Analysis Of Core Components Involved In Small RNA Biogenesis In Phytophthora Sojae And Identification Of Their Defective Mutants

Oomycetes pathogens are a class of eukaryotic microorganism, being fungus-like in morphology and close to algae in evolution. The genus Phytophthora represents typical oomycete pathogens and cause damages on a wide range of important crops, forests and natural ecosystems. Phytophthora infestans is one of the most notorious plant pathogens and caused the Great Irish Famine in the 1840s. It is still the most serious disease problem in potato industry. Phytophthora root rot of soybean is caused by P. sojae, which is a destructive disease of soybean. Small non-coding RNAs, like microRNA and siRNA, are important regulatory factors discovered in recent years. They play an important regulatory role in development and stress responses in plants and animals. Small RNA have been reported in animals, plants, fungi and viruses but not in oomycetes. The identification of key proteins in the small RNA pathway is important for studying the presence of small RNA biogenesis, their function and regulation mechanisms, and facilitates a full understanding of oomycete biology and pathology.P. sojae is homothallic and finished with high quality whole genome sequences. The available large number of EST resources, genetic and molecular methods make P. sojae a model oomycete. We determined the homologs of DCL (Dicer-like protein) and RDR (RNA-dependent RNA polymerases) in P. sojae by bioinformatic analysis. Pfam analysis showed that DCL1 has two RNase III domains, a double-stranded RNA binding domain and a DEAD domain, but lacks PAZ domain and dsrm motif that are present in Dicer / DCL proteins of animals and plants, suggesting that small RNA biogenesis in oomycetesis likely different from that of other eukaryotes. The identification of candidate RDR indicates potential mechanisms of RNA amplificationA mutant library consisting of 640 Ethyl methane sulfonate (EMS) mutants of P. sojae was created, which allows molecular screening for defective mutants of core components involved in small RNA biogenesis. In this study, the reverse-genetics strategy using targeting induced local lesions in genomes (TILLING) technology was employed to screen for P. sojae mutants containg induced mutations in DCL and RDR proteins. A low-cost, simple non-denaturing polyacrylamide gel electrophoresis procedure that uses SYBR GREENⅠwas developed to detect fragments generated by crude celery juice extract digestion of heteroduplexes. The developed method is comparatively sensitive to that of traditional LI-COR based procedure. By using the developed procedure, candidate mutants of DCL1 in were successfully obtained from the generated P. sojae mutant collections.