Search

Xanthan gum is a microbial polysaccharide produced by Xanthomonas and widely used in various industries. To produce xanthan gum, the native Xanthomonas citri- 386 was used in a cheese-whey-based culture medium. The culture conditions were investigated in batch experiments based on the response surface methodology to increase xanthan production and viscosity. Three independent variables in this study included feeding times of acetate, pyruvate, and citrate. The maximum xanthan gum production and viscosity within 120 h by X. citri -386 using Box–Behnken design were 25.7 g/l and 65   500 cP, respectively, with a 151% and 394% increase as compared to the control sample. Overall, the findings of this study recommend the use of X. citri -386 in the cheese-whey-based medium as an economical medium with optimal amounts of acetate, pyruvate, and citrate for commercial production of xanthan gum on an industrial scale. The adjustment of the pyruvate and acetate concentrations optimized xanthan gum production in the environment. [ABSTRACT FROM AUTHOR]

Xanthomonas species infect many important crops and cause huge yield loss. These pathogens deliver transcription activator-like (TAL) effectors into the cytoplasm of plant cells. TAL effectors move to host nuclei, directly bind to the promoters of host susceptible genes, and activate their transcription. However, the molecular mechanisms by which TAL effectors induce host transcription remain unclear. We herein demonstrated that TAL effectors interacted with the SIMILAR TO RCD ONE (SRO) family proteins OsSRO1a and OsSRO1b in nuclei. A transactivation assay using rice protoplasts indicated that OsSRO1a and OsSRO1b enhanced the activation of the OsSWEET14 promoter by the TAL effector AvrXa7. The AvrXa7-mediated expression of OsSWEET14 was significantly reduced in ossro1a mutants. However, the overexpression of OsSRO1a increased disease resistance by up-regulating the expression of defense-related genes, such as WRKY62 and PBZ1. This was attributed to OsSRO1a and OsSRO1b also enhancing the transcriptional activity of WRKY45, a direct regulator of WRKY62 expression. Therefore, OsSRO1a and OsSRO1b appear to positively contribute to transcription mediated by bacterial TAL effectors and rice transcription factors. [ABSTRACT FROM AUTHOR]

Aims Citrus canker caused by Xanthomonas citri subsp. citri (X. citri) is a disease of economic importance. Control of this disease includes the use of metallic copper, which is harmful to the environment and human health. Previous studies showed that the crude extract from the fungus Pseudogymnoascus sp. LAMAI 2784 isolated from Antarctic soil had in vitro antibacterial action against X. citri. The aim of the present study was to expand the applications of this extract. Methods and results In greenhouse assays, the crude extract was able to reduce bacterial infection on citrus leaves from 1.55 lesions/cm2 (untreated plants) to 0.04 lesions/cm2. Bisdechlorogeodin was identified as the main compound of the bioactive fraction produced by Pseudogymnoascus sp. LAMAI 2784, which inhibited bacterial growth in vitro (IC90 ≈ 156 µg ml−1) and permeated 80% of X. citri cells, indicating that the membrane is the primary target. Conclusion The present results showed that the bioactive fraction of the extract is mainly composed of the compound bisdechlorogeodin, which is likely responsible for the biological activity against X. citri , and the main mechanism of action is the targeting of the cell membrane. This study indicates that bisdechlorogeodin has valuable potential for the control of X. citri. [ABSTRACT FROM AUTHOR]

The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads. [ABSTRACT FROM AUTHOR]

Leaf scald is a destructive sugarcane disease caused by the bacterium Xanthomonas albilineans (Ashby) Dowson. This pathogen presents the gene cluster SPI-1 T3SS, a conserved feature in pathogens vectored by animals. In this study, the competence of Mahanarva fimbriolata (Stål), a spittlebug commonly found in sugarcane fields in Brazil, was evaluated for the transmission of X. albilineans. Artificial probing assays were conducted to investigate the ability of M. fimbriolata adults to acquire X. albilineans from artificial diets containing the pathogen with subsequent inoculation of X. albilineans into pathogen-free diets. Plant probing assays with M. fimbriolata adults were conducted to evaluate the acquisition of X. albilineans from diseased source plants and subsequent inoculation of healthy recipient sugarcane plants. The presence of X. albilineans DNA in saliva/diet mixtures of the artificial probing assays and both insects and plants of the plant probing assays were checked using TaqMan assays. The artificial probing assays showed that M. fimbriolata adults were able to acquire and inoculate X. albilineans in diets. Plant probing assays confirmed the competence of M. fimbriolata to transmit X. albilineans to sugarcane. Over the entire experiment, 42% of the insects had acquired the pathogen and successful inoculation of the pathogen occurred in 18% of the recipient-susceptible sugarcane plants at 72 or 96 h of inoculation access period. Assays evidenced the vector competence of M. fimbriolata for transmission of X. albilineans , opening new pathways for investigating the biology and the economic impacts of the interaction between X. albilineans and M. fimbriolata. [ABSTRACT FROM AUTHOR]

Aim: The study was carried out to evaluate three commercial DNA extraction kits, Probe GS, FitoSorb and Sorb GMO, thus identifying the most suitable for isolating the phytopathogenic bacteria Xanthomonas euvesicatoria pv. allii. Materials and Methods: Onion seed samples were prepared which were inoculated with bacterial concentrations ranging from 101 to 107 CFU per ml. Real‐time PCR was performed to determine the efficacy of the isolated DNA in enhancing the sensitivity of the assay. The DNA extracted by Probe GS had the best detectability, having been detected at the lowest concentration used in the study 101 × 3 CFU per ml. FitoSorb and Sorb GMO yielded DNA with a higher and similar limit of detection 103 × 3 CFU per ml. Furthermore, Probe GS had the lowest cycle at every concentration tested as compared to the other methods. Conclusion: Therefore, Probe GS proved to be the most optimized kit for the extraction of X. euvesicatoria pv. allii, hence enhanced degree of sensitivity for the assay. Impact and Significance of the Study: The findings generated in this study can be used by phytosanitary laboratories to develop highly rapid and accurate diagnostic protocols for X. euvesicatoria pv. allii. [ABSTRACT FROM AUTHOR]

Peptidoglycan in bacterial cell walls is a biopolymer consisting of sugars and amino acids and plays important role in maintaining cell integrity from the environment. Its biosynthesis is a major target for antibiotics and the genes and enzymes involved in the biosynthetic pathway have been well studied. However, we recently identified an alternative pathway in the early stage of peptidoglycan biosynthesis in Xanthomonas oryzae, a plant pathogen causing bacterial blight disease of rice. The distribution of the alternative pathway is limited to relatively few bacterial genera that contain many pathogenic species, including Xylella and Stenotrophomonas, besides Xanthomonas. Thus, the alternative pathway is an attractive target for the development of narrow-spectrum antibiotics specific to pathogens. In this minireview, we summarize the discovery of the alternative pathway and identification of its specific inhibitors. [ABSTRACT FROM AUTHOR]

