The molecular interaction between plants and insects is dynamic and multifaceted. We are interested in understanding the molecular mechanism that insects utilize to overcome plant defense proteins, as well as discovering novel plant insecticidal proteins. Three projects were developed. First, we evaluated the effects of soybean cysteine protease inhibitor (soyacystatin N, scN) on the growth and development in southern corn rootworm. Both subtractive suppressed hybridization (SSH) and cDNA microarray analyses were used to uncover the changes of gene expression profiles in southern corn rootworm under the scN challenge. The counterdefense-related genes were identified, suggesting that southern corn rootworm deployed several regulatory mechanisms to overcome the dietary scN. Second, to identify and confirm insecticidal properties of vegetative storage protein 2 in Arabidopsis (AtVSP2), the gene was cloned and expressed in E.coli. This protein showed acid phosphatase activity. Feeding assay indicated that AtVSP increased the mortality and delayed the development of two coleopteran and one dipteran insects. Third, to identify the molecular mechanism of this novel insecticidal protein, P element mutagenesis was utilized to generate AtVSP resistant mutants (VRs). Two balanced VR mutants and their revertants were generated, and can be used to further characterize the genetic loci of P element inserted in the mutants.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / O feijÃo-de-corda [Vigna unguiculata (L.) Walp.] à uma importante leguminosa usada como alimento, sendo cultivada, primariamente, nas savanas secas do Continente Africano, na Ãsia e na AmÃrica do Sul, cobrindo mais de 12 milhÃes de hectares, com produÃÃo anual de cerca de 3 milhÃes de toneladas. O feijÃo-de-corda se constitui numa cultura de extrema importÃncia em zonas semi-Ãridas dos trÃpicos, caracterizadas por baixa precipitaÃÃo pluviomÃtrica, altas temperaturas, solos arenosos e de baixa fertilidade. Dentre os organismos que causam doenÃas nos vegetais, os nematÃides geram prejuÃzos anuais de, aproximadamente, vÃrios bilhÃes de dÃlares na agricultura mundial e as espÃcies Meloidogyne incognita e Meloidogyne javanica sÃo as mais danosas. Uma das principais culturas atacadas pelos nematÃides à o feijÃo-de-corda. Em funÃÃo disso, existe sempre demanda para se buscar melhorar a compreensÃo dessa relaÃÃo entre hospedeiros e patÃgenos objetivando criar novas estratÃgias para minimizar eventuais perdas. O presente trabalho teve como objetivo identificar as proteÃnas que tÃm sua expressÃo diferenciada em raÃzes de feijÃo-de-corda resistente ao Meloidogyne incognita (inoculadas e nÃo inoculadas) usando eletroforese bidimensional (2D) associada à espectrometria de massas de proteÃnas extraÃdas da raiz total e de mitocÃndrias de raiz do cv PitiÃba (CE-31), resistente ao nematÃide, inoculada com este patÃgeno, em comparaÃÃo com plantas nÃo-inoculadas (controle) em conjunto com anÃlise de expressÃo gÃnica semi-quantitativa (PCR semi-quantitativa). Os resultados aqui obtidos demonstraram que houve alteraÃÃo de pelo menos 22 proteÃnas nas amostras de raÃzes inoculadas e mais 22 proteÃnas de mitocÃndrias de raÃzes, quando comparados com seus respectivos controles e que o Ãpice das suas alteraÃÃes ocorriam por volta do 6 dia apÃs a inoculaÃÃo do patÃgeno. As proteÃnas de mitocÃndrias nÃo puderam ser identificadas com confiabilidade. Dentre as proteÃnas de raÃzes totais identificadas podemos destacar as PR-1, 2 e 3, que sÃo reconhecidamente proteÃnas de defesa, ascorbato peroxidase e superÃxido dismutase (enzimas anti-estresse oxidativo) e uma leghemoglobina, que pode ser o primeiro relato dessa proteÃna nesse patossistema. As anÃlises de expressÃo gÃnica concordaram perfeitamente com os achados proteÃmicos. / The cowpea [Vigna unguiculata (L.) Walp.] is an important leguminous used as food, being cultivated, mostly in arid African savannas , Asia and south of America, covering more over 12 millions of hectares, with annual production about 3 millions of tons. The cowpea itâs a culture greatly