Browsing by Author "Zaini, Paulo Adriano"

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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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    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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    Genome-wide profiling and phylogenetic analysis of the SWEET sugar transporter gene family in walnut and their lack of responsiveness to Xanthomonas arboricola pv. juglandis infection.
    (2020) Jiang, Shijiao; Balan, Bipin; Assis, Renata de Almeida Barbosa; Sagawa, Cintia Helena Duarte; Wan, Xueqin; Han, Shan; Wang, Le; Zhang, Lanlan; Zaini, Paulo Adriano; Walawage, Sriema Lalani; Jacobson, Aaron; Lee, Steven H.; Moreira, Leandro Marcio; Leslie, Charles A.; Dandekar, Abhaya M.
    Following photosynthesis, sucrose is translocated to sink organs, where it provides the primary source of carbon and energy to sustain plant growth and development. Sugar transporters from the SWEET (sugar will eventually be exported transporter) family are rate-limiting factors that mediate sucrose transport across concentration gradients, sustain yields, and participate in reproductive development, plant senescence, stress responses, as well as support plant–pathogen interaction, the focus of this study. We identified 25 SWEET genes in the walnut genome and distinguished each by its individual gene structure and pattern of expression in different walnut tissues. Their chromosomal locations, cis-acting motifs within their 50 regulatory elements, and phylogenetic relationship patterns provided the first comprehensive analysis of the SWEET gene family of sugar transporters in walnut. This family is divided into four clades, the analysis of which suggests duplication and expansion of the SWEET gene family in Juglans regia. In addition, tissue-specific gene expression signatures suggest diverse possible functions for JrSWEET genes. Although these are commonly used by pathogens to harness sugar products from their plant hosts, little was known about their role during Xanthomonas arboricola pv. juglandis (Xaj) infection. We monitored the expression profiles of the JrSWEET genes in different tissues of “Chandler” walnuts when challenged with pathogen Xaj417 and concluded that SWEET-mediated sugar translocation from the host is not a trigger for walnut blight disease development. This may be directly related to the absence of type III secretion system-dependent transcription activator-like effectors (TALEs) in Xaj417, which suggests different strategies are employed by this pathogen to promote susceptibility to this major aboveground disease of walnuts.
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    Identification and analysis of seven effector protein families with different adaptive and evolutionary histories in plant-associated members of the Xanthomonadaceae.
    (2017) Assis, Renata de Almeida Barbosa; Polloni, Lorraine Cristina; Patané, José Salvatore Leister; Thakur, Shalabh; Felestrino, Érica Barbosa; Diaz Caballero, Julio; Digiampietri, Luciano Antonio; Goulart Filho, Luiz Ricardo; Almeida Junior, Nalvo Franco de; Nascimento, Rafael; Dandekar, Abhaya M.; Zaini, Paulo Adriano; Setubal, João Carlos; Guttman, David S.; Moreira, Leandro Marcio
    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.
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    Proteome analysis of walnut bacterial blight disease.
    (2020) Sagawa, Cintia Helena Duarte; Assis, Renata de Almeida Barbosa; Zaini, Paulo Adriano; Wilmarth, Phillip A.; Phinney, Brett S.; Moreira, Leandro Marcio; Dandekar, Abhaya M.
    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.
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    A secreted chorismate mutase from Xanthomonas arboricola pv. juglandis attenuates virulence and walnut blight symptoms.
    (2021) Assis, Renata de Almeida Barbosa; Sagawa, Cintia Helena Duarte; Zaini, Paulo Adriano; Saxe, Houston J.; Wilmarth, Phillip A.; Phinney, Brett S.; Salemi, Michelle; Moreira, Leandro Marcio; Dandekar, Abhaya M.
    Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.