Cloning and transient expression of elicitin genes from Phytophthora capsici.

Elicitins are a family of small elicitor proteins secreted by species of the genera Pythium and Phytophthora inducing a hypersensitive reaction and systemic acquired resistance in Nicotiana and Solanum spp. within the Solanaceae family and Brassica spp. within the Cruciferae family. The paper reports the mining of elicitin gene of Phytophthora capsici, their transcription levels at different stages, their cloning and transient expression in plants. These results contribute to better understanding the roles of elicitins in the interactions between P.capsici and its host plants as well as in the developmental stages. Elicitin candidate genes were mined from the recently released genome database of P.capsici using Phytophthora infestans INF1 and our Illumina transcriptome data as tBLASTx queries(an expected value cut-off of 1e-50). The transcriptional level of the predicted elicitin genes was investigated using RT-PCR technique during the development stages(mycelium, sporangium, zoospore and germinating cyst)of P.capsici and its infection phases of Nicotiana benthamiana roots(1.5,3,6,12,24,36,72 h post-inoculation). Primer pairs specific to the genes were employed to amplify their full lengths using high-fidelity DNA polymerase. Phylogenetic tree was constructed based on multiple sequence alignments of them with other Phytophthora and Pythium spp.elicitins. In the end, these elicitin genes were transiently expressed in N.benthamiana leaves using Potato virus X (PVX) agro-infiltration assay. Blasting search yielded 26 P.capsici sequences highly similar to P.infestans INF1 and our previous elicitin data from trans-criptome sequencing. Only 14 elicitin candidate genes with putative signal peptide-encoding sequences and full lengths were obtained. Transcriptional analysis discovered 6 novel ones from these elicitin genes that were differentially expressed during the developmental and infection stages of P.capsici. The transcriptional profiling showed major differences between the infection stages, which can classify the genes' expression into 3 different types:low or no transcripts detected at the early infection stages, but up-regulation at later stages; constant expression at designated time-points; weak expression at the early infection stages, and gradually increasing along with the proceeding time-points. The full lengths of 10 out of 11 elicitin genes(including 6 ones found in this study and 5 previously reported)were successfully cloned, and amino sequences deducted from these genes showed that they are acidic elicitins. Phylogenetic analysis displayed that these 10 elicitins were clustered into class III and I. Ectopic expression indicated that 2 elicitins genes(Pc508763 and Pc508770)can trigger the hypersensitive reaction in N.benthamiana. The results laid an essential foundation not only for elucidating the interaction mechanisms between P.capsici and its hosts but for future engineering of plant Phytophthora disease resistance. [ABSTRACT FROM AUTHOR]