Quorum sensing (QS) helps the Xanthomonas group of phytopathogens to infect several crop plants. The vascular phytopathogen Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease on Brassicaceae leaves, where a typical v-shaped lesion spans both vascular and mesophyll regions with progressive leaf chlorosis. Recently, the role of QS has been elucidated during Xcc early infection stages. However, a detailed insight into the possible role of QS-regulated bacterial invasion in host chlorophagy during late infection stages remains elusive. In this study, using QS-responsive whole-cell bioreporters of Xcc, we present a detailed chronology of QS-facilitated Xcc colonization in the mesophyll region of cabbage (Brassica oleracea) leaves. We report that QS-enabled localization of Xcc to parenchymal chloroplasts triggers leaf chlorosis and promotion of systemic infection. Our results indicate that the QS response in the Xanthomonas group of vascular phytopathogens maximizes their population fitness across host tissues to trigger stage-specific host chlorophagy and establish a systemic infection. [ABSTRACT FROM AUTHOR]

Computational tool-assisted primer design for real-time reverse transcription (RT) PCR (qPCR) analysis largely ignores the sequence similarities between sequences of homologous genes in a plant genome. It can lead to false confidence in the quality of the designed primers, which sometimes results in skipping the optimization steps for qPCR. However, the optimization of qPCR parameters plays an essential role in the efficiency, specificity, and sensitivity of each gene's primers. Here, we proposed an optimized approach to sequentially optimizing primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference (and target) gene. Our approach started with a sequence-specific primer design that should be based on the single-nucleotide polymorphisms (SNPs) present in all the homologous sequences for each of the reference (and target) genes under study. By combining the efficiency calibrated and standard curve methods with the 2−ΔΔCt method, the standard cDNA concentration curve with a logarithmic scale was obtained for each primer pair for each gene. As a result, an R2 ≥ 0.9999 and the efficiency (E) = 100 ± 5% should be achieved for the best primer pair of each gene, which serve as the prerequisite for using the 2−ΔΔCt method for data analysis. We applied our newly developed approach to identify the best reference genes in different tissues and at various inflorescence developmental stages of Tripidium ravennae, an ornamental and biomass grass, and validated their utility under varying abiotic stress conditions. We also applied this approach to test the expression stability of six reference genes in soybean under biotic stress treatment with Xanthomonas axonopodis pv. glycines (Xag). Thus, these case studies demonstrated the effectiveness of our optimized protocol for qPCR analysis. [ABSTRACT FROM AUTHOR]

Aim: To investigate the genetic and pathogenic variability of Xanthomonas oryzae pv. oryzae causing bacterial blight in rice on the remote Andaman Islands, India. Methods and Results: A total of 27 yellow‐pigmented bacterial isolates representing rice fields of Andaman Islands incited blight on the susceptible‐rice cultivar, C14‐8. Phenotypic, pathogenic traits and 16S rRNA gene sequences revealed their identity as X. oryzae pv. oryzae. Virulence profiling indicated the prevalence of seven pathotypes of X. oryzae pv. oryzae on the Islands. Pathotypes‐VI and ‐VII were highly virulent, whereas the pathotype‐I was less virulent. Multilocus sequence typing based on nucleotide sequence polymorphism in nine housekeeping genes dnaK; fyuA; gyrB (two loci): rpoD; fusA; gapA; gltA and lepA clustered 27 isolates into 17 sequence types (STs) segregated into two clonal‐complexes (CC). While CC‐I comprised of isolates from Andaman Island, the CC‐II is a mixture of isolates representing mainland India and Andaman Island. The data revealed trans‐boundary pathogen introduction and a consequent intra‐regional diversification on these islands due to the deployment of different rice cultivars in different regions. Conclusions: Genotyping and pathotyping of sland isolates revealed seven pathotypes distributed in two clonal complexes with strong indications for trans‐boundary movement and consequent diversification of the bacterial pathogen. Highly virulent pathotypes of X. oryzae pv. oryzae that could overcome combinations of R‐genes, xa13+Xa21 as well as xa5+xa13 were found prevalent in the Andaman Islands Significance and Impact of the Study: Genetic and virulence analysis of X. oryzae pv. oryzae in the Andaman Islands revealed introduction and host‐mediated regional diversification and local adaptation of X oryzae pv. oryzae. The study calls for the need of multi‐gene pyramiding for durable disease resistance and establishing stringent quarantine measures for safeguarding island agricultural practices in the future. [ABSTRACT FROM AUTHOR]

Research on a few model plant–pathogen systems has benefitted from years of tool and resource development. This is not the case for the vast majority of economically and nutritionally important plants, creating a crop improvement bottleneck. Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in all regions where cassava (Manihot esculenta Crantz) is grown. Here, we describe the development of cassava that can be used to visualize one of the initial steps of CBB infection in vivo. Using CRISPR-mediated homology-directed repair (HDR), we generated plants containing scarless insertion of GFP at the 3’ end of CBB susceptibility (S) gene MeSWEET10a. Activation of MeSWEET10a-GFP by the transcription activator-like (TAL) effector TAL20 was subsequently visualized at transcriptional and translational levels. To our knowledge, this is the first such demonstration of HDR via gene editing in cassava. [ABSTRACT FROM AUTHOR]

Solanum lycopersicum var. cerasiforme accession PI 114490 has broad-spectrum resistance to bacterial spot caused by several species of Xanthomonas. Resistance is quantitatively inherited, and a common quantitative trait locus QTL-11B on chromosome 11 has been identified previously. In this study, the SlPub24 gene was characterized in QTL-11B. SlPub24 in PI 114490 was upregulated by infection with X. euvesicatoria pv. perforans race T3, but its transcription was low in the susceptible line OH 88119 whether or not it was infected by the pathogen. The differential expression of SlPub24 between PI 114490 and OH 88119 was due to great sequence variation in the promoter region. The promoter of SlPub24 in OH 88119 had very low activity and did not respond to pathogen infection. Transgenic lines of OH 88119 overexpressing SlPub24 isolated from PI 114490 showed significantly enhanced resistance, while mutants of Slpub24 generated by CRISPR/Cas9 editing showed more susceptibility to race T3 and to other races. The mutants also showed spontaneous cell death in leaves. The expression of the salicylic acid (SA) pathway gene phenylalanine ammonia-lyase (PAL) and signaling-related genes pathogenesis-related (PR1) and nonexpresser of PR1 (NPR1) were influenced by SlPub24. The content of SA in tomato plants was consistent with the level of SlPub24 expression. Furthermore, SlPUB24 interacted with the cell wall protein SlCWP and could regulate the degradation of SlCWP. The expression levels of SlCWP and SlCWINV1, a cell wall invertase gene, showed opposite patterns during pathogen infection. The activity of SlCWINV1 was lower in mutants than in PI 114490. The results are discussed in terms of the roles of the abovementioned genes, and a potential model for SlPUB24-mediated resistance to bacterial spot is proposed. [ABSTRACT FROM AUTHOR]