important in tropics zones characterized by low pluviometric precipitation, high temperature , gravely soils and poor fertility. Among the pathogenic organism that cause plant disease, the nematode, mainly Meloydogine sp. generate annual losses about several billions of dollars all over the world and the Meloidogyne incognita and Meloidogyne javanica species be the most damagers. One of the main cultures attacked by nematodes is the cowpea. Regard it, there is always needs to improve the knowledge about the relationship between host and pathogens aimed develop novels strategies to diminish eventual losses. The present work aimed identify the differential expression of proteins in Meloidogyne incognita resistant cowpea total roots and mitochondria roots cv PitiÃba (CE-31), inoculated and mock inoculated, using 2D electrophoresis assay associated with MS identification and gene expression analyses by semi-quantitative PCR. The results obtained showed at least 22 altered proteins in inoculated total roots and more 22 proteins of mitochondria roots, when compared with their respective controls and the top of alterations occurred next to 6 day after inoculation with pathogen. Mitochondria proteins cannot be identified reliability. Among the total roots proteins we can emphasize PR-1, 2 and 3, recognized as defense proteins, ascorbate peroxidase and superoxide dismutase (anti-oxidative brush enzymes) and a leghemoglobin, which can be the first report about this protein in this pathosystem. The gene expression analyses coincided with proteomics finds.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Laticifers plants have been studied by presenting a wide range of proteins related to plant defense in its latex. The aim of this study was to investigate, in tissue cultured in vitro, proteins and activities described for latex laticÃferas two species. Tissue callus and roots of Cryptostegia grandiflora were obtained by in vitro tissues culture protocols and subjected to histological analysis for laticifers characterization. Cultured tissue of Calotropis procera were used as comparative reference in the analysis. There wasnât any laticifer structure in callus or roots of C. grandiflora while in C. procera laticifers are formed in the roots. Soluble proteins were extracted from the cultured tissue and characterized using enzymatic assays, biochemical, immunological techniques and mass spectrometry. The presence of activity against phytopathogenic fungi was investigated and all data obtained were compared with the previously one determined for the plants studied latex. Callus and roots proteins of C. procera showed antifungal activity against pathogenic fungi. The percentage inhibition of the vegetative hyphae growth in the presence of callus and roots C. procera protein respectively were 75.5% and 82.6% for Fusarium solani, 76.7% and 57.1% for Rhizoctonia solani, 88.8% and 79.8% for Fusarium oxysporum, 93.7% and 90.2% for Colletotrichum lindemuthianum and 80.2% and 79.7% for Colletotrichum gloesporioides, however, showed no effect on Mucor sp. Callus and roots proteins of C. grandiflora showed no inhibitory effect on the hyphae growth or spores germination of assayed fungi. Through assays using fluorescent markers, it was demonstrated that proteins extracted from in vitro culture of C. procera interact with the membrane of C. gloesporioides causing leakage of cytoplasmic contents, possibly suggesting that its mechanism of action against fungi is related to the change in plasma membrane permeability. Also oxidative stress was observed in C. gloesporioides spores treated with callus and roots protein C. procera by hydrogen peroxide production. Protease inhibitors, chitinases, osmotins and proteases were detected in the C. procera callus and roots samples, however, osmotins and proteases were not observed in C. grandiflora callus and roots. The activity of antioxidant enzymes APX, G-POD and catalase were observed in tissue cultured in vitro of C. grandiflora. Considering that C. grandiflora laticifers proteases were demonstrated exert action against