Browsing by Author "Varani, Alessandro de Mello"

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    Analyses of seven new genomes of Xanthomonas citri pv. aurantifolii strains, causative agents of Citrus Canker B and C, show a reduced repertoire of pathogenicity-related genes.
    (2019) Fonseca, Natasha Peixoto; Patané, José Salvatore Leister; Varani, Alessandro de Mello; Felestrino, Érica Barbosa; Caneschi, Washington Luiz; Sanchez, Angelica Bianchini; Assis, Renata de Almeida Barbosa; Garcia, Camila Carrião Machado; Belasque Junior, José; Martins Junior, Joaquim; Facincani, Agda Paula
    Xanthomonas citri pv. aurantifolii pathotype B (XauB) and pathotype C (XauC) are the causative agents respectively of citrus canker B and C, diseases of citrus plants related to the better-known citrus canker A, caused by Xanthomonas citri pv. citri. The study of the genomes of strains of these related bacterial species has the potential to bring new understanding to the molecular basis of citrus canker as well as their evolutionary history. Up to now only one genome sequence of XauB and only one genome sequence of XauC have been available, both in draft status. Here we present two new genome sequences of XauB (both complete) and five new genome sequences of XauC (two complete). A phylogenomic analysis of these seven genome sequences along with 24 other related Xanthomonas genomes showed that there are two distinct and wellsupported major clades, the XauB and XauC clade and the Xanthomonas citri pv. citri clade. An analysis of 62 Type III Secretion System effector genes showed that there are 42 effectors with variable presence/absence or pseudogene status among the 31 genomes analyzed. A comparative analysis of secretion-system and surfacestructure genes showed that the XauB and XauC genomes lack several key genes in pathogenicity-related subsystems. These subsystems, the Types I and IV Secretion Systems, and the Type IV pilus, therefore emerge as important ones in helping explain the aggressiveness of the A type of citrus canker and the apparent dominance in the field of the corresponding strain over the B and C strains.
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    A comparative genomic analysis of Xanthomonas arboricola pv. juglandis strains reveal hallmarks of mobile genetic elements in the adaptation and accelerated evolution of virulence.
    (2021) Assis, Renata de Almeida Barbosa; Varani, Alessandro de Mello; Sagawa, Cintia Helena Duarte; Patané, José Salvatore Leister; Setubal, João Carlos; Uceda-Campos, Guillermo; Silva, Aline Maria da; Zaini, Paulo Adriano; Almeida Junior, Nalvo Franco de; Moreira, Leandro Marcio; Dandekar, Abhaya M.
    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 reper- toire 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.
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    Complete genome sequence and analysis of Alcaligenes faecalis strain Mc250, a new potential plant bioinoculant.
    (2020) Felestrino, Érica Barbosa; Sanchez, Angelica Bianchini; Caneschi, Washington Luiz; Lemes, Camila Gracyelle de Carvalho; Assis, Renata de Almeida Barbosa; Cordeiro, Isabella Ferreira; Fonseca, Natasha Peixoto; Villa, Morghana Marina; Vieira, Izadora Tabuso; Kamino, Luciana Hiromi Yoshino; Carmo, Flávio Fonseca do; Silva, Aline Maria da; Thomas, Andrew Maltez; Patané, José Salvatore Leister; Ferreira, Fernanda Carla; Freitas, Leandro Grassi de; Varani, Alessandro de Mello; Ferro, Jesus Aparecido; Silva, Robson Soares; Almeida Junior, Nalvo Franco de; Garcia, Camila Carrião Machado; Setubal, João Carlos; Moreira, Leandro Marcio
    Here we present and analyze the complete genome of Alcaligenes faecalis strain Mc250 (Mc250), a bacterium isolated from the roots of Mimosa calodendron, an endemic plant growing in ferruginous rupestrian grasslands in Minas Gerais State, Brazil. The genome has 4,159,911 bp and 3,719 predicted protein-coding genes, in a single chromosome. Comparison of the Mc250 genome with 36 other Alcaligenes faecalis genomes revealed that there is considerable gene content variation among these strains, with the core genome representing only 39% of the protein-coding gene repertoire of Mc250. Mc250 encodes a complete denitrification pathway, a network of pathways associated with phenolic compounds degradation, and genes associated with HCN and siderophores synthesis; we also found a repertoire of genes associated with metal internalization and metabolism, sulfate/sulfonate and cysteine metabolism, oxidative stress and DNA repair. These findings reveal the genomic basis for the adaptation of this bacterium to the harsh environmental conditions from where it was isolated. Gene clusters associated with ectoine, terpene, resorcinol, and emulsan biosynthesis that can confer some competitive advantage were also found. Experimental results showed that Mc250 was able to reduce (~60%) the virulence phenotype of the plant pathogen Xanthomonas citri subsp. citri when co-inoculated in Citrus sinensis, and was able to eradicate 98% of juveniles and stabilize the hatching rate of eggs to 4% in two species of agricultural nematodes. These results reveal biotechnological potential for the Mc250 strain and warrant its further investigation as a biocontrol and plant growth-promoting bacterium.
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    Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.
    (2019) Fonseca, Natasha Peixoto; Felestrino, Érica Barbosa; Caneschi, Washington Luiz; Sanchez, Angelica Bianchini; Cordeiro, Isabella Ferreira; Lemes, Camila Gracyelle de Carvalho; Assis, Renata de Almeida Barbosa; Carvalho, Flávia Maria de Souza; Ferro, Jesus Aparecido; Varani, Alessandro de Mello; Belasque Junior, José; Setubal, João Carlos; Telles, Guilherme Pimentel; Aguena, Deiviston da Silva; Almeida Junior, Nalvo Franco de; Moreira, Leandro Marcio
    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.
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    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 Carlos
    Xanthomonas 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.
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    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 Marcio
    Serratia 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.
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    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 Mello
    Members 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.
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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.