The present work intended to evaluate the applicability of photodynamic inactivation (PDI) of Xanthomonas citri subsp. citri with toluidine blue O (TBO), a commercial photosensitizer, as a strategy to control citrus canker. Assays were conducted with cell suspensions and biofilms, constructed either on polypropylene microtubes (in vitro assays) or on the surface of orange leaves (ex vivo assays), in the presence of TBO and under irradiation with artificial white light or natural sunlight. PDI assays using TBO alone caused a maximum 5·8 log10 reduction of X. citri viable cells in suspensions, and a much smaller inactivation (1·5 log10) in biofilms. However, concomitant use of KI potentiated the TBO photosensitization. Biofilms were inactivated down to the detection limit (>6 log10 reduction) with 5·0 µmol l−1 TBO + 10 mmol l−1 KI (in vitro) or 5·0 µmol l−1 TBO + 100 mmol l−1 KI (ex vivo) after artificial white light irradiation. Under natural sunlight, a reduction down to the detection limit of the Miles–Misra method was achieved with 50 µmol l−1 TBO and 100 mmol l−1 KI. PDI has potential to be applied in the control of citrus canker in field conditions although further studies are needed to show that there are no risks to plant physiology or fruit quality. Significance and Impact of the Study: Xanthomonas citri subsp. citri is a major cause of disease in citrus orchards. Because of the low efficacy and high environmental toxicity of copper‐based treatments, there is growing interest on more sustainable phytosanitary approaches. Photodynamic inactivation (PDI) is being successfully used to control infectious agents and literature reports indicate that it is effective against some fungi and bacteria attacking fruit crops. The results of the present work open the perspective of using a low‐cost photosensitizer and sunlight, as energy source, to control of the causative agent of citrus canker. [ABSTRACT FROM AUTHOR]

The deferred antagonism technique has been utilized for several decades for detecting antibiosis activity. Most protocols require the elimination of antibiotic‐producing cells by exposing them to chloroform vapour, UV radiation or filter sterilizing the filtrate steps that require additional time and expense to complete. We provide a modified approach to current soft agar overlay practices, which involves addition of antibiotics to the soft agar overlay to inhibit growth of the producer but not the indicator strain. This technique can be used to reproducibly and efficiently screen for antibiotic production with ease. We demonstrate the effectiveness of this technique with three bacterial systems: inhibition of the bacterial spot of tomato pathogen, Xanthomonas euvesicatoria, by its pathogenic competitor Xanthomonas perforans; and inhibition of the fire blight pathogen, Erwinia amylovora, by Pantoea vagans C9‐1 or Pseudomonas fluorescens A506. Significance and Impact of the Study: Deferred antagonism assays are used commonly to observe antibiotic production by micro‐organisms. Killing or removing the producer cells prior to introduction of the indicator strain is a standard practice but requires additional time and special handling procedures. We evaluated a modification of the assay, where the overlay medium is amended with an antibiotic to which the indicator strain is resistant and the producer strain is sensitive. This modification obviates extra steps to kill the producer strain prior to overlaying with the indicator strain and provides a rapid, consistent and cost‐effective method to detect antibiosis. [ABSTRACT FROM AUTHOR]

Xanthomonas citri subsp. citri (Xcc) is the bacteria responsible for citrus canker. During its life cycle Xcc is found on leaves as epiphyte, where desiccation conditions may occur. In this work, two Xcc genes, XAC0100 and XAC4007, predicted in silico to be involved in general stress response, were studied under salt, osmotic, desiccation, oxidative and freezing stress, and during plant-pathogen interaction. Expression of XAC0100 and XAC4007 genes was induced under these stress conditions. Disruption of both genes in Xcc caused decreased bacterial culturability under desiccation, freezing, osmotic and oxidative stress. Importantly, the lack of these genes impaired Xcc epiphytic fitness. Both Xac0100 and Xac4007 recombinant proteins showed protective effects on Xanthomonas cells subjected to drought stress. Also, Escherichia coli overexpressing Xac4007 showed a better performance under standard culture, saline and osmotic stress and were more tolerant to freezing and oxidative stress than wild type E. coli. Moreover, both Xac0100 and Xac4007 recombinant proteins were able to prevent the freeze-thaw-induced inactivation of L-Lactate dehydrogenase. In conclusion, Xac0100 and Xac4007 have a relevant role as bacteria and protein protectors; and these proteins are crucial to bacterial pathogens that must face environmental stressful conditions that compromise the accomplishment of the complete virulence process. [ABSTRACT FROM AUTHOR]