fungi, the results observed in this study suggest that the absence of antifungal activity in C. grandiflora cultured tissue is due to the absence of proteases in these tissues as well exclude chitinases and proteases inhibitors as antifungal proteins. The study concludes that the use of cultured tissues that do not differentiate laticifers is an interesting model to study activities associated to proteins founded in latex. Antifungal proteases present in C. grandiflora latex were not found in the tissues without laticifer formation. / Plantas laticÃferas tÃm sido estudadas por apresentarem uma grande diversidade de proteÃnas relacionadas à defesa vegetal em seu lÃtex. O objetivo deste trabalho foi pesquisar em tecidos cultivados in vitro, proteÃnas e atividades descritas para o lÃtex de duas espÃcies laticÃferas. Tecidos de calos e raÃzes de Cryptostegia grandiflora foram obtidos atravÃs de protocolos de cultura in vitro de tecidos e submetidos à anÃlise histolÃgica para caracterizaÃÃo de laticÃferos. Tecidos cultivados de Calotropis procera foram utilizados como referencial comparativo nas anÃlises. NÃo foi detectada qualquer estrutura laticÃfera em calos ou raÃzes de C. grandiflora enquanto que em C. procera laticÃferos se formam nas raÃzes. ProteÃnas solÃveis foram extraÃdas dos tecidos cultivados e caracterizadas por meio de ensaios enzimÃticos, tÃcnicas bioquÃmicas, imunolÃgicas e espectrometria de massas. A presenÃa de atividade contra fungos fitopatogÃnicos foi investigada e todos os dados obtidos foram comparados com dados previamente determinados para os lÃtex das espÃcies estudadas. ProteÃnas dos calos e raÃzes de C. procera, apresentaram atividade antifÃngica sobre fungos fitopatogÃnicos. Os percentuais de inibiÃÃo do crescimento vegetativo de hifas na presenÃa de proteÃnas de calos e raÃzes de C. procera, respectivamente, foram: 75,5% e 82,6% para Fusarium solani, 76,7% e 57,1% para Rhizoctonia solani, 88,8% e 79,8% para Fusarium oxysporum, 93,7% e 90,2% para Colletotrichum lindemuthianum e 80,2% e 79,7% para Colletotrichum gloesporioides, no entanto, nÃo demonstraram nenhum efeito sobre Mucor sp. As proteÃnas de calos e raÃzes de C. grandiflora nÃo apresentaram qualquer efeito inibitÃrio sobre o crescimento de hifas ou germinaÃÃo de esporos dos fungos avaliados. Por meio de ensaios com marcadores de fluorescÃncia, foi possÃvel demonstrar que as proteÃnas extraÃdas da cultura in vitro de C. procera interagem com a membrana de C. gloesporioides causando extravasamento do conteÃdo citoplasmÃtico, sugerindo que possivelmente seu mecanismo de aÃÃo contra fungos esteja relacionado à alteraÃÃo na permeabilidade da membrana plasmÃtica. TambÃm foi observado estresse oxidativo em esporos de C. gloesporioides tratados com proteÃnas de calos e raÃzes de C. procera atravÃs da produÃÃo de perÃxido de hidrogÃnio. Inibidores de proteases, quitinases, osmotinas e proteases foram detectados nas amostras de calos e raÃzes de C. procera, porÃm, osmotinas e proteases nÃo foram observadas em calos e raÃzes de C. grandiflora. A atividade das enzimas antioxidantes APX, G-POD e catalase foram observadas nos tecidos cultivados in vitro de C. grandiflora. Considerando que proteases laticÃferas de C. grandiflora foram demonstradas exercer aÃÃo contra fungos, os resultados observados nesta pesquisa sugerem que a ausÃncia de atividade antifÃngica em tecidos cultivados de C. grandiflora deve-se a ausÃncia de proteases nestes tecidos e ainda excluem quitinases e inibidores de proteases presentes como proteÃnas antifÃngicas. Em calos e raÃzes de C. procera, alÃm de proteases, outras proteÃnas tais como, quitinases, inibidores de proteases e osmotinas detectadas podem estar envolvidas na atividade antifÃngica observada, e/ou agir sinergicamente na defesa contra fungos. O estudo conclui que o uso de tecidos cultivados que nÃo diferenciam laticÃferos à um interessante modelo para estudar atividades associadas Ãs proteÃnas encontradas no lÃtex. Proteases antifÃngicas presentes no lÃtex de C. grandiflora nÃo foram encontradas nos tecidos sem formaÃÃo laticÃfera.