicits no response from protoplasts, which lack walls (13). However, HrpZ of P. syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore (16). The N-terminal 109 amino acids and the C-terminal 216 amino acids of HrpZ are able to elicit HR to a level similar to full-length HrpZ (2). Kim et al. and Charkowski et al. showed that the HrpW harpins of E. amylovora and P. syringae pv. tomato are composed of two domains-the N-terminal harpin domain and C-terminal Pel (pectate lyase) domain-and proposed that HrpW acts in the cell wall (8,14).We previously published the first report of a harpin from Xanthomonas species, HpaG (15). At 13.4 kDa, HpaG is smaller than other known harpins (15). Four additional...

Elicitins, extracellular proteins from Phytophthora fungi, elicit a hypersensitivity response (HR), including systemic acquired resistance, in some plants. The elicitin capsicein (~10 kDa) was purified by FPLC from culture filtrates of P. capsici. Purified native and recombinant capsicein induced a hypersensitive response in leaves of the non-host plants Nicotiana glutinosa and Brassica rapa subsp. pekinensis. To search for candidate capsicein-interacting proteins from N. glutinosa, a yeast two-hybrid assay was used. We identified a protein interactor that is homologous to a serine/threonine kinase of the plant receptor-like kinase (RLK) group and designated it NgRLK1. The ORF of NgRLK1 encodes a polypeptide of 832 amino acids (93,490 Da). A conserved domain analysis revealed that NgRLK1 has structural features typical of a plant RLK. NgRLK1 was autophosphorylated, with higher activity in the presence of Mn2+ than Mg2+.

Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60°C. Glycinecin A is a heterodimer consisting of 39-and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes, glyA and glyB, encoding the 39-and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of glyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with either glyA or glyB separately did not show the bacteriocin activity.Bacteriocins are bactericidal compounds, usually proteinaceous, whose activities are often restricted to bacterial strains that are closely related to the producing bacterium (6, 23). Bacteriocins are produced in all major groups of Eubacteria and Archaebacteria (22). Some bacteriocins, the halocins from Halobacteria, have no protein sequence homology to any known bacteriocins (20), whereas others, such as the S-type pyocins of Pseudomonas aeruginosa, some of the colicins of Escherichia coli, and a cloacin of Enterobacter cloacae, reveal protein sequence homologies (16,17,19).Despite their diversity, the bacteriocins share several characteristics (6, 23). They are generally high-molecular-weight proteins that gain entry into target cells by binding to cell surface receptors. Their bactericidal mechanisms vary and include pore formation, degradation of cellular DNA, disruption of specific cleavage of 16S rRNA, and inhibition of peptidoglycan synthesis.Many phytopathogenic bacteria, including members of the corynebacteria, erwinias, pseudomonads, and xanthomonads, produce proteinaceous bacteriocins (1, 2, 4, 5, 21, 24, 25). Since these bacteriocins are highly specific, can be produced at low cost, and are likely to be safe for both users and the environment, they appear to be excellent candidates for agricultural use in controlling plant pathogens. However, little is known about the chemical compositions, structures, modes of action, and genetics of most bacteriocins. Only a few isolated and/or purified bacteriocins have been reported from Agrobacterium radiobacter (18), Corynebacterium ulcerans (1), Erwinia carotovora subsp. carotovora (4), and Pseudomonas syringae pv. syringae (21).Thus far, bacteriocin production by xanthomonads has received little attention. Bacteriocin production by X...

Burkholderia glumae produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the toxABCDE (toxoflavin biosynthesis genes) and toxFGHI (toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for tox gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both tox operons. ToxR and ToxJ both bound to the toxA and toxF regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both tox operons possessed T-N 11 -A motifs. Following random mutagenesis of toxR, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (toxRA::⍀ toxF::Tn3-gusA34) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred toxF expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that B. glumae cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR.

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