Aims: To evaluate the inhibitory effect of five structurally different imidazolium salts on the in vitro growth of plant pathogenic bacteria that belong to divergent taxonomic genera as well as their ability to reduce the severity of common bacterial blight of common bean caused by Xanthomonas axonopodis pv. phaseoli and bacterial speck of tomato caused by Pseudomonas syringae pv. tomato. Methods and Results: Growth inhibition of Xanthomonas, Pseudomonas, Erwinia, Pectobacterium and Dickeya strains by imidazolium salts was assessed in vitro by radial diffusion on agar medium and by ressazurin reduction in liquid medium. The reduction of common bacterial blight and bacterial speck symptoms and the area under de disease progress curves were determined by spraying two selected imidazolium salts on healthy plants 48 h prior to inoculation with virulent strains of the bacterial pathogens. All imidazolium salts inhibited the growth of all plant pathogenic bacteria when tested by radial diffusion on agar medium. The strength of inhibition differed among imidazolium salts when tested on the same bacterial strain and among bacterial strains when tested with the same imidazolium salt. In liquid medium, most imidazolium salts presented the same minimum inhibitory concentration (MIC) and minimum bactericidal concentration values (200 µmol l−1), the most notable exception of which was the MIC (at least 1000 µmol l−1) for the dicationic MImC10MImBr2. The imidazolium salts C16MImBr and C16MImCl caused significant reductions in the severity of common bacterial blight symptoms when compared with nontreated plants. Conclusion: Imidazolium salts inhibit the in vitro growth of plant pathogenic bacteria and reduce plant disease symptoms to levels comparable to an authorized commercial antibiotic product. Significance and Impact of the Study: New compounds exhibiting broad‐spectrum antibacterial activity with potential use in agriculture were identified. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC) is a disease resulting from Xanthomonas citri subsp. citri (Xcc) infection and poses a grave threat to citrus production worldwide. Wall-associated receptor-like kinases (WAKLs) are proteins with a central role in resisting a range of fungal and bacterial diseases. The roles of WAKLs in the context of CBC resistance, however, remain unclear. Here, we explored the role of CsWAKL08, which confers resistance to CBC, and we additionally analyzed the molecular mechanisms of CsWAKL08-mediated CBC resistance. Based on systematic annotation and induced expression analysis of the CsWAKL family in Citrus sinensis, CsWAKL08 was identified as a candidate that can be upregulated by Xcc infection in the CBC-resistant variety. CsWAKL08 can also be induced by the phytohormones salicylic acid (SA) and methyl jasmonic acid (MeJA) and spans the plasma membrane. Overexpression of CsWAKL08 resulted in strong CBC resistance in transgenic sweet oranges, whereas silencing of CsWAKL08 resulted in susceptibility to CBC. The peroxidase (POD) and superoxide dismutase (SOD) activities were significantly enhanced in the CsWAKL08-overexpressing plants compared to the control plants, thereby mediating reactive oxygen species (ROS) homeostasis in the transgenic plants. Moreover, the JA levels and the expression of JA biosynthesis and JA responsive genes were substantially elevated in the CsWAKL08 overexpression plants relative to the controls upon Xcc infection. Based on these findings, we conclude that the wall-associated receptor-like kinase CsWAKL08 positively regulates CBC resistance through a mechanism involving ROS control and JA signaling. These results further highlight the importance of this kinase family in plant pathogen resistance. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae (Xoo) is a serious pathogen of rice which displays tremendous interstrain variation. The emergence of highly-virulent strains of Xoo is a major threat to rice cultivation. Evolutionary insights into genome dynamics of highly virulent strains as compared with the less-virulent ones are crucial for understanding the molecular basis of exceptional success of Xoo as a highly evolved plant pathogen. In the present study, we report complete genome sequence of Xoo strains with extreme-virulent pathotypes (XVPs) characterized based on their reaction toward ten resistance (Xa) genes. One strain, IXO1088, can overcome resistance mediated by all the ten resistance genes while the other strain IXO704 cannot overcome any of them. Interestingly, our investigation revealed that XVPs display dramatic variation in the genome structure with numerous rearrangements/inversions. Moreover, XVPs also possess distinct transposon content and prophage elements that may provide genomic flux required for the acquisition of novel gene cassettes and structural changes in the genome. Interestingly, analysis of transcription activator-like effector proteins, which are major virulence determinants of Xanthomonas pathogen show marked variation in the transcription activator-like effector content and DNA binding domain of tal genes. Overall, the present study indicates the possible role of mobilomes and repetitive elements in major structural and sequence alterations, which may be leading to the emergence of novel and extreme pathotypes. The knowledge and resource of XVPs will be invaluable in the further systematic understanding of evolution and management of variant pathotypes of Xoo. [ABSTRACT FROM AUTHOR]

Citrus canker type A is a serious disease caused by Xanthomonas citri subsp. citri (X. citri), which is responsible for severe losses to growers and to the citrus industry worldwide. To date, no canker-resistant citrus genotypes are available, and there is limited information regarding the molecular and genetic mechanisms involved in the early stages of the citrus canker development. Here, we present the CitrusKB knowledge base. This is the first in vivo interactome database for different citrus cultivars, and it was produced to provide a valuable resource of information on citrus and their interaction with the citrus canker bacterium X. citri. CitrusKB provides tools for a user-friendly web interface to let users search and analyse a large amount of information regarding eight citrus cultivars with distinct levels of susceptibility to the disease, with controls and infected plants at different stages of infection by the citrus canker bacterium X. citri. Currently, CitrusKB comprises a reference citrus genome and its transcriptome, expressed transcripts, pseudogenes and predicted genomic variations (SNPs and SSRs). The updating process will continue over time by the incorporation of novel annotations and analysis tools. We expect that CitrusKB may substantially contribute to the field of citrus genomics. CitrusKB is accessible at http://bioinfo.deinfo.uepg.br/citrus. Users can download all the generated raw sequences and generated datasets by this study from the CitrusKB website. [ABSTRACT FROM AUTHOR]

Xanthomonas is a well-studied genus of bacterial plant pathogens whose members cause a variety of diseases in economically important crops worldwide. Genomic and functional studies of these phytopathogens have provided significant understanding of microbial-host interactions, bacterial virulence and host adaptation mechanisms including microbial ecology and epidemiology. In addition, several strains of Xanthomonas are important as producers of the extracellular polysaccharide, xanthan, used in the food and pharmaceutical industries. This polymer has also been implicated in several phases of the bacterial disease cycle. In this review, we summarise the current knowledge on the infection strategies and regulatory networks controlling virulence and adaptation mechanisms from Xanthomonas species and discuss the novel opportunities that this body of work has provided for disease control and plant health. [ABSTRACT FROM AUTHOR]

Enzymes in the thiolase superfamily catalyze carbon–carbon bond formation for the biosynthesis of polyhydroxyalkanoate storage molecules, membrane lipids and bioactive secondary metabolites. Natural and engineered thiolases have applications in synthetic biology for the production of high-value compounds, including personal care products and therapeutics. A fundamental understanding of thiolase substrate specificity is lacking, particularly within the OleA protein family. The ability to predict substrates from sequence would advance (meta)genome mining efforts to identify active thiolases for the production of desired metabolites. To gain a deeper understanding of substrate scope within the OleA family, we measured the activity of 73 diverse bacterial thiolases with a library of 15 p -nitrophenyl ester substrates to build a training set of 1095 unique enzyme–substrate pairs. We then used machine learning to predict thiolase substrate specificity from physicochemical and structural features. The area under the receiver operating characteristic curve was 0.89 for random forest classification of enzyme activity, and our regression model had a test set root mean square error of 0.22 (R 2 = 0.75) to quantitatively predict enzyme activity levels. Substrate aromaticity, oxygen content and molecular connectivity were the strongest predictors of enzyme–substrate pairing. Key amino acid residues A173, I284, V287, T292 and I316 in the Xanthomonas campestris OleA crystal structure lining the substrate binding pockets were important for thiolase substrate specificity and are attractive targets for future protein engineering studies. The predictive framework described here is generalizable and demonstrates how machine learning can be used to quantitatively understand and predict enzyme substrate specificity. [ABSTRACT FROM AUTHOR]

To compare overall genome structure and transcription activator-like effector content, we completely sequenced Xanthomonas axonopodis pv. glycines strain 12-2, isolated in 1992 in Thailand, and strain EB08, isolated in 2008 in the United States (Iowa) using PacBio technology. We reassembled the genome sequence for a second US strain, 8ra, derived from a 1980 Iowa isolate, from existing PacBio reads. Despite geographic and temporal separation, the three genomes are highly syntenous, and their transcription activator-like effector repertoires are highly conserved. [ABSTRACT FROM AUTHOR]

