Browsing by Author "Souza, Robson Francisco de"

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    Development and validation of a Xanthomonas axonopodis pv.citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome.
    (2010) Moreira, Leandro Marcio; Laia, Marcelo Luiz de; Souza, Robson Francisco de; Zaini, Paulo Adriano; Silva, Ana C. R. da; Silva, Aline M. da; Ferro, Jesus Aparecido
    Background: From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings: The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions: Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.
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    Genomics and walnut hull proteomics of Xanthomonas arboricola pv. juglandis 417 for the development of new disease control.
    (2021) Assis, Renata de Almeida Barbosa; Moreira, Leandro Marcio; Dandekar, Abhaya M.; Moreira, Leandro Marcio; Ferro, Jesus Aparecido; Souza, Robson Francisco de; Cruz, Izinara Rosse da; Borges, Wiliam de Castro
    Xanthomonas arboricola pv. juglandis 417 (Xaj417) is the causal agent of walnut bacterial blight, the most significant above-ground disease of walnuts (Juglans regia L.). Walnut producers have registered losses of up to 40% in local production annually. Disease management uses copper-based pesticides which induce pathogen resistance despite being harmful for the environment. Our aim was to evaluate the genome content of the pathogen, dissect the host-pathogen response to define determinants that regulate the host susceptibility and assess the mutation effect of a conserved secreted protein among plant-associated Xanthomonadaceae. Our study focused on Xaj417 to understand the proteo-genomics attributes to colonize its host. We investigated the genome sequence and proteome of this plant pathogen by performing a comparative analysis with other sequenced Xaj and inoculating walnut fruits with Xaj417. The comparison of 32 Xaj genomes revealed that the adaptive evolution generated by intensive spray application to control bacterial diseases possibly led to selection of resistant bacteria and emergence of pathogenic strains (Chapter I). The results revealed that bacterial virulence and copper resistance emerged by the acquisition of specific sets of pathogenesis-related genes commonly transferred among the members of the Xanthomonas genus on mobile genetic elements. This was evidenced for the reference strain Xaj417, a copper-resistant Californian isolate, that acquired a new copper resistance cassette by HGT associated with a new transposon family in Xanthomonas (TnXaj417). The expansion of mobile genetic elements in the most virulent strains influence the repertoire of virulence effectors and adaptation strategies shaping the evolution of pathogenic strains. On Chapter II, we dissected this pathosystem using tandem mass tag quantitative proteomics. This is the first proteome study of this pathosystem examining the molecular responses during the disease development by comparing the proteomes of infected fruit hulls to healthy tissue. Xaj proteins detected in infected tissues demonstrated its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Finally, on Chapter III the secreted monofunctional chorismate mutase mutant (XajCM) was created in Xaj417 and showed increased virulence in walnut nuts. The bacterial morphology was characterized and IX changes in the protein profile of the mutant in planta were tested. The proteomic results suggested intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Overall, this study offers new insights into the emergence of virulence, adaptation, and tolerance to disease management strategies used in orchard ecosystems. It also provides knowledge into molecular mechanisms highlighting potential molecular tools for early detection and disease control strategies.
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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.