Expressão temporal e comparativa de genes efetores intracelulares de Phytopythora parasitica durante a interação com citros.

Abstract

Phytophthora species, such as many other pathogens, secrete effector proteins that are able to module the plant innate immunity to promote the host colonization. The Oomicet effector proteins can it divided into two classes: apoplastics/extracellular and citosolics/intracellular effectors. The objective of this work was to analyse the temporal and comparative expression of intracellular effectors from Phytophthora parasitica during the citrus interaction, by using RT-qPCR (reverse transcriptase-quantitative PCR). Then, interaction bioassays were performed using leaf discs of acidic orange (Citrus aurantium) and micelia of Phytophthora parasitica IAC095 isolate for inoculate treatment and only leaf discs form uninoculated treatment, in times 0, 12, 24, 36 e 48 hours, with 5 biological repetitions for each time. Total RNAs from each sample of all treatmens were isolated by using a SDS/fenol method and analyzed in relation to quantity and quality by electrophoresis in agarose gel and spectrophotometry. Single strand cDNAs were synthesized for 3 biological repeticions from each studied time/treatment. The cultive and manuser conditions of the plant material and Phytophthora parasitica during the bioassay were fundamentals to obtaining of RNAs of quality and to biological responses to be analysed. Th employed method to isolation of citrus leaf RNAs and mix of citrus leaf and Phytophthora parasitica RNAs was successfully. The quantity and quality of extracted RNAs varied among samples in different studied times. Phytophthora parasitica DNA amplified with the CRN primers was a good positive control to comparing with sample RNAs digested with DNase I, in order to eliminate contaminant genomic DNAs. The quantity of synthesized cDNAs estimated by single strand DNA spectrophotometry was diagnostic for presence of cDNAs into the samples. The expression of RXLR effector genes was visualized into the I0h and I24h samples, according to quantitative analysis.