Hrp Gene Cluster Research Articles

Harnessing the Potential of Harpin Proteins: Elicitation Strategies for Enhanced Secondary Metabolite Accumulation in Grapevine Callus Cultures

Grapes and grape products are rich in secondary metabolites such as phenolic compounds and anthocyanins, which have antioxidant properties. These compounds possess health-promoting attributes, including cardioprotective, antimicrobial, and anticancer effects. In recent years, biotechnological methods have been employed to produce high quantities and purity of secondary metabolites under in vitro conditions, aiming to elucidate their complex functions and optimize production methods. However, the potential effects of harpin proteins on the accumulation of secondary compounds in callus cultures have not been investigated thus far. Harpin proteins, encoded by the hrp gene clusters in Gram-negative phytopathogens, are known to trigger defense responses in various plant species by promoting the accumulation of secondary compounds. These findings suggest that harpin proteins may have the potential to enhance secondary metabolite accumulation in callus cultures. This study therefore investigated the potential of applying different concentrations of harpin protein (0, 0.1, 1, 10, and 100 ppm) to increase secondary metabolite production in calluses derived from petioles of the “Horoz Karası” grape cultivar. Our findings revealed that 1 and 10 ppm harpin treatments resulted in the highest anthocyanin accumulations, with 17.21 and 16.57 CV/g, respectively, representing 1.95- and 1.87-fold increases compared to control treatments, respectively. Total phenolic content peaked at 0.39 mg GAE g−1 FW with the 1 ppm harpin treatment, representing a 4.33-fold increase over the control. Total flavonol levels reached their highest levels at 0.027 mg CE g−1 FW with 1 and 10 ppm harpin concentrations, resulting in a 2.25-fold increase compared to the control. The highest averages for total flavonol content were recorded at 0.024 and 0.021 mg RE g−1 FW with 1 and 10 ppm harpin concentrations, respectively, representing 1.5- and 1.3-fold increases over the control. Principal component analysis (PCA) corroborated the results obtained from the heatmap analysis, indicating that harpin applications at 1 and 10 ppm were the most effective concentration range for maximizing secondary metabolite synthesis, while very low or high concentrations diminished these effects. These findings offered valuable insights for optimizing the production of high-value bioactive compounds, which can be utilized in various fields such as medicine, pharmaceuticals, food, and cosmetics. These results are expected to serve as a valuable reference for elucidating the mechanisms by which harpin proteins, rarely used in vitro, exert their effects on grapevine calluses, contributing to the literature in this domain.

Antibacterial Activity and Mechanism of Three Root Exudates from Mulberry Seedlings against Ralstonia pseudosolanacearum.

Bacterial wilt is a significant soil-borne disease that poses a threat to mulberry production yield and quality of agricultural production worldwide. However, the disease resistance mechanisms dependent on root exudates are not well understood. In this present study, we investigated the antibacterial mechanisms of the main active substances (erucamide, oleamide, and camphor bromide) present in mulberry root exudates (MRE) against Ralstonia pseudosolanacearum (Rp), the causal agent of bacterial wilt. Our findings revealed that these three active substances inhibited the growth activity of Rp by affecting the cell morphology and extracellular polysaccharide content, as well as triggering a burst of reactive oxygen species. The active substances induced oxidative stress, leading to a decrease in Rp growth. Additionally, the expression levels of key genes in the hrp gene cluster (hrpB, hrpX, and hrpF) and other virulence-related genes (such as ripAW, ripAE, Rs5-4819, Rs5-4374, ace, egl3, and pehB) were significantly reduced upon treatment with the active substances. Further pathogenicity experiments demonstrated that root exudates (at a concentration of 1.5 mg·mL-1) delayed or slowed down the occurrence of bacterial wilt in mulberry. These findings provide valuable insight into the antimicrobial mechanisms of MRE against Rp and lay a theoretical foundation for the development and application of biocontrol agents to control mulberry bacterial wilt.

