Search

Made available in DSpace on 2021-06-25T11:01:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 Xanthomonas arboricola pv. juglandis (Xaj) is the most significant aboveground walnut bacterial pathogen. Disease management uses copper-based pesticides which induce pathogen resistance. We examined the genetic repertoire associated with adaptation and virulence evolution in Xaj. Comparative genomics of 32 Xaj strains reveal the possible acquisition and propagation of virulence factors via insertion sequences (IS). Fine-scale annotation revealed a Tn3 transposon (TnXaj417) encoding copper resistance genes acquired by horizontal gene transfer and associated with adaptation and tolerance to metal-based pesticides commonly used to manage pathogens in orchard ecosystems. Phylogenomic analysis reveals IS involvement in acquisition and diversification of type III effector proteins ranging from two to eight in non-pathogenic strains, 16 to 20 in pathogenic strains, besides six other putative effectors with a reduced identity degree found mostly among pathogenic strains. Yersiniabactin, xopK, xopAI, and antibiotic resistance genes are also located near ISs or inside genomic islands and structures resembling composite transposons. Center of Research in Biological Science Federal University of Ouro Preto Department of Plant Sciences University of California Faculty of Agricultural and Veterinary Sciences of Jaboticabal (FCAV) Universidade Estadual Paulista (UNESP) Department of Technology Cell Cycle Laboratory Butantan Institute Department of Biochemistry Chemistry Institute University of Sao Paulo School of Computing Federal University of Mato Grosso do Sul Department of Biological Science Institute of Exact and Biological Science Federal University of Ouro Preto Faculty of Agricultural and Veterinary Sciences of Jaboticabal (FCAV) Universidade Estadual Paulista (UNESP) Department of Technology

The interaction between the plant host, walnut (Juglans regia, Jr), and a deadly pathogen (Xanthomonas arboricola pv. juglandis 417, Xaj) can lead to walnut bacterial blight (WB), which depletes walnut productivity by degrading the nut quality. Here, we dissect this pathosystem using tandem mass tag quantitative proteomics. Walnut hull tissues inoculated with Xaj were compared to mock-inoculated tissues, and 3972 proteins were identified, of which 3296 are from Jr and 676 from Xaj. Proteins with differential abundance include oxidoreductases, proteases, and enzymes involved in energy metabolism and amino acid interconversion pathways. Defense responses and plant hormone biosynthesis were also increased. Xaj proteins detected in infected tissues demonstrate its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Secreted proteases and extracellular secretion apparatus such as type IV pilus for twitching motility and type III secretion effectors indicate putative factors recognized by the host. Taken together, these results suggest intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Our results provide insights into molecular mechanisms and highlight potential molecular tools for early detection and disease control strategies.

A mutant library of the bacterium Xanthomonas citri subsp. citri strain 306 pathotype A (Xac), the causative agent of most aggressive Asiatic type A citrus canker, was screened regarding altered canker symptoms after inoculations into Citrus sinensis and Citrus limonia host leaves. Twenty-six mutants have shown phenotypic virulence changes and have respectively knocked out gene identified by sequencing. In vivo growth curves were obtained for nine mutants to quantify how the mutations could affect pathogen’s adaptability to growth inside and attack host plant infected tissue. Among identified genes in mutated strains, we could find those that until now had not been reported as being involved in Xac adaptation and/or virulence, such as predicted to encode for xylose repressor-like protein (XACΔxylR), Fe-S oxidoredutase (XACΔaslB), helicase IV (XACΔhelD), ubiquinol cytochrome c oxidoreductase iron-sulfur subunit (XACΔpetA), chromosome partitioning protein (XACΔparB) and cell division protein FtsB (XACΔftsB), in addition to genes predicted to encode for hypothetical proteins. The new genes found in this study as being relevant to adaptation and virulence, improve the understanding of Xac fitness during citrus plant attack and canker symptoms development.

