Transcriptomics and systems biology analysis in identification of specific pathways involved in cacao resistance and susceptibility to witches' broom disease

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Utilização de análise de segregantes agrupados na identificação de marcadores ligados a genes que controlam a resistência à ferrugem (Puccinia psidii Winter) em Eucalyptus sp.

Devido a grande importância da cultura de Eucalyptus no Brasil, empresas do setor florestal têm buscado através de programas de melhoramento genético, reduzir as perdas de produção e atender a demanda do mercado de papel e celulose. Um exemplo, é a busca por genes de resistência a doenças, principalmente a ferrugem causada por Puccinia psidii Winter, que resulta em redução da produtividade em plantas altamente suscetíveis. No presente trabalho, mudas de Eucalyptus pertencentes a uma geração F1, provenientes do cruzamento controlado entre parentais híbridos E. grandis X E. urophylla, sendo eles resistente e suscetível, foram inoculadas com Puccinia psidii em casa de vegetação e acompanhadas até o aparecimento dos sintomas da ferrugem. Foram classificadas, em dois grupos: resistentes (ausência de sintomas) e suscetíveis (presença de sintomas e esporulação). As amostras de DNA foram comparadas com o uso de marcadores moleculares associado ao método de BSA (Bulked Segregant Analysis). O polimorfismo entre os grupos foi geneticamente relacionado ao loco que determina a característica de resistência ou sucetibilidade. Dentre os 720 primers testados, 19 foram polimórficos, porém, apenas o marcador AK 01 manteve-se presente, quando testado em todos os indivíduos da população, mostrando-se a uma distância genética estimada de 20 cM em repulsão ao gene de resistência.

Peanut genes identified during initial phase of Cercosporidium personatum infection

Late leaf spot (LLS), caused by the fun.-us Cercosporidium personatum, is one of the most severe diseases in peanut (Arachis hypogaea). The vast majority of commercial cultivars do not exhibit satisfactory levels of resistance to the pathogen, whereas non-commercial genotypes cv. 850 and cv. 909 are resistant to LLS and show symptoms similar to hypersensitive response (HR) lesions. In the present study, we investigated the molecular components of the initial stages of the resistance by gene expression profiling using suppression subtractive hybridization and differential screening of cDNA macroarray techniques. Gene expression analyses have allowed us to identify more than 700 peanut unique expressed sequence taus (EST) involved in several aspects of the early stages of C. personatum pathogenesis, such as components of defense signaling pathways, gene expression regulators, cell cycle controlling genes and components of the biosynthesis of transducer and antimicrobial compounds. The most significantly induced gene corresponds to a novel O'-methyltranferase, suggesting its involvement in the production of local lesions in C. personatum-resistant A. hypogea genotypes. Taken together, our results contribute to elucidate the defense strategies of peanut and provide the framework for the generation of pathogen-resistant peanut cultivars. (C) 2007 Elsevier B.V. All rights reserved.

Genetic basis of the resistance to Strongyloides venezuelensis (Nematoda, Rhabdiasidae) infection in mice (Mus musculus)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila

Insecticide resistance in laboratory selected Drosophila strains has been associated with upregulation of a range of different cytochrome P450s, however in recent field isolates of D. melanogaster resistance to DDT and other compounds is conferred by one P450 gene, Cyp6g1. Using microarray analysis of all Drosophila P450 genes, here we show that different P450 genes such as Cyp12d1 and Cyp6a8 can also be selected using DDT in the laboratory. We also show, however, that a homolog of Cyp6g1 is over-expressed in a field resistant strain of D. simulans. In order to determine why Cyp6g1 is so widely selected in the field we examine the pattern of cross-resistance of both resistant strains and transgenic flies over-expressing Cyp6g1 alone. We show that all three DDT selected P450s can confer resistance to the neonicotinoid imidacloprid but that Cyp6a8 confers no cross-resistance to malathion. Transgenic flies over-expressing Cyp6g1 also show cross-resistance to other neonicotinoids such as acetamiprid and nitenpyram. We suggest that the broad level of cross-resistance shown by Cyp6g1 may have facilitated its selection as a resistance gene in natural Drosophila populations. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

High Prevalence of bla(CTX-M) Extended Spectrum Beta-Lactamase Genes in Klebsiella pneumoniae Isolates from a Tertiary Care Hospital: First report of bla(SHV-12), bla(SHV-31), bla(SHV-38), and bla(CTX-M-15) in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The copper resistance operon copAB from Xanthomonas axonopodis pathovar citri: gene inactivation results in copper sensitivity

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CUSO4 concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.

Genetics of resistance to soybean cyst nematode, Heterodera glycines ichione (Race 3), in a brazilian soybean population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Copper resistance of biofilm cells of the plant pathogen Xylella fastidiosa

Xylella fastidiosa is a phytopathogen that causes diseases in different plant species. The development of disease symptoms is associated to the blockage of the xylem vessels caused by biofilm formation. In this study, we evaluated the sensitivity of biofilm and planktonic cells to copper, one of the most important antimicrobial agents used in agriculture. We measured the exopolysaccharides (EPS) content in biofilm and planktonic cells and used real-time reverse transcription polymerase chain reaction to evaluate the expression of the genes encoding proteins involved in cation/multidrug extrusion (acrA/B, mexE/czcA, and metI) and others associated with different copper resistance mechanisms (copB, cutA1, cutA2, and cutC) in the X. fastidiosa biofilm formed in two different media. We confirmed that biofilms are less susceptible to copper than planktonic cells. The amount of EPS seems to be directly related to the resistance and it varies according to the media where the cells are grown. The same was observed for gene expression. Nevertheless, some genes seem to have a greater importance in biofilm cells resistance to copper. Our results suggest a synergistic effect between diffusion barriers and other mechanisms associated with bacterial resistance in this phytopathogen. These mechanisms are important for a bacterium that is constantly under stress conditions in the host.