Xanthomonas, a complex group of pathogens, infects more than 400 plants, which is expanding to new hosts causing serious diseases. Genome-based studies are transforming our understanding on diversity and relationship of host-specific members, known as pathovars. In this study, we report complete genome sequence of a novel pathovar Xanthomonas axonopodis pv. commiphorae (Xcom) from India. It causes gumming disease of Commiphora wightii, a medicinally important plant. Genome-based phylogenetic and taxonomic investigation revealed that the pathovar belongs to Xanthomonas euvesicatoria and not X. axonopodis as reported earlier. Interestingly, it is a novel host and novel geographic origin for a X. euvesicatoria pathovar. A core-genome-based phylogenetic analysis resolved the pathovar complex of this species on the basis of their hosts. Interestingly, this pathovar harbors a unique 35-kb plasmid encoding type III effectors and toxin–antitoxin gene that is absent in other X. euvesicatoria pathovars and infects tomato, pepper, rose, onion, philodendron, alfalfa, and citrus plants. The pathovar contains two TAL (transcription activator-like) genes, one on plasmid and another on genomic region with an additional pseudo TAL gene flanked by IS elements in the plasmid. Further, Xcom has acquired a novel set of lipopolysaccharide biosynthesis genes after its divergence from the closely related pathovar that infects rose and supports the role of horizontal gene transfer in hypervariation at this locus in the species. Complete genome sequence of this variant pathovar has provided novel insights into evolution of an emerging pathovar in Xanthomonas and will be valuable resource in pathogenomics of X. euvesicatoria. [ABSTRACT FROM AUTHOR]

Plant meristems were previously thought to be sterile. Today, meristem-associated shoot endophytes are mainly reported as contaminants from plant tissue cultures, the number of observed species being very low. However, the few strains characterized have the capacity for infecting host cells and affecting plant growth and development. Here we studied the communities of endophytic bacteria in the buds of mountain birch (Betula pubescens ssp. czerepanovii (N. I. Orlova) Hämet-Ahti) exposed to winter moth (Operophtera brumata L.) herbivory, to identify differences between sprouts and branches of mature birch trees. Mountain birch of the high subarctic is cyclically exposed to winter moth and produces sprouts to generate new trees as a survival mechanism. The majority (54%) of operational taxonomic units belonged to Xanthomonadaceae and Pseudomonales of Proteobacteria. Most of the observed species were classified as Xanthomonas (28%). Sprout buds had the highest diversity, containing approximately three times more species, and significantly more (43%) Pseudomonas species than the mature trees (14%). Our results demonstrate that endophytic communities of buds are richer than previously thought. We suggest that the meristem-associated endophytes should be studied further for a possible role in sprouting and aiding regeneration of trees. [ABSTRACT FROM AUTHOR]

Abstract: This study aims to obtain secondary metabolites extracts from filamentous fungi isolated from soil and marine sediments from Antarctica and assess its potential antibacterial activity on Xanthomonas citri subsp. citri, the agent of citrus canker. Metabolites production was conducted in Malt 2% broth at 15°C for 20 days after which intracellular and extracellular extracts were obtained. The extracts were evaluated by cell viability assays through Resazurin Microtitre Assay. From 158 fungal extracts, 33 hampered bacterial growth in vitro. The average inhibition of the extracts obtained from terrestrial (soil) and marine (sediments) fungi was 94 and 97% respectively. These inhibition values were close to the average of 90% cell death for the positive control. MIC90 and MBC for the bioactive extracts were established. Isolates that produced active metabolites against the phytopathogen were identified using molecular taxonomy (ITS‐rRNA sequencing) as: Pseudogymnoascus, Penicillium, Cadophora, Paraconiothyrium and Toxicocladosporium. Antarctic fungal strains isolated from terrestrial and marine sediments were able to produce secondary metabolites with antimicrobial activity against X. citri subsp. citri, highlighting the importance of these microbial genetic resources. These metabolites have potential to be used as alternatives for the control of this plant pathogen. Significance and Impact of the Study: This manuscript makes an impact on the study of micro‐organisms from extreme habitats and their possible contribution in discovering new active molecules against pathogens of agricultural interest. Studies on the Antarctic continent and its communities have attracted the scientific community due to the long period of isolation and low levels of disturbance that surrounds the region. Knowing the potential of fungi in this region to produce active secondary metabolites, we aim to contribute to the discovery of compounds with antibacterial action in Xanthomonas citri subsp. citri, a plant pathogen present in several regions around the globe. [ABSTRACT FROM AUTHOR]

In the ongoing battle between microbial pathogens and their plant hosts, many phytopathogenic bacteria secrete effector proteins into the host cell cytoplasm to alter host physiology and promote virulence. In yeast two-hybrid (Y2H) analyses using the highly conserved barley Exo70 integrated domain, which acts as a decoy to detect pathogens, plus a series of effector proteins from I Xanthomonas i , they identified several interacting effectors, including XopP. Recent work presented now by Michalopoulou et al. shows that the XopP effector protein secreted by I Xanthomonas campestris i can suppress host defenses and promote virulence by directly targeting the exocyst ([4]). [Extracted from the article]

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses. [ABSTRACT FROM AUTHOR]

Transcription activator-like effectors (TALEs) are secreted by plant-pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, aremajor drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess ahighly repetitiveDNA-bindingdomain of typically34amino acid(AA) tandemrepeats, where AA12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on ricepathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. Wefind strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters ismediated by an integron-likemechanismin Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity. [ABSTRACT FROM AUTHOR]

Xanthomonas translucens pv. translucens (Xtt) is a Gram-negative pathogen of crops from the plant family Poaceae. The lipopolysaccharide (LPS) of Xtt was isolated and chemically characterized. The analyses revealed the presence of rhamnose, xylose, mannose, glucose, galacturonic acid, phosphates, 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) and fatty acids (10:0, 11:0, 11:0(3-OH) i/a, 11:0(3-OH), 12:0(3-OH) i/a, 12:0(3-OH), 12:0, 13:0(3-OH) i, 13:0(3-OH) a, 13:0(3-OH), 14:0(3-OH) i/a, 14:0(3-OH) and 16:0). The rough type of LPS (lipooligosaccharides; LOS) was isolated and its composition determined utilizing mass spectrometry. The structure of core-lipid A backbone was revealed by nuclear magnetic resonance (NMR) spectroscopy performed on O-deacylated LOS sample, and was shown to be: α-D-Manp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→4)-α-D-Manp-(1→5)-α-Kdo-(2→6)-β-D-GlcpN-(1→6)-α-D-GlcpN. 4-α-Man and Kdo were further substituted via phosphodiester groups by two galactopyranuronic acids. Xtt LPS elicited a stress response in Nicotiana tabacum suspension cell cultures, namely a transient calcium signal and the generation of H2O2 was observed. Pharmacological studies indicated the involvement of plasma membrane calcium channels, kinases and phospholipase C as key factors in Xtt LPS induced pathogen signaling. [ABSTRACT FROM AUTHOR]