Whole-genome sequencing and comparative genome analysis of Xanthomonas fragariae YM2 causing angular leaf spot disease in strawberry.

Angular leaf spot disease caused by plant pathogenic bacterium Xanthomonas fragariae seriously threatens strawberry crop production globally. In this study, we sequenced the whole genome of X. fragariae YM2, isolated from Yunnan Province, China. In addition, we performed a comparative genome analysis of X. fragariae YM2 with two existing strains of X. fragariae YL19 and SHQP01 isolated from Liaoning and Shanghai, respectively. The results of Nanopore sequencing showed that X. fragariae YM2 comprises one single chromosome with a contig size of 4,263,697 bp, one plasmid contig size of 0.39 Mb, a GC content ratio of 62.27%, and 3,958 predicted coding genes. The genome of YM2 comprises gum, hrp, rpf, and xps gene clusters and lipopolysaccharide (LPS), which are typical virulence factors in Xanthomonas species. By performing a comparative genomic analysis between X. fragariae strains YM2, YL19, and SHQP01, we found that strain YM2 is similar to YL19 and SHQP01 regarding genome size and GC contents. However, there are minor differences in the composition of major virulence factors and homologous gene clusters. Furthermore, the results of collinearity analysis demonstrated that YM2 has lower similarity and longer evolutionary distance with YL19 and SHQP01, but YL19 is more closely related to SHQP01. The availability of this high-quality genetic resource will serve as a basic tool for investigating the biology, molecular pathogenesis, and virulence of X. fragariae YM2. In addition, unraveling the potential vulnerabilities in its genetic makeup will aid in developing more effective disease suppression control measures.

Discovery of the Hrp Type III Secretion System in Phytopathogenic Bacteria: How Investigation of Hypersensitive Cell Death in Plants Led to a Novel Protein Injector System and a World of Inter-Organismal Molecular Interactions Within Plant Cells.

In the early 1960s, Pseudomonas syringae and other host-specific phytopathogenic proteobacteria were discovered to elicit a rapid, resistance-associated death when infiltrated at high inoculum levels into nonhost tobacco leaves. This hypersensitive reaction (or response; HR) was a useful indicator of basic pathogenic ability. Research over the next 20 years failed to identify an elicitor of the HR but revealed that its elicitation required contact between metabolically active bacterial and plant cells. Beginning in the early 1980s, molecular genetic tools were applied to the HR puzzle, revealing the presence in P. syringae of clusters of hrp genes, so named because they are required for the HR and pathogenicity, and of avr genes, so named because their presence confers HR-associated avirulence in resistant cultivars of a host plant species. A series of breakthroughs over the next two decades revealed that (i) hrp gene clusters encode a type III secretion system (T3SS), which injects Avr (now "effector") proteins into plant cells, where their recognition triggers the HR; (ii) T3SSs, which are typically present in pathogenicity islands acquired by horizontal gene transfers, are found in many bacterial pathogens of plants and animals and inject many effector proteins, which are collectively essential for pathogenicity; and (iii) a primary function of phytopathogen effectors is to subvert non-HR defenses resulting from recognition of conserved microbial features presented outside of plant cells. In the 2000s, Hrp system research shifted to extracellular components enabling effector delivery across plant cell walls and plasma membranes, regulation, and tools for studying effectors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Molecular genetic detection and differentiation of <i>Xanthomonas oryzae</i> pv. <i>oryzicola</i>, bacterial leaf streak agents of rice

