Identificação de genes que codificam proteínas secretadas por Puccinia psidii, agente etiológico da ferrugem das mirtáceas

• Autor(a) principal: Silva, Ricardo Roberto da
• Data de Publicação: 2010
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/4714
• topic: Eucalyptus; Puccinia psidii; Efetor; Resistência; Eucalyptus; Puccinia psidii; Effector; Resistance; CNPQ::CIENCIAS AGRARIAS::RECURSOS FLORESTAIS E ENGENHARIA FLORESTAL::SILVICULTURA::GENETICA E MELHORAMENTO FLORESTAL

The rust caused by the basidiomycete Puccinia psidii is one of the most destructive diseases of eucalipty plantations in Brazil, and cause for sanitary barriers where this disease does not occur in Asia and Oceania. During the interaction with the host the biotrophic fungi, such as rusts, secrete effector proteins involved in the suppression of the defense response and in the manipulation of the host metabolism. A strategy that can be used to discovery of these proteins is the in silico prediction of cellular sequences export in open reading frames deduced from cDNA sequencing. This study aimed to identify and characterize cDNA sequences of P. psidii that encode secreted proteins through sequence analysis of a cDNA library constructed from mRNA isolated from Eucalyptus grandis leaves infected by P. psidii. We analyzed 9,864 expressed sequence tags (ESTs). From 1,675 transcripts that encode proteins with no similarity to protein sequences of non-redundant GenBank/NCBI database and lacked identity to cDNA or genome sequences deposited in the eucalipty databases, 173 ORFs that showed positive prediction in five or more parameters of the export signal sequence prediction algorithm SignalP were obtained. A subgroup of 31 clones with better predictive value of cell export in different algorithms was selected for characterization. Twenty-nine of the clones had the ORF amplified from the genome of P. psidii generating equal or larger fragments than those amplified from the cDNA. Eight clones showed sequences similarity to Puccinia graminis and five Melampsora laricis sequences, among which, four have similar in both banks. The selected clones were also compared with each other using the program BlastP, and presented similarity, ranging from small motifs shared to extensive identity. Secretion of proteins encoded for 24 clones was confirmed in vitro by the yeast secretion trap (YST). Twenty clones had their ORF cloned into a vector for Agrobacterium-mediated transient expression. Analysis of transient expression and expression monitoring of secreted proteins are being optimized to investigate the effector role of hypothetical proteins identified.