Bioassay guidance was used along the whole process including method development, isolation and identification of antibacterial neem (Azadirachta indica) oil compounds. The biomonitoring was performed by direct bioautography (DB), a combination of thin-layer chromatography (TLC) and antimicrobial detection. DB of neem oil showed one antibacterial zone that was not UV-active; therefore, the TLC separation was improved under DB control. The chromatographic zone that exhibited activity against, Bacillus subtilis, Xanthomonas euvesicatoria, Aliivibrio fischeri Staphylococcus aureus and methicillin-resistant Staphylococcus aureus was characterized by TLC reagents, indicating a lipophilic, fatty acid-like chemical feature. Two compounds were found and identified in the active zone by high-performance liquid chromatography-electrospray ionization mass spectrometry as linoleic and oleic acids. Both fatty acids inhibited B. subtilis, but A. fischeri was sensitive only against linoleic acid. [ABSTRACT FROM AUTHOR]

Plant pathogenic bacteria disseminate and survive mainly in association with seeds. This study addresses whether seeds are passive carriers or engage a molecular dialogue with pathogens during their development. We developed two pathosystems using Medicago truncatula with Xanthomonas alfalfae subsp. alfalfae (Xaa), the natural Medicago sp. pathogen and Xanthomonas campestris pv. campestris (Xcc), a Brassicaceae pathogen. Three days after flower inoculation, the transcriptome of Xcc-infected pods showed activation of an innate immune response that was strongly limited in Xcc mutated in the type three secretion system, demonstrating an incompatible interaction of Xcc with the reproductive structures. In contrast, the presence of Xaa did not result in an activation of defence genes. Transcriptome profiling during development of infected seeds exhibited time-dependent and differential responses to Xcc and Xaa. Gene network analysis revealed that the transcriptome of Xcc-infected seeds was mainly affected during seed filling whereas that of Xaa-infected seeds responded during late maturation. The Xcc-infected seed transcriptome exhibited an activation of defence response and a repression of targeted seed maturation pathways. Fifty-one percent of putative ABSCISIC ACID INSENSITIVE3 targets were deregulated by Xcc, including oleosin, cupin, legumin and chlorophyll degradation genes. At maturity, these seeds displayed decreased weight and increased chlorophyll content. In contrast, these traits were not affected by Xaa infection. These findings demonstrate the existence of a complex molecular dialogue between xanthomonads and developing seeds and provides insights into a previously unexplored trade-off between seed development and pathogen defence. [ABSTRACT FROM AUTHOR]

Xanthomonas citri subsp. citri (Xcc) is a bacterial pathogen that causes citrus canker in susceptible Citrus spp. The Xcc genome contains genes encoding enzymes from three separate pathways of trehalose biosynthesis. Expression of genes encoding trehalose-6-phosphate synthase (otsA) and trehalose phosphatase (otsB) was highly induced during canker development, suggesting that the two-step pathway of trehalose biosynthesis via trehalose-6-phosphate has a function in pathogenesis. This pathway was eliminated from the bacterium by deletion of the otsA gene. The resulting XccΔotsA mutant produced less trehalose than the wild-type strain, was less resistant to salt and oxidative stresses, and was less able to colonize plant tissues. Gene expression and proteomic analyses of infected leaves showed that infection with XccΔotsA triggered only weak defence responses in the plant compared with infection with Xcc, and had less impact on the host plant’s metabolism than the wild-type strain. These results suggested that trehalose of bacterial origin, synthesized via the otsA–otsB pathway, in Xcc, plays a role in modifying the host plant’s metabolism to its own advantage but is also perceived by the plant as a sign of pathogen attack. Thus, trehalose biosynthesis has both positive and negative consequences for Xcc. On the one hand, it enables this bacterial pathogen to survive in the inhospitable environment of the leaf surface before infection and exploit the host plant’s resources after infection, but on the other hand, it is a tell-tale sign of the pathogen’s presence that triggers the plant to defend itself against infection. [ABSTRACT FROM AUTHOR]

• Background Cereal diseases cause tens of billions of dollars of losses annually and have devastating humanitarian consequences in the developing world. Increased understanding of the molecular basis of cereal host-pathogen interactions should facilitate development of novel resistance strategies. However, achieving this in most cereals can be challenging due to large and complex genomes, long generation times and large plant size, as well as quarantine and intellectual property issues that may constrain the development and use of community resources. Brachypodium distachyon (brachypodium) with its small, diploid and sequenced genome, short generation time, high transformability and rapidly expanding community resources is emerging as a tractable cereal model. • Scope Recent research reviewed here has demonstrated that brachypodium is either susceptible or partially susceptible to many of the major cereal pathogens. Thus, the study of brachypodium-pathogen interactions appears to hold great potential to improve understanding of cereal disease resistance, and to guide approaches to enhance this resistance. This paper reviews brachypodium experimental pathosystems for the study of fungal, bacterial and viral cereal pathogens; the current status of the use of brachypodium for functional analysis of cereal disease resistance; and comparative genomic approaches undertaken using brachypodium to assist characterization of cereal resistance genes. Additionally, it explores future prospects for brachypodium as a model to study cereal-pathogen interactions. • Conclusions The study of brachypodium-pathogen interactions appears to be a productive strategy for understanding mechanisms of disease resistance in cereal species. Knowledge obtained from this model interaction has strong potential to be exploited for crop improvement. [ABSTRACT FROM AUTHOR]

We announce the genome sequence for Xanthomonas species strain Nyagatare, isolated from beans showing unusual disease symptoms in Rwanda. This strain represents the first sequenced genome belonging to an as-yet undescribed Xanthomonas species known as species-level clade 1. It has at least 100 kb of genomic sequence that shows little or no sequence similarity to other xanthomonads, including a unique lipopolysaccharide synthesis gene cluster. At least one genomic region appears to have been acquired from relatives of Agrobacterium or Rhizobium species. The genome encodes homologues of only three known type-three secretion system effectors: AvrBs2, XopF1 and AvrXv4. Availability of the genome sequence will facilitate development of molecular tools for detection and diagnostics for this newly discovered pathogen of beans and facilitate epidemiological investigations of a potential causal link between this pathogen and the disease outbreak. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBs T-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants. [ABSTRACT FROM AUTHOR]

Xanthomonas axonopodis pathovar vasculorum strain NCPPB 900 was isolated from sugarcane on Reunion island in 1960. Consistent with its belonging to fatty-acid type D, multi-locus sequence analysis confirmed that NCPPB 900 falls within the species X. axonopodis. This genome harbours sequences similar to plasmids pXCV183 from X. campestris pv. vesicatoria 85-10 and pPHB194 from Burkholderia pseudomallei. Its repertoire of predicted effectors includes homologues of XopAA, XopAD, XopAE, XopB, XopD, XopV, XopZ, XopC and XopI and transcriptional activator-like effectors and it is predicted to encode a novel phosphonate natural product also encoded by the genome of the phylogenetically distant X. vasicola pv. vasculorum. Availability of this novel genome sequence may facilitate the study of interactions between xanthomonads and sugarcane, a host-pathogen system that appears to have evolved several times independently within the genus Xanthomonas and may also provide a source of target sequences for molecular detection and diagnostics. [ABSTRACT FROM AUTHOR]