The genus Xanthomonas comprises phytopathogenic bacteria which infect about 400 host species, including a wide variety of economically important plants. Xanthomonas oryzae pv. oryzicola (Fang et al., 1957) Swings et al., 1990 is the causal agent of bacterial leaf streak (BLS) being one of the most destructive bacterial diseases of rice. BLS symptoms are very similar to those of bacterial blight caused by closely related Xanthomonas oryzae pv. oryzae. X. o. pv. oryzae and X. o. pv. oryzicola and often occur in rice f ields simultaneously, so separate leaves may show symptoms of both diseases. The quarantine status and high severity of the pathogen require a highly eff icient, fast and precise diagnostic method. We have developed an assay for Xanthomonas oryzae pv. oryzicola detection using real-time polymerase chain reaction (qPCR) and PCR amplicon sequencing. The DNA samples of X. o. pv. oryzae and X. o. pv. oryzicola were obtained from the collection of CIRM-CFBR (France). To evaluate the analytical sensitivity of the assay, a vector construct based on the pAL2-T plasmid was created through the insertion of X. o. pv. oryzicola target fragment (290 bp). Primers and a probe for qPCR were selected for the hpa1 gene site. They allowed identifying all the strains the sequences of which had been loaded in the GenBank NCBI Nucleotide database before November 11, 2021. The SeqX.o.all sequencing primers were selected for the hrp gene cluster sequence, namely for the nucleotide sequence encoding the Hpa1 protein, the sequencing of which allows for eff icient differentiation of X. oryzae species. The analytical specif icity of the system was tested using the DNAs of 53 closely related and accompanying microorganisms and comprised 100 % with no false-positive or false-negative results registered. The system’s analytical sensitivity was not less than 25 copies per PCR reaction. Its eff icacy has been conf irmed using f ive different qPCR detection systems from different manufacturers, so it can be recommended for diagnostic and screening studies.

Primers for specific detection and identification of Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis.

Bacterial leaf spot and bacterial leaf blight are global threats to the cultivation of cruciferous vegetables, and it is necessary to develop methods to easily detect, identify, and distinguish the causative pathogens Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca). Here, we used the sequence specificity of the exchangeable effector loci flanking the hrp gene cluster to design primers that can help detect and discriminate between Psm and Pca. Primers common to both bacteria (hrpK_fw1 and hrpK_fw2) were designed within hrpK at the end of the hrp gene cluster. Psm-specific primers (MAC_rv1 and MAC_rv2) were designed in hopPtoB1 and Pca-specific primers (ALS_rv1 and ALS_rv2) were designed in hopX1 adjacent to hrpK. PCR using hrpK_fw1 and MAC_rv1 or hrpK_fw2 and MAC_rv2 amplified DNA fragments of only Psm, P. syringae pv. tomato (causal agent of tomato bacterial speck), and P. syringae pv. spinaciae (causal agent of spinach bacterial leaf spot), among 76 strains of phytopathogenic bacteria. PCR using hrpK_fw1 and ALS_rv1 or hrpK_2 and ALS_rv2 amplified DNA fragments of only Pca. Multiplex PCR with these primers could easily distinguish Psm and Pca from bacterial colonies isolated on growth media and detect the pathogen in symptomatic leaves. Multiplex nested PCR with the primers detected contamination in one Psm- and/or one Pca-infected seeds in 1000 seeds. These results suggest that these PCR primers could help detect and discriminate Psm and Pca. KEY POINTS: • We investigated Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis. • Novel primers common to both bacteria were designed following genome comparison. • Multiplex PCR with new primers could discriminate Psm and Pca.

Comparative Genomics, Pangenome, and Phylogenomic Analyses of Brenneria spp., and Delineation of Brenneria izadpanahii sp. nov.

Brenneria species are bacterial plant pathogens mainly affecting woody plants. Association of all members with devastating disorders (e.g., acute oak decline in Iran and United Kingdom) are due to adaptation and pathogenic behavior in response to host and environmental factors. Some species, including B. goodwinii, B. salicis, and B. nigrifluens, also show endophytic residence. Here we show that all species including novel Brenneria sp. are closely related. Gene-based and genome/pangenome-based phylogeny divide the genus into two distinct lineages, Brenneria clades A and B. The two clades were functionally distinct and were consistent with their common and special potential activities as determined via annotation of functional domains. Pangenome analysis demonstrated that the core pathogenicity factors were highly conserved, an hrp gene cluster encoding a type III secretion system was found in all species except B. corticis. An extensive repertoire of candidate virulence factors was identified. Comparative genomics indicated a repertoire of plant cell wall degrading enzymes, metabolites/antibiotics, and numerous prophages providing new insights into Brenneria-host interactions and appropriate targets for further characterization. This work not only documented the genetic differentiation of Brenneria species but also delineates a more functionally driven understanding of Brenneria by comparison with relevant Pectobacteriaceae thereby substantially enriching the extent of information available for functional genomic investigations.

