Small RNA Profiling And The Function Of Dicer Genes In Phytophthora Infestans

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is the most serious potato disease in the world. Currently, application of chemical fungicide is the only preventative treatment for late blight control. However, fungicide application is a costly approach, which is not realistic in the developing countries, and is environmentally unfriendly. P. infestans can infect potato leaves, stems and tubers, which can potentially wipe out the entire crop. The complete genome of P. infestans strain T30-4 has recently been sequenced. This key resource has made it possible to conduct genome-wide genomic studies in this important plant pathogen. This study aims to profile the microRNAs and their target genes in the P. infestans genome using high throughput sequencing. We also investigate the number and function of the Dicer genes, which are associated with microRNA biogenesis.Two P. infestans strains, YN-YJ54.1 and IIA-4, which are differently virulent, were used in small RNA profiling. We discovered 331 novel miRNAs and 1449 target genes,1805 conserved miRNAs and 15300 target genes in the mycelia tissue of YN-YJ54.1; 29 novel miRNAs and 71 target genes,1527 conserved miRNAs and 10892 target genes in germination of YN-YJ54.1; 275 novel miRNAs and 180 target genes,1852 conserved miRNAs and 16540 target genes in sporangia of YN-YJ54.1. Similarly, we identified 4 novel miRNAs and 9 target genes,1046 conserved miRNAs and 9328 target genes in germination of IIA-4. The target genes regulated by miRNAs account approximately 80% of the P. infestans genes that span a wide range of function of catalytic activity, hydrolase activity and metabolic process. Approximately 85% of the P. infestans miRNA have homologues in other eukaryotes, including C. elegance, Arabidopsis thaliana, Drosophila melaogaster and mammalian species. These results show that many ancient miRNAs have been retained in P. infestans. Thus, our data provide strong evidence that miRNAs originated very early during eukaryote evolution and different sets of miRNAs have been selectively maintained in plant and animal species.P. infestans contains two genes, DCLl and DCL2, encoding for the Dicer proteins. Using in vitro siRNA feeding, we silenced DCL1 and DCL2 individually as well as simultaneously. Silencing of DCL1 resulted in drastic phenotypic changes, including decrease of germination and shortening of germ tubes. Simultaneous silencing of both DCL1 and DCL2 resulted in a similar phenotype as silencing DCL1 alone. In contrast, silencing DCL2 alone did not result in unambiguous phenotype. These results suggest that DCL1 plays an important role in P. infestans germination. We used stem-loop RT-PCR to investigate the expression of several miRNA genes in P. infestans after feeding of DCL1-targeted siRNA. We observed a decreased expression of miR393 and miR-2476 genes. However, the expression of two other genes, miR-2132 and miR-1626, was not changed in DCL1-silenced P. infestans. These results confirmed that DCL1 plays a role of biogenesis of miRNA in P. infestans, which is consistent with the fact that silencing of DCL1 can result in a drastic phenotype change.We successfully demonstrate that siRNA-based in vitro feeding can effectively silence endogenic P. infestans genes. Thus, if a potato plant is engineered to produce sufficient amount of siRNA that target critical P. infestans genes, such siRNA could potentially be absorbed by P. infestans during infection, which may ultimately interfere the expression of the target P. infestans genes. Thus, our work provides a potential foundation for designing novel strategies of RNAi-based potato late blight control.