Browsing by Author "Oliveira, Julio Cezar Franco de"

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    Comparative proteomic analysis reveals that T3SS, Tfp, and xanthan gum are key factors in initial stages of Citrus sinensis infection by Xanthomonas citri subsp. citri.
    (2013) Facincani, Agda Paula; Moreira, Leandro Marcio; Soares, Márcia Regina; Ferreira, Cristiano Barbalho; Ferreira, Rafael Marini; Ferro, Maria Inês Tiraboschi; Ferro, Jesus Aparecido; Gozzo, Fabio Cesar; Oliveira, Julio Cezar Franco de
    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.
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    Identification of new genes related to virulence of xanthomonas axonopodis pv. citri during citrus host interactions.
    (2017) Ferreira, Cristiano Barbalho; Moreira, Leandro Marcio; Brigati, Joice Bissoloti; Lima, Lonjoré Leocádio de; Ferro, Jesus Aparecido; Ferro, Maria Inês Tiraboschi; Oliveira, Julio Cezar Franco de
    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.
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    New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library.
    (2009) Laia, Marcelo Luiz de; Moreira, Leandro Marcio; Dezajacomo, Juliana; Brigati, Joice Bissoloti; Ferreira, Cristiano Barbalho; Ferro, Maria Inês Tiraboschi; Silva, Ana Cristina Simões e; Ferro, Jesus Aparecido; Oliveira, Julio Cezar Franco de
    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.
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    Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii.
    (2010) Moreira, Leandro Marcio; Almeida Junior, Nalvo Franco de; Potnis, Neha; Digiampietri, Luciano Antonio; Adi, Said Sadique; Bortolossi, Julio Cesar; Silva, Ana C.; Silva, Aline M. da; Moraes, Fabrício E. de; Oliveira, Julio Cezar Franco de; Souza, Robson Francisco de; Facincani, Agda Paula; Ferraz, André L.; Ferro, Maria Inês Tiraboschi; Furlan, Luiz Roberto; Gimenez, Daniele F.; Jones, Jeffrey B.; Kitajima, Elliot Watanabe; Laia, Marcelo Luiz de; Leite Junior, Rui P.; Nishyama, Milton Yutaka; Rodrigues Neto, Julio; Nociti, Letícia A.; Norman, David J.; Ostroski, Eric Hainer; Pereira Junior, Haroldo Alves; Staskawicz, Brian J.; Tezza, Renata Izabel; Ferro, Jesus Aparecido; Vinatzer, Boris A.; Setubal, João Carlos
    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.
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    Proposal for the inclusion of the factor “production of electricity by SHPs” in the “ecological sales tax” distribution criteria in the state of Minas Gerais.
    (2013) Oliveira, Julio Cezar Franco de; Oliveira, Delly; Martins, José Helvecio; Monteiro, Paulo Marcos de Barros; Moraes, Maria Joselma de; Altoé, Leandra
    Vários mecanismos legais têm sido criados para compensar territórios que promovam ações ambientais. Este trabalho foi desenvolvido com o objetivo de analisar os impactos da inserção do critério “produção de energia elétrica por Pequenas Centrais Hidrelétricas” na distribuição do ICMS Ecológico no Estado de Minas Gerais. Foi realizada uma simulação para verificar o impacto da adoção deste critério para a região de abrangência da Associação dos Municípios da Microrregião do Vale do Rio Piranga. O município de Diogo de Vasconcelos, por exemplo, poderia arrecadar R$ 199.411,00 por ano com a inclusão do critério “produção de energia elétrica por Pequenas Centrais Hidrelétricas” na legislação estadual. O recurso poderia ser aplicado em ações socioambientais, como plantio de áreas verdes, construção de escolas e investimentos em saúde. Portanto, recomenda-se a inclusão do critério analisado neste trabalho na Lei de ICMS Ecológico no Estado de Minas Gerais.
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    Proteome of the phytopathogen Xanthomonas citri subsp. citri : a global expression profile.
    (2010) Soares, Márcia Regina; Fancicani, Agda Paula; Ferreira, Rafael Marini; Moreira, Leandro Marcio; Oliveira, Julio Cezar Franco de; Ferro, Jesus Aparecido; Ferro, Maria Inês Tiraboschi; Meneghini, Rogério; Gozzo, Fabio Cesar
    Background: Citrus canker is a disease caused by Xantomonas citri subsp.citri (Xac), and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of Xac was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS). Results: In order to gain insight into the metabolism of Xac, cells were grown on two different media (NB - Nutrient Broth and TSE - Tryptone Sucrose broth enriched with glutamic acid), and proteins were proteolyzed with trypsin and examined by 2D LC-MS/MS. Approximately 39% of all predicted proteins by annotation of Xac were identified with their component peptides unambiguously assigned to tandem mass spectra. The proteins, about 1,100, were distributed in all annotated functional categories. Conclusions: This is the first proteomic reference map for the most aggressive strain of Xanthomonas pathogen of all orange varieties. The compilation of metabolic pathways involved with bacterial growth showed that Xac expresses a complete central and intermediary metabolism, replication, transcription and translation machineries and regulation factors, distinct membrane transporters (ABC, MFS and pumps) and receptors (MCP, TonB dependent and metabolites acquisition), two-component systems (sensor and regulatory components) and response regulators. These data corroborate the growth curve in vitro and are the first reports indicating that many of these genome annotated genes are translated into operative in Xac. This proteomic analysis also provided information regarding the influence of culture medium on growth and protein expression of Xac.
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    Proteomics-based identification of differentially abundant proteins reveals adaptation mechanisms of Xanthomonas citri subsp. citri during Citrus sinensis infection.
    (2017) Moreira, Leandro Marcio; Silva, Marcia Regina Soares da; Facincani, Agda Paula; Ferreira, Cristiano Barbalho; Ferreira, Rafael Marini; Ferro, Maria Inês Tiraboschi; Gozzo, Fabio Cesar; Felestrino, Érica Barbosa; Assis, Renata de Almeida Barbosa; Garcia, Camila Carrião Machado; Setubal, João Carlos; Ferro, Jesus Aparecido; Oliveira, Julio Cezar Franco de
    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.
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    Unravelling potential virulence factor candidates in Xanthomonas citri. subsp. citri by secretome analysis.
    (2016) Ferreira, Rafael Marini; Moreira, Leandro Marcio; Ferro, Jesus Aparecido; Silva, Marcia Regina Soares da; Laia, Marcelo Luiz de; Varani, Alessandro de Mello; Oliveira, Julio Cezar Franco de; Ferro, Maria Inês Tiraboschi
    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 (1hrpB4) 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 XAM1. 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 the1hrpB4 mutant. Furthermore, Peptidoglycan-associated outer membrane protein, Regulator of pathogenicity factor, Outer membrane proteins, Endopolygalacturonase, Chorismate mutase, Peptidyl-prolyl cis-trans isomerase and 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.