Type IV pili (Tfp) are widely distributed adhesins of bacterial surfaces. In plant pathogenic bacteria, Tfp are involved in host colonization and pathogenesis. Xanthomonas citri subsp. citri (Xcc) is the phytopathogen responsible for citrus canker disease. In this work, three Tfp structural genes, fimA, fimA1, and pilA from Xcc were studied. A pilA mutant strain from Xcc (XccΔpilA) was constructed and differences in physiological features, such as motilities, adhesion, and biofilm formation, were observed. A structural study of the purified Tfp fractions from Xcc wild-type and Xcc∆pilA showed that pilins are glycosylated in both strains and that FimA and FimA1 are the main structural components of the pili. Furthermore, smaller lesion symptoms and reduced bacterial growth were produced by Xcc∆pilA in orange plants compared to the wild-type strain. These results indicate that the minor pilin-like gene, pilA, is involved in Tfp performance during the infection process. Fil: Petrocelli, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Arana, Maite Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Cabrini, Marcela N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Casabuono, Adriana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Moyano, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Beltramino, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Moreira, Leandro M.. Universidade Federal de Ouro Preto; Brasil Fil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Made available in DSpace on 2020-12-12T01:43:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Fundo de Defesa da Citricultura Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Background. In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas. Results. Using comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/µl in vitro and 1.5 × 104 CFU ml−1 in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study. Conclusions. We demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism. Núcleo de Pesquisas em Ciências Biológicas Universidade Federal de Ouro Preto Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Universidade Estadual Paulista-Unesp Departamento de Fitopatologia e Nematologia Escola Superior de Agricultura ‘‘Luiz de Queiroz’’ Departamento de Bioquímica Instituto de Química Universidade de São Paulo Instituto de Computação Universidade Estadual de Campinas Faculdade de Computação Universidade Federal de Mato Grosso do Sul Departamento de Ciências Biológicas Universidade Federal de Ouro Preto Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Universidade Estadual Paulista-Unesp Fundo de Defesa da Citricultura: 007/2015 SIAFEM 025139 CAPES: 3385/2013 CNPq: 481226/2013-3 FAPEMIG: APQ-02387-14 CAPES: CFP 51/2013

Made available in DSpace on 2020-12-10T19:41:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fund for Citrus Plant Protection (FUNDECITRUS) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Background: Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results: Under infective conditions, 446 genes were up-and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up-and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions: Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac. (C) 2019 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved. Univ Fed Vales do Jequitirthonha e Mucuri, Fac Ciencias Agr, Dept Engn Florestal, Diamantina, MG, Brazil Univ Fed Ouro Preto, Inst Ciencias Exatas & Biol, Dept Ciencias Biol, Ouro Preto, MG, Brazil Univ Fed Ouro Preto, Ouro Preto, Nucleo Pesquisas Ciencias Biol, Ouro Preto, MG, Brazil Univ Fed Vales do Jequitirthonha e Mucuri, Inst Ciencias Agr, Unai, MG, Brazil Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, Campus Jaboticabal, Jaboticabal, SP, Brazil Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, Campus Jaboticabal, Jaboticabal, SP, Brazil FAPESP: 02/13862-6 FAPEMIG: CBB-APQ-04425-10 CAPES: 001

The genome of Xanthomonas citri subsp. Citri strain 306 pathotype A (Xac) was completely sequenced more than 10 years; to date, few studies involving functional genomics Xac and its host compatible have been developed, specially related to adaptive events that allow the survival of Xac within the plant. Proteomic analysis of Xac showed that the processes of chemotactic signal transduction and phosphate metabolism are key adaptive strategies during the interaction of a pathogenic bacterium with its plant host. The results also indicate the importance of a group of proteins that may not be directly related to the classical virulence factors, but that are likely fundamental to the success of the initial stages of the infection, such as methyl-accepting chemotaxis protein (Mcp) and phosphate specific transport (Pst). Furthermore, the analysis of the mutant of the gene pstB which codifies to an ABC phosphate transporter subunit revealed a complete absence of citrus canker symptoms when inoculated in compatible hosts. We also conducted an in silico analysis which established the possible network of genes regulated by two-component systems PhoPQ and PhoBR (related to phosphate metabolism), and possible transcriptional factor binding site (TFBS) motifs of regulatory proteins PhoB and PhoP, detaching high degree of conservation of PhoB TFBS in 84 genes of Xac genome. This is the first time that chemotaxis signal transduction and phosphate metabolism were therefore indicated to be fundamental to the process of colonization of plant tissue during the induction of disease associated with Xanthomonas genus bacteria.

