Caracterização do secretoma de Phakopsora pachyrhizi expresso durante a interação com a soja

• Autor(a) principal: Zaramela, Lívia Soares
• Data de Publicação: 2011
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/4749
• topic: Ferrugem asiática da soja; Phakopsora pachyrhizi; Secretoma; Efetores; Asian soybean rust; Phakopsora pachyrhizi; Secretome; Effectors; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

During the interaction with host plants rust fungi secrete an arsenal of effector proteins involved with suppressing host defense responses and redirecting normal host metabolism to facilitate infection. These effector proteins are directed to the apoplast and/or into plant cell cytoplasm by a unknown mechanism. Some effector proteins, called Avr proteins, are recognized by proteins encoded by resistance R genes. This recognition triggers defense responses that lead to a resistance phenotype. Seven R genes (Rpp genes) that confer resistance to Asian soybean rust caused by P. pachyrhizi have been described. However, the Avr proteins recognized by the proteins encoded by these Rpp genes have not yet been identified. Also, P. pachyrhizi genes that encode proteins secreted during the interaction with soybean have not yet been described. Therefore, the objective of this work was to identify genes of P. pachyrhizi that encode proteins secreted during the interaction with soybean using the Yeast Secretion Trap system (YST system), aiming the future identification of Avr genes. From the screening of one YST library constructed using cDNA synthesized from mRNA isolated from infected soybean leaves, 2054 positive clones were selected. From these clones, 1777 single pass 5´-end cDNA sequences with a Phred quality score > 20 were obtained, which were grouped into 95 contigs and 66 singlets. Comparative analyses with sequences deposited in soybean ESTs (BlastN) and Genbank/NCBI non-redundant protein databases (BlastX) resulted in the identification of 48 contigs and 16 singlets no hits that are potential P. pachyhrizi genes. Corroborating this hypothesis, 20 contigs and 4 singlets were similar to trace genome sequences from P. pachyrhizi (1X coverage) deposited at NCBI. Deduced ORFs from twelve genes encode proteins with similarity to Puccinia proteins. Signal peptide prediction by SignalP algorithm was positive for 88.9% of the deduced proteins. In addition to, 87.7% of these deduced proteins showed prediction for secretion by TargetP algorithm, and 92.6% did not showed transmembrane domain prediction. Two deduced proteins have a motif similar to oomycete RxLx-dEER translocation domain, and one protein has a motif similar to the Y/F/WxC motif found in powdery mildew's effector candidates. Three new conserved protein regions, not yet reported in the literature, were also identified. Disulfide bond prediction was positive for 14 deduced proteins. The complete sequence of 29 genes that were selected for functional characterization was obtained, and yeast secretion of proteins encoded by nine selected genes has been confirmed. Secretion assays for proteins encoded by the other genes are in progress. Time course expression analyses and transient expression studies in resistant genotypes will be used to determine the function of the identified genes during the pathogenesis.