Characterization, Phylogeny, and Genome Analyses of Nonpathogenic Xanthomonas campestris Strains Isolated from Brassica Seeds.

Xanthomonads were detected by using the Xan-D(CCF) medium from the brassica seeds, and their pathogenicity was determined by plant inoculation tests. It was found that some seed lots were infested with Xanthomonas campestris pv. campestris, some with X. campestris pv. raphani, and some with nonpathogenic xanthomonads. The nonpathogenic xanthomonad strains were identified as X. campestris, and the multilocus sequence analysis showed that the nonpathogenic X. campestris strains were grouped together with pathogenic X. campestris, but not with nonpathogenic strains of X. arboricola. In addition, all isolated X. campestris pv. campestris and X. campestris pv. raphani strains were positive in the hrpF-PCR, but the nonpathogenic strains were negative. It was further found that nonpathogenic X. campestris strain nE1 does not contain the entire pathogenicity island (hrp gene cluster; type III secretion system) and all type III effector protein genes based on the whole genome sequence analyses. The nonpathogenic X. campestris strain nE1 could acquire the entire pathogenicity island from the endemic X. campestris pv. campestris and X. campestris pv. raphani strains by conjugation, but type III effector genes were not cotransferred. The studies showed that the nonpathogenic X. campestris strains indeed exist on the brassica seeds, but it could be differentiated by the PCR assays on the hrp and type III effector genes. Nevertheless, the nonpathogenic X. campestris strains cannot be ignored because they may be potential gene resources to increase genetic diversity in the endemic pathogenic X. campestris pv. campestris and X. campestris pv. raphani strains.

Synthesis and biological evaluation of 1,3,4-thiadiazole derivatives as type III secretion system inhibitors against Xanthomonas oryzae

Xanthomonas oryzae pv. oryzae (Xoo) infection directly leads to a severe disease known as leaf blight, which is a major cause of yield loss of rice. Use of traditional bactericides has resulted in severe resistance in pathogenic bacteria. A new approach screening compounds that target the virulence factors rather than killing bacterial pathogens is imperative. In gram-negative bacteria, the type III secretion system (T3SS) is a conserved and significant virulence factor considered as a target for drug development. Therefore, we designed and synthesized a new series of 5-phenyl-2-furan carboxylic acid derivatives stitched with 2-mercapto-1,3,4-thiadiazole. Bioassays revealed that the title candidates attenuated the hypersensitive response through suppressing the promoter activity of a harpin gene hpa1 without affecting bacterial growth. Quantitative real time polymerase chain reaction (qRT-PCR) analysis demonstrated reduced the expression of several genes associated with T3SS, when title compounds were applied. Additionally, hrp gene cluster members, including hrpG and hrpX, had reduced mRNA levels. In vivo greenhouse tests showed that candidate compounds could alleviate the effects of Xoo infection in rice (Oryza sativa) and possess better protective activity against rice bacterial leaf blight than bismerthiazol and thiodiazole copper. All tested compounds were safe to rice. This work suggests there are new safe options for Xoo control in rice from these 1,3,4-thiadiazole derivatives.

Systematic Functional Analysis of Sigma (σ) Factors in the Phytopathogen Xanthomonas campestris Reveals Novel Roles in the Regulation of Virulence and Viability.

