Identificação in silico de genes efetores citosólicos a partir do secretoma de Phytophthora parasítica dos citros.

Abstract

During plant–pathogen interactions, pathogens secrete effectors toward apoplast and cytoplasm of plants that modulate disease or defense host responses. Due to the importance of Phytophthora parasitica as causal agent of the citros gummosis, the main objective of this work was to identify cytosolic effector genes from Phytophthora parasitica secretome, using computational tools and biological assays. By using the Netglycate Program, a total of total of 117 P. parasitica genes were identified into the CitEST/PP database, which putatively encode secreted glycoproteins, sharing homologies with other Phytophthora spp proteins. Among of them, six genes were selected as candidates to future studies of gene expression, and reason that specific primers to quantitative real time PCR amplification were designed and sintesized. These primers were validated by the PCR amplification using P. parasitica genomic DNA, since it has been generated amplicons of expected sizes. Aiming gene expression studies, in vitro assexual growth phases of the pathogen were obtained in large scale and the culture conditions were different for each phase. Hyphae (mycelia) and chlamydospores abundant production was obtained on liquid carrot culture medium under constant light at 25°C during 12 and 45 days, respectively. P. parasitica sporangia were in vitro produced at 5 days after the immersion of mycelia-agar in sterile distilled water. In order to activate the pathogenicity of the pathogen, P. parasitica was successfully re-isolated from orange leaf pieces. Total RNA from mycelia was extracted using a phenol protocol modified, trizol and RNeasy Kit (Qiagen). The identified glycoprotein-like effectors are strong candidates for the quantitative expression studies, as well as to understand and control of the citrus gummosis.