Beef Carcass Contamination by Shiga Toxin-Producing Escherichia coli Strains in an Abattoir in Brazil: Characterization and Resistance to Antimicrobial Drugs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressed sequence tags from Atta laevigata and identification of candidate genes for the control of pest leaf-cutting ants.

Leafcutters are the highest evolved within Neotropical ants in the tribe Attini and model systems for studying caste formation, labor division and symbiosis with microorganisms. Some species of leafcutters are agricultural pests controlled by chemicals which affect other animals and accumulate in the environment. Aiming to provide genetic basis for the study of leafcutters and for the development of more specific and environmentally friendly methods for the control of pest leafcutters, we generated expressed sequence tag data from Atta laevigata, one of the pest ants with broad geographic distribution in South America. Results: The analysis of the expressed sequence tags allowed us to characterize 2,006 unique sequences in Atta laevigata. Sixteen of these genes had a high number of transcripts and are likely positively selected for high level of gene expression, being responsible for three basic biological functions: energy conservation through redox reactions in mitochondria; cytoskeleton and muscle structuring; regulation of gene expression and metabolism. Based on leafcutters lifestyle and reports of genes involved in key processes of other social insects, we identified 146 sequences potential targets for controlling pest leafcutters. The targets are responsible for antixenobiosis, development and longevity, immunity, resistance to pathogens, pheromone function, cell signaling, behavior, polysaccharide metabolism and arginine kynase activity. Conclusion: The generation and analysis of expressed sequence tags from Atta laevigata have provided important genetic basis for future studies on the biology of leaf-cutting ants and may contribute to the development of a more specific and environmentally friendly method for the control of agricultural pest leafcutters.

Pheno and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of São Paulo State, Brazil, were compared pheno and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms. © 2004 NRC Canada.

Multiplex PCR use for Staphylococcus aureus identification and oxacillin and mupirocin resistance evaluation

Oxacillin-resistant Staphylococcus aureus represents a serious problem in hospitals worldwide, increasing infected patients' mortality and morbidity and raising treatment costs and internment time. In this study, the results of using the Multiplex PCR technique to amplify fragments of the genes femA (specific-species), mecA (oxacillin resistance) and ileS-2 (mupirocin resistance) were compared with those of tests conventionally used to identify S. aureus isolates and ascertain their resistance to drugs. Fifty S. aureus strains were isolated from patients receiving treatment at UNOESTE University Hospital in Presidente Prudente, SP, Brazil. The 686 bp fragment corresponding to the gene femA was amplified and detected in all the isolates. On the other hand, the 310 bp fragment corresponding to the mecA gene was amplified in 29 (58%) of the isolates. All of the isolates showed sensitivity to mupirocin in the agar diffusion test, which was corroborated by the lack of any amplicon of the 456 bp fragment corresponding to the ileS-2 gene, in the PCR bands. The conventional tests to identify S. aureus and detect resistance to oxacillin and mupirocin showed 100% agreement with the PCR Multiplex results. The use of techniques for rapid and accurate identification of bacteria and assessment of their resistance may be valuable in the control of infection by resistant strains, allowing the rapid isolation and treatment of an infected patient. However, the results demonstrate that traditional phenotypic tests are also reliable, though they take more time.

Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR

Background: Rust caused by Puccinia psidii Winter has been limiting for the establishment of new Eucalyptus plantations, as well as for resprouting of susceptible genetic materials. Identifying host genes involved in defense responses is important to elucidate resistance mechanisms. Reverse transcription-quantitative PCR is the most common method of mRNA quantitation for gene expression analysis. This method generally employs a reference gene as an internal control to normalize results. A good endogenous control transcript shows minimal variation due to experimental conditions. Findings. We analyzed the expression of 13 genes to identify transcripts with minimal variation in leaves of 60-day-old clonal seedlings of two Eucalyptus clones (rust-resistant and susceptible) subjected to biotic (P. psidii) and abiotic (acibenzolar-S-methyl, ASM) stresses. Conclusions. For tissue samples of clones that did not receive any stimulus, a combination of the eEF2 and EglDH genes was the best control for normalization. When pathogen-inoculated and uninoculated plant samples were compared, eEF2 and UBQ together were more appropriate as normalizers. In ASM-treated and untreated leaves of both clones, transcripts of the CYP and elF4B genes combined were the ones with minimal variation. Finally, when comparing expression in both clones for ASM-treated leaves, P. psidii-inoculated leaves, ASM-treated plus P. psidii-inoculated leaves, and their respective controls, the genes with the most stable expression were EgIDH and UBQ. The chitinase gene, which is highly expressed in studies on plant resistance to phytopathogens, was used to confirm variation in gene expression due to the treatments. © 2010 Laia et al; licensee BioMed Central Ltd.

Caracterização da resistência à oxacilina em Staphylococcus aureus isolados de feridas de pacientes atendidos em Unidades Básicas de Saúde da cidade de Botucatu

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)