The black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is a model organism for the study of plant bacterial pathogenesis mechanisms. In bacteria, σ factors serve as important regulatory elements that respond to various environmental signals and cues. Though Xcc encodes 15 putative σ factors little is known about their roles. As an approach to identify the potential role of each σ factor, we constructed mutations in each of the σ-factor genes as well as generating mutants deficient in multiple σ factors to assess these regulators potential additive functions. The work identified two σ70 factors essential for growth. Furthermore, the work discovered a third σ70 factor, RpoE1, important for virulence. Further studies revealed that RpoE1 positively regulates the expression of the hrp gene cluster that encodes the type III secretion system (T3SS) which determines the pathogenicity and hypersensitive response of Xcc on plants. In vivo and in vitro studies demonstrated that RpoE1 could bind to the promoter region and promote transcription of hrpX, a gene encoding a key regulator of the hrp genes. Overall, this systematic analysis reveals important roles in Xcc survival and virulence for previously uncharacterized σ70 factors that may become important targets for disease control.

Involvement of hrpX and hrpG in the Virulence of Acidovorax citrulli Strain Aac5, Causal Agent of Bacterial Fruit Blotch in Cucurbits.

Acidovorax citrulli causes bacterial fruit blotch, a disease that poses a global threat to watermelon and melon production. Despite its economic importance, relatively little is known about the molecular mechanisms of pathogenicity and virulence of A. citrulli. Like other plant-pathogenic bacteria, A. citrulli relies on a type III secretion system (T3SS) for pathogenicity. On the basis of sequence and operon arrangement analyses, A. citrulli was found to have a class II hrp gene cluster similar to those of Xanthomonas and Ralstonia spp. In the class II hrp cluster, hrpG and hrpX play key roles in the regulation of T3SS effectors. However, little is known about the regulation of the T3SS in A. citrulli. This study aimed to investigate the roles of hrpG and hrpX in A. citrulli pathogenicity. We found that hrpG or hrpX deletion mutants of the A. citrulli group II strain Aac5 had reduced pathogenicity on watermelon seedlings, failed to induce a hypersensitive response in tobacco, and elicited higher levels of reactive oxygen species in Nicotiana benthamiana than the wild-type strain. Additionally, we demonstrated that HrpG activates HrpX in A. citrulli. Moreover, transcription and translation of the type 3-secreted effector (T3E) gene Aac5_2166 were suppressed in hrpG and hrpX mutants. Notably, hrpG and hrpX appeared to modulate biofilm formation. These results suggest that hrpG and hrpX are essential for pathogenicity, regulation of T3Es, and biofilm formation in A. citrulli.

Comparative Genomics Identifies a Novel Conserved Protein, HpaT, in Proteobacterial Type III Secretion Systems that Do Not Possess the Putative Translocon Protein HrpF.

Xanthomonas translucens is the causal agent of bacterial leaf streak, the most common bacterial disease of wheat and barley. To cause disease, most xanthomonads depend on a highly conserved type III secretion system, which translocates type III effectors into host plant cells. Mutagenesis of the conserved type III secretion gene hrcT confirmed that the X. translucens type III secretion system is required to cause disease on the host plant barley and to trigger a non-host hypersensitive response (HR) in pepper leaves. Type III effectors are delivered to the host cell by a surface appendage, the Hrp pilus, and a translocon protein complex that inserts into the plant cell plasma membrane. Homologs of the Xanthomonas HrpF protein, including PopF from Ralstonia solanacearum and NolX from rhizobia, are thought to act as a translocon protein. Comparative genomics revealed that X. translucens strains harbor a noncanonical hrp gene cluster, which rather shares features with type III secretion systems from Ralstonia solanacearum, Paraburkholderia andropogonis, Collimonas fungivorans, and Uliginosibacterium gangwonense than other Xanthomonas spp. Surprisingly, none of these bacteria, except R. solanacearum, encode a homolog of the HrpF translocon. Here, we aimed at identifying a candidate translocon from X. translucens. Notably, genomes from strains that lacked hrpF/popF/nolX instead encode another gene, called hpaT, adjacent to and co-regulated with the type III secretion system gene cluster. An insertional mutant in the X. translucens hpaT gene, which is the first gene of a two-gene operon, hpaT-hpaH, was non-pathogenic on barley and did not cause the HR or programmed cell death in non-host pepper similar to the hrcT mutant. The hpaT mutant phenotypes were partially complemented by either hpaT or the downstream gene, hpaH, which has been described as a facilitator of translocation in Xanthomonas oryzae. Interestingly, the hpaT mutant was also complemented by the hrpF gene from Xanthomonas euvesicatoria. These findings reveal that both HpaT and HpaH contribute to the injection of type III effectors into plant cells.

