Navegando por Autor "Ferreira, Rafael Marini"
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Item 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 deThe 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.Item Origin and diversification of Xanthomonas citri subsp. citri pathotypes revealed by inclusive phylogenomic, dating, and biogeographic analyses.(2019) Patané, José Salvatore Leister; Martins Junior, Joaquim; Rangel, Luiz Thiberio; Belasque Junior, José; Digiampietri, Luciano Antonio; Facincani, Agda Paula; Ferreira, Rafael Marini; Jaciani, Fabrício José; Zhang, Yunzeng; Varani, Alessandro de Mello; Almeida Junior, Nalvo Franco de; Wang, Nian; Ferro, Jesus Aparecido; Moreira, Leandro Marcio; Setubal, João CarlosXanthomonas citri subsp. citri pathotypes cause bacterial citrus canker, being responsible for severe agricultural losses worldwide. The A pathotype has a broad host spectrum, while A* and Aw are more restricted both in hosts and in geography. Two previous phylogenomic studies led to contrasting well-supported clades for sequenced genomes of these pathotypes. No extensive biogeographical or divergence dating analytic approaches have been so far applied to available genomes. Results: Based on a larger sampling of genomes than in previous studies (including six new genomes sequenced by our group, adding to a total of 95 genomes), phylogenomic analyses resulted in different resolutions, though overall indicating that A + AW is the most likely true clade. Our results suggest the high degree of recombination at some branches and the fast diversification of lineages are probable causes for this phylogenetic blurring effect. One of the genomes analyzed, X. campestris pv. durantae, was shown to be an A* strain; this strain has been reported to infect a plant of the family Verbenaceae, though there are no reports of any X. citri subsp. citri pathotypes infecting any plant outside the Citrus genus. Host reconstruction indicated the pathotype ancestor likely had plant hosts in the family Fabaceae, implying an ancient jump to the current Rutaceae hosts. Extensive dating analyses indicated that the origin of X. citri subsp. citri occurred more recently than the main phylogenetic splits of Citrus plants, suggesting dispersion rather than host-directed vicariance as the main driver of geographic expansion. An analysis of 120 pathogenic-related genes revealed pathotype-associated patterns of presence/absence. Conclusions: Our results provide novel insights into the evolutionary history of X. citri subsp. citri as well as a sound phylogenetic foundation for future evolutionary and genomic studies of its pathotypes.Item 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 CesarBackground: 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.Item 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 deBackground: 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.Item Serratia liquefaciens FG3 isolated from a metallophyte plant sheds light on the evolution and mechanisms of adaptive traits in extreme environments.(2019) Caneschi, Washington Luiz; Sanchez, Angelica Bianchini; Felestrino, Érica Barbosa; Lemes, Camila Gracyelle de Carvalho; Cordeiro, Isabella Ferreira; Fonseca, Natasha Peixoto; Villa, Morghana Marina; Vieira, Izadora Tabuso; Moraes, Lauro Ângelo Gonçalves de; Assis, Renata de Almeida Barbosa; Carmo, Flávio Fonseca do; Kamino, Luciana Hiromi Yoshino; Silva, Robson Soares; Ferro, Jesus Aparecido; Ferro, Maria Inês Tiraboschi; Ferreira, Rafael Marini; Santos, Vera Lúcia; Silva, Ubiana de Cássia Mourão; Almeida Junior, Nalvo Franco de; Varani, Alessandro de Mello; Garcia, Camila Carrião Machado; Setubal, João Carlos; Moreira, Leandro MarcioSerratia liquefaciens strain FG3 (SlFG3), isolated from the flower of Stachytarpheta glabra in the Brazilian ferruginous fields, has distinctive genomic, adaptive, and biotechnological potential. Herein, using a combination of genomics and molecular approaches, we unlocked the evolution of the adaptive traits acquired by S1FG3, which exhibits the second largest chromosome containing the largest conjugative plasmids described for Serratia. Comparative analysis revealed the presence of 18 genomic islands and 311 unique protein families involved in distinct adaptive features. S1FG3 has a diversified repertoire of genes associated with Nonribosomal peptides (NRPs/PKS), a complete and functional cluster related to cellulose synthesis, and an extensive and functional repertoire of oxidative metabolism genes. In addition, S1FG3 possesses a complete pathway related to protocatecuate and chloroaromatic degradation, and a complete repertoire of genes related to DNA repair and protection that includes mechanisms related to UV light tolerance, redox process resistance, and a laterally acquired capacity to protect DNA using phosphorothioation. These findings summarize that SlFG3 is well-adapted to different biotic and abiotic stress situations imposed by extreme conditions associated with ferruginous fields, unlocking the impact of the lateral gene transfer to adjust the genome for extreme environments, and providing insight into the evolution of prokaryotes.Item A TALE of transposition : Tn3-like transposons play a major role in the spread of pathogenicity determinants of Xanthomonas citri and other Xanthomonads.(2015) Ferreira, Rafael Marini; Oliveira, Amanda Carolina Paulino de; Moreira, Leandro Marcio; Belasque Junior, José; Gourbeyre, Edith; Siguier, Patricia; Ferro, Maria Inês Tiraboschi; Ferro, Jesus Aparecido; Chandler, Michael; Varani, Alessandro de MelloMembers of the genus Xanthomonas are among the most important phytopathogens. A key feature of Xanthomonas pathogenesis is the translocation of type III secretion system (T3SS) effector proteins (T3SEs) into the plant target cells via a T3SS. Several T3SEs and a murein lytic transglycosylase gene (mlt, required for citrus canker symptoms) are found associated with three transposition-related genes in Xanthomonas citri plasmid pXAC64. These are flanked by short inverted repeats (IRs). The region was identified as a transposon, TnXax1, with typical Tn3 family features, including a transposase and two recombination genes. Two 14-bp palindromic sequences within a 193-bp potential resolution site occur between the recombination genes. Additional derivatives carrying different T3SEs and other passenger genes occur in different Xanthomonas species. The T3SEs include transcription activator-like effectors (TALEs). Certain TALEs are flanked by the same IRs as found in TnXax1 to form mobile insertion cassettes (MICs), suggesting that they may be transmitted horizontally. A significant number of MICs carrying other passenger genes (including a number of TALE genes) were also identified, flanked by the same TnXax1 IRs and delimited by 5-bp target site duplications. We conclude that a large fraction of T3SEs, including individual TALEs and potential pathogenicity determinants, have spread by transposition and that TnXax1, which exhibits all of the essential characteristics of a functional transposon, may be involved in driving MIC transposition. We also propose that TALE genes may diversify by fork slippage during the replicative Tn3 family transposition. These mechanisms may play a crucial role in the emergence of Xanthomonas pathogenicity.Item 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 TiraboschiCitrus 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.