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo Fundacao de Amparo a Pesquisa do Estado de Minas Gerais BIGA grant-CAPES Background: Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. A proteomic analysis under in planta infectious and non-infectious conditions was conducted in order to increase our knowledge about the adaptive process of Xac during infection. Results: For that, a 2D-based proteomic analysis of Xac at 1, 3 and 5 days after inoculation, in comparison to Xac growth in NB media was carried out and followed by MALDI-TOF-TOF identification of 124 unique differentially abundant proteins. Among them, 79 correspond to up-regulated proteins in at least one of the three stages of infection. Our results indicate an important role of proteins related to biofilm synthesis, lipopolysaccharides biosynthesis, and iron uptake and metabolism as possible modulators of plant innate immunity, and revealed an intricate network of proteins involved in reactive oxygen species adaptation during Plants'Oxidative Burst response. We also identified proteins previously unknown to be involved in Xac-Citrus interaction, including the hypothetical protein XAC3981. A mutant strain for this gene has proved to be non-pathogenic in respect to classical symptoms of citrus canker induced in compatible plants. Conclusions: This is the first time that a protein repertoire is shown to be active and working in an integrated manner during the infection process in a compatible host, pointing to an elaborate mechanism for adaptation of Xac once inside the plant. Univ Fed Ouro Preto, Inst Ciencias Exatas & Biol, Dept Ciencias Biol DECBI, Ouro Preto, MG, Brazil Univ Fed Ouro Preto, Nucleo Pesquisas Ciencias Biol NUPEB, Ouro Preto, MG, Brazil Univ Fed Rio de Janeiro, Inst Quim, Dept Bioquim DBq, Rio De Janeiro, RJ, Brazil Univ Estadual Paulista, UNESP, Dept Tecnol, Fac Ciencias Agr & Vet Jaboticabal, Jaboticabal, SP, Brazil Univ Estadual Campinas, UNICAMP, Inst Quim, Campinas, SP, Brazil Univ Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, SP, Brazil Univ Fed Sao Paulo UNIFESP, Dept Ciencias Biol, Diadema, SP, Brazil Virginia Tech, Biocomplex Inst, Blacksburg, VA USA Univ Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, SP, Brazil Univ Fed Sao Paulo UNIFESP, Dept Ciencias Biol, Diadema, SP, Brazil CAPES FAPESP: 04/02006-7 Fundacao de Amparo a Pesquisa do Estado de Minas Gerais: CBB-APQ-04425-10 BIGA grant-CAPES: 3385/2013 Web of Science

The Xanthomonadaceae family consists of species of non-pathogenic and pathogenic γ-proteobacteria that infect different hosts, including humans and plants. In this study, we performed a comparative analysis using 69 fully sequenced genomes belonging to this family, with a focus on identifying proteins enriched in phytopathogens that could explain the lifestyle and the ability to infect plants. Using a computational approach, we identified seven phytopathogen-enriched protein families putatively secreted by type II secretory system: PheA (CM-sec), LipA/LesA, VirK, and four families involved in N-glycan degradation, NixE, NixF, NixL, and FucA1. In silico and phylogenetic analyses of these protein families revealed they all have orthologs in other phytopathogenic or symbiotic bacteria, and are involved in the modulation and evasion of the immune system. As a proof of concept, we performed a biochemical characterization of LipA from Xac306 and verified that the mutant strain lost most of its lipase and esterase activities and displayed reduced virulence in citrus. Since this study includes closely related organisms with distinct lifestyles and highlights proteins directly related to adaptation inside plant tissues, novel approaches might use these proteins as biotechnological targets for disease control, and contribute to our understanding of the coevolution of plant-associated bacteria.