Genome Sequencing of Ralstonia solanacearum CQPS-1, a Phylotype I Strain Collected from a Highland Area with Continuous Cropping of Tobacco.

Ralstonia solanacearum, an agent of bacterial wilt, is a highly variable species with a broad host range and wide geographic distribution. As a species complex, it has extensive genetic diversity and its living environment is polymorphic like the lowland and the highland area, so more genomes are needed for studying population evolution and environment adaptation. In this paper, we reported the genome sequencing of R. solanacearum strain CQPS-1 isolated from wilted tobacco in Pengshui, Chongqing, China, a highland area with severely acidified soil and continuous cropping of tobacco more than 20 years. The comparative genomic analysis among different R. solanacearum strains was also performed. The completed genome size of CQPS-1 was 5.89 Mb and contained the chromosome (3.83 Mb) and the megaplasmid (2.06 Mb). A total of 5229 coding sequences were predicted (the chromosome and megaplasmid encoded 3573 and 1656 genes, respectively). A comparative analysis with eight strains from four phylotypes showed that there was some variation among the species, e.g., a large set of specific genes in CQPS-1. Type III secretion system gene cluster (hrp gene cluster) was conserved in CQPS-1 compared with the reference strain GMI1000. In addition, most genes coding core type III effectors were also conserved with GMI1000, but significant gene variation was found in the gene ripAA: the identity compared with strain GMI1000 was 75% and the hrpII box promoter in the upstream had significantly mutated. This study provided a potential resource for further understanding of the relationship between variation of pathogenicity factors and adaptation to the host environment.

Expression of the dspA/E gene of Erwinia amylovora in non-host plant Arabidopsis thaliana

ABSTRACTIn the Erwinia amylovora genome, the hrp gene cluster containing the dspA/E/EB/F operon plays a crucial role in mediating the pathogenicity and the hypersensitive response (HR) in the host plant. The role of the dspA/E gene derived from E. amylovora was investigated by monitoring the expression of the β-glucuronidase (GUS) reporter system in transgenic Arabidopsis thaliana cv. Pri-Gus seedlings. A mutant ΔdspA/E strain of E. amylovora was generated to contain a deletion of the dspA/E gene for the purpose of this study. Two-week-old seedlings of GUS transgenic Arabidopsis were vacuum-infiltrated with the wild-type and the mutant (ΔdspA/E) E. amylovora strains. The Arabidopsis seedlings were fixed and stained for GUS activity after 3–5 days following infiltration. The appearance of dense spots with blue staining on the Arabidopsis leaves indicated the typical characteristic of GUS activity. This observation indicated that the wild-type E. amylovora strain had induced a successful and efficient infection on the A. thaliana Pri-Gus leaves. In contrast, there was no visible GUS expression on leaf tissues which were inoculated with the ΔdspA/E mutant E. amylovora strain. These results indicate that the dspA/E gene is required by the bacterial cells to induce HR in non-host plants.

Occurrence of copper-resistant strains and a shift in Xanthomonas spp. causing tomato bacterial spot in Ontario.