Xanthomonas phytopathogenic bacteria produce unique transcription activator-like effector (TALE) proteins that recognize and activate specific plant promoters through a set of tandem repeats. A unique TALE-DNA-binding code uses two polymorphic amino acids in each repeat to mediate recognition of specific nucleotides. The order of repeats determines effector’s specificity toward the cognate nucleotide sequence of the sense DNA strand. Artificially designed TALE-DNA-binding domains fused to nuclease or activation and repressor domains provide an outstanding toolbox for targeted gene editing and gene regulation in research, biotechnology and gene therapy. Gene editing with custom-designed TALE nucleases (TALENs) extends the repertoire of targeted genome modifications across a broad spectrum of organisms ranging from plants and insect to mammals. [ABSTRACT FROM AUTHOR]

Xylem sap (XS) is the first environment that xylem phytopathogens meet in planta during the early infection steps. Xanthomonas campestris pv. campestris ( Xcc), the causative agent of Brassicaceae black rot, colonizes the plant xylem vessels to ensure its multiplication and dissemination. Besides suppression of plant immunity, Xcc has to adapt its metabolism to exploit plant-derived nutrients present in XS. To study Xcc behaviour in the early infection steps, we used cabbage XS to analyse bacterial growth. Mineral and organic composition of XS were determined. Significant growth of Xcc in XS was allowed by the rapid catabolism of amino acids, sugars and organic acids, and it was accompanied by the formation of biofilm-like structures. Transcriptome analysis of Xcc cultivated in XS using cDNA microarrays revealed a XS-specific transcriptional reprogramming compared to minimal or rich media. More specifically, up-regulation of genes encoding transporters such as TonB-dependent transporters (TBDTs), that could be associated with nutrient acquisition and detoxification, was observed. In agreement with the aggregation phenotype, expression of genes important for twitching motility and adhesion was up-regulated in XS. Taken together, our data show specific responses of Xcc to colonization of cabbage XS that could be important for the pathogenesis process and establish XS as a model medium to study mechanisms important for the early infection events. [ABSTRACT FROM AUTHOR]

As part of their immune system, plants have pattern recognition receptors (PRRs) that can detect a broad range of microbe-associated molecular patterns (MAMPs). Here, we identified a PRR of Arabidopsis thaliana with specificity for the bacterial MAMP eMax from xanthomonads. Response to eMax seems to be restricted to the Brassicaceae family and also varied among different accessions of Arabidopsis. In crosses between sensitive accessions and the insensitive accession Shakhdara, eMax perception mapped to RECEPTOR-LIKE PROTEIN1 (RLP1). Functional complementation of rlp1 mutants required gene constructs that code for a longer version of RLP1 that we termed ReMAX (for receptor of eMax). ReMAX/RLP1 is a typical RLP with structural similarity to the tomato (Solanum lycopersicum) RLP Eix2, which detects fungal xylanase as a MAMP. Attempts to demonstrate receptor function by interfamily transfer of ReMAX to Nicotiana benthamiana were successful after using hybrid receptors with the C-terminal part of ReMAX replaced by that of Eix2. These results show that ReMAX determines specificity for eMax. They also demonstrate hybrid receptor technology as a promising tool to overcome problems that impede interfamily transfer of PRRs to enhance pathogen detection in crop plants. [ABSTRACT FROM AUTHOR]

We report the use of designer transcription activator-like (TAL) effectors (dTALEs) for gene activation and function analysis in plants. Seven dTALEs tested all induce their corresponding target genes and some trigger disease susceptibility or resistance to the bacteria expressing them in rice.TAL (transcription activator-like) effectors from Xanthomonas bacteria activate the cognate host genes, leading to disease susceptibility or resistance dependent on the genetic context of host target genes. The modular nature and DNA recognition code of TAL effectors enable custom-engineering of designer TAL effectors (dTALE) for gene activation. However, the feasibility of dTALEs as transcription activators for gene functional analysis has not been demonstrated. Here, we report the use of dTALEs, as expressed and delivered by the pathogenic Xanthomonas oryzae pv. oryzae (Xoo), in revealing the new function of two previously identified disease-related genes and the potential of one developmental gene for disease susceptibility in rice/Xoo interactions. The dTALE gene dTALE-xa27, designed to target the susceptible allele of the resistance gene Xa27, elicited a resistant reaction in the otherwise susceptible rice cultivar IR24. Four dTALE genes were made to induce the four annotated Xa27 homologous genes in rice cultivar Nipponbare, but none of the four induced Xa27-like genes conferred resistance to the dTALE-containing Xoo strains. A dTALE gene was also generated to activate the recessive resistance gene xa13, an allele of the disease-susceptibility gene Os8N3 (also named Xa13 or OsSWEET11, a member of sucrose efflux transporter SWEET gene family). The induction of xa13 by the dTALE rendered the resistant rice IRBB13 (xa13/xa13) susceptible to Xoo. Finally, OsSWEET12, an as-yet uncharacterized SWEET gene with no corresponding naturally occurring TAL effector identified, conferred susceptibility to the Xoo strains expressing the corresponding dTALE genes. Our results demonstrate that dTALEs can be delivered through the bacterial secretion system to activate genes of interest for functional analysis in plants. [ABSTRACT FROM PUBLISHER]

Aims To establish protocols for the simultaneous detection and identification of Xanthomonas species causing tomato bacterial spot. Methods and Results We verified the specificity and sensitivity of the previously reported sets of primers designed for strains of the four species of Brazilian tomato bacterial spot xanthomonads, consisting of 30 of Xanthomonas euvesicatoria, 30 of X. vesicatoria, 50 of X. perforans and 50 of X. gardneri. Furthermore, we tested a multiplex PCR protocol for the purpose of concurrent species identification. The possibility of direct detection of the pathogens in diseased leaf samples was also verified. The primers were highly specific, amplifying only target DNA. The sensitivity of the primers in conventional PCR was 50 pg μl−1 for purified DNA and ranged from 5 × 102 to 5 × 104 CFU ml−1 when bacterial suspensions were analysed. The multiplex PCR was suitable for the detection of all four species and showed similar sensitivity to conventional PCR when tested on purified DNA. When using bacterial suspensions, its sensitivity was similar to conventional PCR only when a biological amplification step (Bio- PCR) was included. Both methods were able to detect the pathogens in symptomatic tomato leaves. Conclusions Brazilian Xanthomonas strains causing tomato bacterial spot can be differentiated and identified at species level by a PCR-based method and by a multiplex PCR. Significance and Impact of the Study This protocol may be a feasible alternative tool for the identification and detection of these pathogens in plant material and may be used for routine diagnostic purposes in plant pathology laboratories. [ABSTRACT FROM AUTHOR]