Made available in DSpace on 2018-11-26T16:27:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-02-23 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Citrus canker is a major disease affecting citrus production in Brazil. It's mainly caused by Xanthomonas citri subsp. citri strain 306 pathotype A (Xac). We analysed the differential expression of proteins secreted by wild type Xac and an asymptomatic mutant for hrpB4 (Delta hrpB4) grown in Nutrient Broth (NB) and a medium mimicking growth conditions in the plant (XAM1). This allowed the identification of 55 secreted proteins, of which 37 were secreted by both strains when cultured in XAMI. In this secreted protein repertoire, the following stand out: Virk, Polyphosphate-selective porin, Cellulase, Endoglucanase, Histone-like protein, Ribosomal proteins, five hypothetical proteins expressed only in the wild type strain, Lytic murein transglycosylase, Lipoprotein, Leucyl-tRNA synthetase, Co-chaperonin, Toluene tolerance, C-type cytochrome biogenesis membrane protein, Aminopeptidase and two hypothetical proteins expressed only in the Delta hrpB4 mutant. Furthermore, Peptidoglycan-associated outer membrane protein, Regulator of pathogenicity factor, Outer membrane proteins, Endopolygalacturonase, Chorismate mutase, Peptidyl-prolyl cis-trans isornerase land seven hypothetical proteins were detected in both strains, suggesting that there was no relationship with the secretion mediated by the type III secretory system, which is not functional in the mutant strain. Also worth mentioning is the Elongation factor Tu (EF-Tu), expressed only the wild type strain, and Type IV pilus assembly protein, Flagellin (FliC) and Flagellar hook-associated protein, identified in the wild-type strain secretome when grown only in NB. Noteworthy, that FliC, EF-Tu are classically characterized as PAMPs (Pathogen-associated molecular patterns), responsible for a PAMP-triggered immunity response. Therefore, our results highlight proteins potentially involved with the virulence. Overall, we conclude that the use of secretome data is a valuable approach that may bring more knowledge of the biology of this important plant pathogen, which ultimately can lead to the establishment of new strategies to combat citrus canker. Univ Estadual Paulista, Dept Tecnol, Jaboticabal, SP, Brazil Univ Fed Ouro Preto, Dept Ciencias Biol, Nucleo Pesquisas Ciencias Biol NUPEB, Ouro Preto, MG, Brazil Univ Fed Rio de Janeiro, Dept Bioquim, Rio De Janeiro, Rio De Janeiro, Brazil Univ Fed Vales Jequitinhonha & Mucuri, Dept Engn Florestal, Diamantina, MG, Brazil Univ Fed Sao Paulo, Dept Ciencias Biol, Diadema, SP, Brazil Univ Estadual Paulista, Dept Tecnol, Jaboticabal, SP, Brazil

The bacteria Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. The disease symptoms are characterized by localized host cell hyperplasia followed by tissue necrosis at the infected area. An arsenal of bacterial pathogenicity- and virulence-related proteins is expressed to ensure a successful infection process. At the post-genomic stage of Xac, we used a proteomic approach to analyze the proteins that are displayed differentially over time when the pathogen attacks the host plant. Protein extracts were prepared from infectious Xac grown in inducing medium (XAM1) for 24 h or from host citrus plants for 3 or 5 days after infection, detached times to evaluate the adaptation and virulence of the pathogen. The protein extracts were proteolyzed, and the peptides derived from tryptic digestion were investigated using liquid chromatography and tandem mass spectrometry. Changes in the protein expression profile were compared with the Xac genome and the proteome recently described under non-infectious conditions. An analysis of the proteome of Xac under infectious conditions revealed proteins directly involved in virulence such as the type III secretion system (T3SS) and effector proteins (T3SS-e), the type IV pilus (Tfp), and xanthan gum biosynthesis. Moreover, four new mutants related to proteins detected in the proteome and with different functions exhibited reduced virulence relative to the wild-type proteins. The results of the proteome analysis of infectious Xac define the processes of adaptation to the host and demonstrate the induction of the virulence factors of Xac involved in plant-pathogen interactions.

Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis ( Xam ), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies.