Field strains of tomato bacterial spot pathogen (Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri) were characterized for sensitivity to copper and species composition. A total of 98 strains were isolated from symptomatic leaf and fruit samples collected from 18 tomato fields in Ontario. In greenhouse pathogenicity tests, most of the field strains caused severe (37 strains) to highly severe (23 strains) symptoms on 'Bonny Best' tomato plants, whereas 38 strains caused moderate symptoms. In MGY agar plates amended with various concentrations of copper sulfate, 11 strains were completely sensitive (no growth) and 87 strains were resistant (grew on 1.0 mmol/L or higher copper concentration). PCR analysis of the hrp gene cluster followed by restriction digestion with HaeIII and sequencing identified X. gardneri (35 strains) and X. perforans (26 strains) as predominant species and X. euvesicatoria and X. vesicatoria as less common species in Ontario tomato fields. Separation of field strains into various species was also confirmed with starch hydrolysis activity on agar medium. Moreover, 72 field strains produced shiny greenish-yellow colonies surrounded by a milky zone on xanthomonad differential (Xan-D) medium, and the colonies of 26 strains did not produce a milky zone. Thirty-four strains could not be clustered into any species and 25 of those strains were negative for the hrp gene PCR and also did not produce a milky zone around colonies on Xan-D medium. Our results suggest a widespread existence of copper-resistant strains and an increase in X. perforans strains of bacterial spot pathogen in Ontario. This information on copper resistance and species composition within bacterial spot pathogens in Ontario will be helpful for developing effective disease management strategies, making cultivar selection, and breeding new tomato cultivars.

The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection

Xanthomonas translucens pv. graminis (Xtg) is a gammaproteobacterium that causes bacterial wilt on a wide range of forage grasses. To gain insight into the host-pathogen interaction and to identify the virulence factors of Xtg, we compared a draft genome sequence of one isolate (Xtg29) with other Xanthomonas spp. with sequenced genomes. The type III secretion system (T3SS) encoding a protein transport system for type III effector (T3E) proteins represents one of the most important virulence factors of Xanthomonas spp. In contrast with other Xanthomonas spp. assigned to clade 1 on the basis of phylogenetic analyses, we identified an hrp (hypersensitive response and pathogenicity) gene cluster encoding T3SS components and a representative set of 35 genes encoding putative T3Es in the genome of Xtg29. The T3SS was shown to be divergent from the hrp gene clusters of other sequenced Xanthomonas spp. Xtg mutants deficient in T3SS regulating and structural genes were constructed to clarify the role of the T3SS in forage grass colonization. Italian ryegrass infection with these mutants led to significantly reduced symptoms (P < 0.05) relative to plants infected with the wild-type strain. This showed that the T3SS is required for symptom evocation. In planta multiplication of the T3SS mutants was not impaired significantly relative to the wild-type, indicating that the T3SS is not required for survival until 14 days post-infection. This study represents the first major step to understanding the bacterial colonization strategies deployed by Xtg and may assist in the identification of resistance (R) genes in forage grasses.

Xanthomonas campestris pv. campestris (cause of black rot of crucifers) in the genomic era is still a worldwide threat to brassica crops

Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson is a Gram-negative bacterium that causes black rot, the most important disease of vegetable brassica crops worldwide. Intensive molecular investigation of Xcc is gaining momentum and several whole genome sequences are available. Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadacea; Genus Xanthomonas; Species X. campestris. Xcc can cause disease in a large number of species of Brassicaceae (ex-Cruciferae), including economically important vegetable Brassica crops and a number of other cruciferous crops, ornamentals and weeds, including the model plant Arabidopsis thaliana. Black rot is a systemic vascular disease. Typical disease symptoms include V-shaped yellow lesions starting from the leaf margins and blackening of the veins. Collections of Xcc isolates have been differentiated into physiological races based on the response of several brassica species lines. Black rot is a seed-borne disease. The disease is favoured by warm, humid conditions and can spread rapidly from rain dispersal and irrigation water. The control of black rot is difficult and relies on the use of pathogen-free planting material and the elimination of other potential inoculum sources (infected crop debris and cruciferous weeds). Major gene resistance is very rare in B. oleracea (brassica C genome). Resistance is more readily available in other species, including potentially useful sources of broad-spectrum resistance in B. rapa and B. carinata (A and BC genomes, respectively) and in the wild relative A. thaliana. The reference genomes of three isolates have been released. The genome consists of a single chromosome of approximately 5 100 000 bp, with a GC content of approximately 65% and an average predicted number of coding DNA sequences (CDS) of 4308. Three different secretion systems have been identified and studied in Xcc. The gene clusters xps and xcs encode a type II secretion system and xps genes have been linked to pathogenicity. The role of the type IV secretion system in pathogenicity is still uncertain. The hrp gene cluster encodes a type III secretion system that is associated with pathogenicity. An inventory of candidate effector genes has been assembled based on homology with known effectors. A range of other genes have been associated with virulence and pathogenicity, including the rpf, gum and wxc genes involved in the regulation of the synthesis of extracellular degrading enzymes, xanthan gum and lipopolysaccharides. http://www.xanthomonas.org/

