Browsing by Author "Laia, Marcelo Luiz de"

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    Bactérias cultiváveis obtidas a partir de Langsdorffia hypogaea como potenciais controladores biológicos de fungos saprofíticos associados com deterioração de tomate pós-colheita.
    (2021) Silva, Ana Karla da; Moreira, Leandro Marcio; Sanchez, Angelica Bianchini; Moreira, Leandro Marcio; Garcia, Camila Carrião Machado; Laia, Marcelo Luiz de
    Grandes quantidades de frutos e vegetais são perdidos por consequência da ação de fungos que os deterioram na fase de pós-colheita. Fatores como cultivo inapropriado, vasto uso de agrotóxicos, armazenamento, manuseio, transporte inadequados e grandes variações de temperatura podem favorecer o aparecimento e a propagação de doenças fúngicas alavancando a fase de deterioração. O tomate, por exemplo, por ser uma hortaliça de alta perecividade, pode incorrer em perdas de até metade da produção na fase pós-colheita. Atualmente, a forma mais comumente utilizada para controle destes microrganismos está na aplicação de fungicidas sintéticos (agrotóxicos), porém, com o agravo de seu uso ser altamente prejudicial à saúde. Diante desse cenário, o controle biológico surge como uma alternativa limpa de controlar microrganismos indesejáveis, por meio da utilização de seus próprios inimigos naturais. Neste trabalho, propomos o controle de espécies fúngicas causadoras de doenças pós-colheita e deterioração de tomates, por meio da utilização de isolados bacterianos cultiváveis obtidos previamente a partir de rizosfera e raízes de Langsdorffia hypogaea, uma planta holoparasita obrigatória. Um total de 36 isolados foram diretamente confrontados com quatro espécies fúngicas bioprospectadas diretamente de tomates, nomeadas como F8", "F11", "F7.2" e "F12". Além disso, os isolados de melhor eficiência nesta etapa, foram posteriormente avaliados quanto a capacidade de autoagregação e produção de biofilme, características fundamentais para fixação em tecido vegetal. Deste total, 33 isolados foram capazes de inibir in vitro o crescimento de F8 com taxas de até 61,27%, enquanto que, para o fungo F11, os 36 isolados apresentaram taxas de inibição positiva, sendo a maior delas 76,81%. O fungo F7.2 se demonstrou o mais difícil de ser contido, 33 isolados bacterianos foram capazes de inibi-lo e a maior taxa alcançada foi 56,04%, entretanto, diferente dos demais, apenas um isolado bacteriano apresentou taxa de inibição superior a 50%. Finalmente, todos os 36 isolados bacterianos foram capazes de inibir o fungo F12 alcançando a maior faixa de inibição, com valores de até 93,83%. Dos 36 isolados testados, 14 melhores passaram para a próxima fase, dentre estes, 8 isolados foram capazes de se autoagregar com taxas baixas de até 20,1%. Os 14 isolados bacterianos, foram capazes de produzir biofilme, 7 deles se enquadraram como produtores moderados, enquanto os 7 demais foram classificados como fortes produtores. Concluímos que bactérias provenientes do solo do QF e da planta Langsdorffia hypogaea são potenciais agentes biocontroladores de fungos deterioradores, entretanto, serão necessários estudos mais aprofundados para verificar a atividade diretamente no fruto.
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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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    Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri.
    (2019) Laia, Marcelo Luiz de; Moreira, Leandro Marcio; Gonçalves, Janaína Fernandes; Ferro, Maria Inês Tiraboschi; Rodrigues, Any Caroliny Pinto; Santos, Jéssica Naiara dos; Felestrino, Érica Barbosa; Ferro, Jesus Aparecido
    Background: Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results: Under infective conditions, 446 genes were up- and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up- and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions: Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac.
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    Identificação de proteínas exclusivas de fitopatógenos da família Xanthomonadaceae : uso de genômica comparativa para identificação de novos alvos de combate.
    (2016) Assis, Renata de Almeida Barbosa; Moreira, Leandro Marcio; Guttman, David S.; Moreira, Leandro Marcio; Garcia, Camila Carrião Machado; Laia, Marcelo Luiz de
    A família Xanthomonadaceae compreende espécies diferentes de proteobactérias não patogênicas e patogênicas que infectam diferentes hospedeiros, incluindo humanos e plantas. Nesse estudo, foi realizado uma análise comparativa usando o genoma completo de 69 cepas bacterianas pertencentes à família Xanthomonadaceae com foco na identificação de famílias de proteínas exclusivas de fitopatógenos que poderiam justificar o estilo de vida e a capacidade desses microrganismos infectarem hospedeiros compatíveis. Foram identificadas sete famílias de proteínas fitopatógeno-específicas, todas supostamente secretadas pelo sistema secretório tipo II: PheA (CMs), LipA/LesA, VirK, e quatro famílias de proteínas envolvidas na degradação de N-glicanos, NixE, NixF, NixL e FucA1. Análises filogenéticas e in silico dessas famílias de proteínas revelaram que todas elas possuem ortólogos em outras bactérias simbióticas ou patogênicas de plantas e estão envolvidas na modulação e evasão do sistema imune. Uma vez que esse estudo inclui microrganismos estreitamente relacionados, com estilos de vida diferenciados e evidencia proteínas diretamente relacionadas com adaptação dentro dos tecidos vegetais, abordagens inovadoras podem ser implementadas visando não somente a utilização destas proteínas como alvos biotecnológicos para o controle da doença, mas também contribuindo para a nossa compreensão da coevolução de bactérias associadas a plantas.
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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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    Type IV secretion system is not involved in infection process in citrus.
    (2014) Jacob, Tiago Rinaldi; Laia, Marcelo Luiz de; Moreira, Leandro Marcio; Gonçalves, Janaína Fernandes; Carvalho, Flavia Maria de Souza; Ferro, Maria Inês Tiraboschi; Ferro, Jesus Aparecido
    The type IV secretion system (T4SS) is used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Xanthomonas citri subsp. citri contains two copies of the T4SS, one in the chromosome and the other is plasmid-encoded. To understand the conditions that induce expression of the T4SS in Xcc, we analyzed, in vitro and in planta, the expression of 18 ORFs from the T4SS and 7 hypothetical flanking genes by RT-qPCR. As a positive control, we also evaluated the expression of 29 ORFs from the type III secretion system (T3SS), since these genes are known to be expressed during plant infection condition, but not necessarily in standard culture medium. From the 29 T3SS genes analyzed by qPCR, only hrpA was downregulated at 72 h after inoculation. All genes associated with the T4SS were downregulated on Citrus leaves 72 h after inoculation. Our results showed that unlike the T3SS, the T4SS is not induced during the infection process.
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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.