Made available in DSpace on 2013-08-28T14:10:23Z (GMT). No. of bitstreams: 1 WOS000279860700001.pdf: 3485552 bytes, checksum: 6658bf0186a402df6a667de3557552a4 (MD5) Made available in DSpace on 2013-09-30T18:27:02Z (GMT). No. of bitstreams: 2 WOS000279860700001.pdf: 3485552 bytes, checksum: 6658bf0186a402df6a667de3557552a4 (MD5) WOS000279860700001.pdf.txt: 108690 bytes, checksum: 4e4070122fbef38b47035beace12b14d (MD5) Previous issue date: 2010-04-13 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:40:29Z No. of bitstreams: 2 WOS000279860700001.pdf: 3485552 bytes, checksum: 6658bf0186a402df6a667de3557552a4 (MD5) WOS000279860700001.pdf.txt: 108690 bytes, checksum: 4e4070122fbef38b47035beace12b14d (MD5) Made available in DSpace on 2014-05-20T13:40:29Z (GMT). No. of bitstreams: 2 WOS000279860700001.pdf: 3485552 bytes, checksum: 6658bf0186a402df6a667de3557552a4 (MD5) WOS000279860700001.pdf.txt: 108690 bytes, checksum: 4e4070122fbef38b47035beace12b14d (MD5) Previous issue date: 2010-04-13 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) FUNDECITRUS Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control. Virginia Polytech Inst & State Univ, Virginia Bioinformat Inst, Blacksburg, VA 24061 USA Univ Fed Ouro Preto, Inst Ciencias Exatas & Biol, Dept Ciencias Biol, Ouro Preto, MG, Brazil Univ São Paulo, Inst Quim, Dept Bioquim, BR-01498 São Paulo, Brazil Univ Florida, Dept Plant Pathol, Gainesville, FL 32611 USA Virginia Polytech Inst & State Univ, Dept Plant Pathol Physiol & Weed Sci, Blacksburg, VA 24061 USA Univ Estadual Paulista, Fac Ciencias Agr & Vet Jaboticabal, Dept Tecnol, Jaboticabal, SP, Brazil Univ Fed São Paulo, Dept Ciencias Biol, São Paulo, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia Botucatu, Dept Melhoramento & Nutr Anim, Botucatu, SP, Brazil Univ Estado Santa Catarina, Ctr Ciencias Agrovet, Dept Engn Florestal, Lages, SC, Brazil Allelyx Appl Genom, Campinas, SP, Brazil Univ São Paulo, Escola Super Agr Luiz de Queiroz, Nucleo Apoio Pesquisa Microscopia Eletron Aplicad, Piracicaba, SP, Brazil Inst Biol Campinas, Lab Bacteriol Vegetal, Campinas, SP, Brazil Univ São Paulo, Escola Artes Ciencias & Humanidades, São Paulo, Brazil Univ Fed Mato Grosso do Sul, Fac Computacao, Campo Grande, MS, Brazil Univ Estadual Campinas, Inst Computacao, Lab Bioinformat, Campinas, SP, Brazil Virginia Polytech Inst & State Univ, Dept Comp Sci, Blacksburg, VA 24061 USA Inst Agron Parana, Londrina, Parana, Brazil Univ Florida, Inst Food & Agr Sci, Mid Florida Res & Educ Ctr, Gainesville, FL 32611 USA Univ Calif Berkeley, Dept Plant & Microbial Biol, Berkeley, CA 94720 USA Univ Estadual Paulista, Fac Ciencias Agr & Vet Jaboticabal, Dept Tecnol, Jaboticabal, SP, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia Botucatu, Dept Melhoramento & Nutr Anim, Botucatu, SP, Brazil

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa , constituting an important step towards understanding the molecular basis of the disease.

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

Made available in DSpace on 2013-08-28T14:10:11Z (GMT). No. of bitstreams: 1 WOS000264158500001.pdf: 2073598 bytes, checksum: 1b34ea00cdecd061a7e6523efc9fda61 (MD5) Made available in DSpace on 2013-09-30T18:00:21Z (GMT). No. of bitstreams: 2 WOS000264158500001.pdf: 2073598 bytes, checksum: 1b34ea00cdecd061a7e6523efc9fda61 (MD5) WOS000264158500001.pdf.txt: 78494 bytes, checksum: 67bc62d4f386ee061ca41da6854d57ee (MD5) Previous issue date: 2009-01-16 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:17:43Z No. of bitstreams: 2 WOS000264158500001.pdf: 2073598 bytes, checksum: 1b34ea00cdecd061a7e6523efc9fda61 (MD5) WOS000264158500001.pdf.txt: 78494 bytes, checksum: 67bc62d4f386ee061ca41da6854d57ee (MD5) Made available in DSpace on 2014-05-20T13:17:43Z (GMT). No. of bitstreams: 2 WOS000264158500001.pdf: 2073598 bytes, checksum: 1b34ea00cdecd061a7e6523efc9fda61 (MD5) WOS000264158500001.pdf.txt: 78494 bytes, checksum: 67bc62d4f386ee061ca41da6854d57ee (MD5) Previous issue date: 2009-01-16 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundo de Defesa da Citricultura (FUNDECITRUS) Background: Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.Results: Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands.Conclusion: The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker. Univ Estadual Paulista, UNESP, Dept Technol, BR-14884900 Jaboticabal, SP, Brazil Univ São Paulo, Inst Quim, Dept Bioquim, BR-05509900 São Paulo, Brazil Alellyx Appl Genom, BR-13069380 Campinas, SP, Brazil Univ Fed São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, Brazil Univ Estadual Paulista, UNESP, Dept Technol, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 04/02006-7