A key feature of the virulence of many bacterial pathogens is the ability to deliver effector proteins into eukaryotic cells via a dedicated type three secretion system (T3SS). Many bacterial pathogens, including species of Chlamydia, Xanthomonas, Pseudomonas, Ralstonia, Shigella, Salmonella, Escherichia and Yersinia, depend on the T3SS to cause disease. T3SS effectors constitute a large and diverse group of virulence proteins that mimic eukaryotic proteins in structure and function. A salient feature of bacterial effectors is their modular architecture, comprising domains or motifs that confer an array of subversive functions within the eukaryotic cell. These domains/motifs therefore represent a fascinating repertoire of molecular determinants with important roles during infection. This review provides a snapshot of our current understanding of bacterial effector domains and motifs where a defined role in infection has been demonstrated. [ABSTRACT FROM AUTHOR]

The complete DNA sequence of the 41 102-bp plasmid pXap41 from the invasive plant pathogen Xanthomonas arboricola pv. pruni CFBP 5530 was determined and its 44 coding regions were annotated. Comparative analysis with 15 Xanthomonas plasmids and 19 complete genomes revealed that nearly one-fourth of this plasmid has high sequence identity to plasmid pXAC64 and an 8.8-kb chromosomal region of Xanthomonas axonopodis pv. citri strain 306 carrying genes that encode type III effectors and helper proteins. The presence of pXap41 in all X. arboricola pv. pruni genotypes was confirmed for eight strains by plasmid profiling and for 35 X. arboricola pv. pruni isolates with a new plasmid multiplex PCR assay. This plasmid was not detected in any other X. arboricola pathovars ( n=12), indicating the potential for the application of the pXap41 PCR method as a pathovar-level detection and identification tool. [ABSTRACT FROM AUTHOR]

Plant and animal pathogens inject type III effectors (T3Es) into host cells to suppress host immunity and promote successful infection. XopD, a T3E from Xanthomonas campestris pv vesicatoria , has been proposed to promote bacterial growth by targeting plant transcription factors and/or regulators. Here, we show that XopD from the B100 strain of X. campestris pv campestris is able to target MYB30, a transcription factor that positively regulates Arabidopsis thaliana defense and associated cell death responses to bacteria through transcriptional activation of genes related to very-long-chain fatty acid (VLCFA) metabolism. XopD specifically interacts with MYB30, resulting in inhibition of the transcriptional activation of MYB30 VLCFA-related target genes and suppression of Arabidopsis defense. The helix-loop-helix domain of XopD is necessary and sufficient to mediate these effects. These results illustrate an original strategy developed by Xanthomonas to subvert plant defense and promote development of disease. [ABSTRACT FROM AUTHOR]

Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CARING1, from pepper (Capsicum anmtum). In pepper, CARING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRINGI contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CARING1. CARING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CARING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS- RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CARING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaR1NG1. By contrast, overexpression of CARING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CARING1 is involved in the induction of ceil death and the regulation of ubiquitination during the defense response to microbial pathogens. [ABSTRACT FROM AUTHOR]

Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CARING1, from pepper (Capsicum anmtum). In pepper, CARING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRINGI contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CARING1. CARING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CARING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS- RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CARING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaR1NG1. By contrast, overexpression of CARING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CARING1 is involved in the induction of ceil death and the regulation of ubiquitination during the defense response to microbial pathogens. [ABSTRACT FROM AUTHOR]

hrp genes encode components of a type III secretion (T3S) system and play crucial roles in the pathogenicity of the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). A histone-like nucleoid-structuring (H-NS) protein binds DNA and acts as a global transcriptional repressor. Here, we investigated the involvement of an h-ns-like gene, named xrvB, in the expression of hrp genes in Xoo. Under the hrp-inducing culture condition, the expression of a key hrp regulator HrpG increased in the XrvB mutant, followed by activation of the downstream gene expression. Also, in planta, the secretion of a T3S protein (XopR) was activated by the mutation in xrvB. Gel retardation assay indicated that XrvB has DNA-binding activity, but without a preference for the promoter region of hrpG. The results suggest that XrvB negatively regulates hrp gene expression and that an unknown factor(s) mediates the regulation of hrpG expression by XrvB. [ABSTRACT FROM AUTHOR]

Copper (Cu)-based biocides are important chemical controls for both fungal and bacterial diseases in crop fields. Here, we showed that Cu ions at a concentration of 100 μM enhanced t-butyl hydroperoxide ( tBOOH) and hydrogen peroxide (HO) killing of Xanthomonas campestris pv. campestris through different mechanisms. The addition of an antilipid peroxidation agent (α-tocopherol) and hydroxyl radical scavengers (glycerol and dimethyl sulphoxide) partially protected the bacteria from the Cu-enhanced tBOOH and HO killing, respectively. Inactivation of the alkyl hydroperoxide reductase gene rendered the mutant vulnerable to lethal doses of copper sulphate, which could be alleviated by the addition of an HO scavenger (pyruvate) and α-tocopherol. Taken together, the data suggest that Cu ions influence the killing effect of tBOOH through the stimulation of lipid peroxidation, while hydroxyl radical production is the underlying mechanism responsible for the Cu-ion-enhanced HO killing effects. [ABSTRACT FROM AUTHOR]

Xanthomonas campestris pv. glycines (Xcg), an etiological agent of the bacterial pustule disease of soybean, displayed nutritionally regulated caspase-dependent programmed cell death (PCD). Experiments showed that Xcg was under metabolic stress during PCD, as evident from the intracellular accumulation of NADH and ATP. Further, the accumulation of reactive oxygen species (ROS), as confirmed by 2′,7′-dichlorofluorescein diacetate labeling, electron spin resonance spectroscopy, and scopoletin assay, was also observed along with the activation of caspase-3. ROS scavengers such as dimethylsulfoxide, glutathione, n-propyl gallate, and catalase significantly inhibited caspase biosynthesis as well as its activity, eventually leading to the inhibition of PCD. The presence of a sublethal concentration of an electron transport chain uncoupler, 2,4-dinitrophenol, was found to reduce the ROS generation and the increase in the cell survival. These results indicated that Xcg cells grown in a protein-rich medium experienced metabolic stress due to electron leakage from the electron transport chain, leading to the generation of ROS and the expression as well as the activation of caspase-3, and resulting in PCD. A bacterial DNA gyrase inhibitor, nalidixic acid, was also found to inhibit PCD. Gyrase, which regulates DNA superhelicity, and consequently DNA replication and cell multiplication, appears to be involved in the process. [ABSTRACT FROM AUTHOR]

Aims: Natural rubber (poly-[ cis-1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant γ-Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed. Methods and Results: A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose-inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained. Conclusions and Significance and Impact of the Study: Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose-induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell-free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA. [ABSTRACT FROM AUTHOR]