Simple Deletion: a vector- and marker-free method to generate and isolate site-directed deletion mutants

We have designed a new vector- and marker-free site-directed deletion system for gram-negative bacteria. In this system, a specific DNA fragment is amplified from a parental strain by using polymerase chain reaction (PCR), then circularized and introduced back into the parental strain for homologous recombination. The recombinant mutant is then detected and isolated by PCR-based sib selection. Unlike conventional methods, our Simple Deletion method requires no cloning procedures, and no foreign genes such as antibiotic-resistance genes are introduced as selection markers. The resulting mutant is, therefore, the same as the parental strain except for the lack of the target region. This method is categorized as a type of "self-cloning," and the resulting mutant can be used for laboratory research without restrictions. Using this method, we generated a mutant of a plant pathogenic bacterium, Xanthomonas campestris pv. campestris, in which the 20.4-kb hrp gene cluster involved in the type III secretion system and in pathogenicity was deleted. In addition, we proved that this method can also be used to delete smaller DNA regions of X. campestris pv. campestris and to generate deletion mutants of the bacterium Ralstonia solanacearum.

Genetic Organization of thehrpGene Cluster inAcidovorax avenaeStrain N1141 and a Novel Effector Protein That Elicits Immune Responses in Rice (Oryza sativaL.)

The immune system of plants consists of two main arms, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). The multiple effectors that trigger ETI are translocated into plant cells by the type III secretion system (T3SS) of pathogenic bacteria. The rice-avirulent N1141 strain of Acidovorax avenae causes ETI in rice, including hypersensitive response (HR) cell death. Sequence analysis indicated that the N1141 genome contains the hrp gene cluster (35.3 kb), including genes encoding the T3SS apparatus. The T3SS-defective N1141 mutant (NΔT3SS) did not cause HR cell death, suggesting that ETI is caused by translocation of effector proteins into rice cells via T3SS. Computational sequence analysis predicted that Lrp, HrpW, and HrpY are secreted by T3SS. The hrpY deletion mutant (NΔhrpY) did not cause ETI, suggesting that HrpY is an important effector of ETI in the interaction between A. avenae N1141 and rice.

Pseudomonas syringae pv. tabaci 에서 식물세포접촉에 의한 병원성 유전자의 조절

The hrp gene cluster in the plant pathogen Pseudomonas syringae is a key determinant of pathogenicity. Recent studies have demonstrated that specific host cell induction of the Ralstonia solanacearum hrp gene cluster is controlled by the PrhA (plant regulator of hrp) receptor. To characterize the role that P. syringae PrhA plays in the virulence of plant cells, a prhA homolog was isolated from P. syringae pv. tabaci and a ΔprhA mutant was constructed by allelic exchange. The ΔprhA mutant had reduced virulence in the host plant, and co-culture of P. syringae pv. tabaci and plant cell suspensions induced a much higher level of hrpA gene transcription than culture in hrp-inducing minimal medium. These results indicate that PrhA of P. syringae is a putative pathogen-plant cell contact sensor, therefore, we used a hrpA-gfp reporter fusion to monitor the in situ expression of PrhA. The results of this study demonstrated that PrhA induces hrp gene expression in P. syringae pv. tabaci in the presence of plant cells.