Bioinformatic Characterization Of Oomycetes And Fungi Argonaute Family And Functional Analysis Of AGOl In Phytophthora Sojae

RNA interference is a universal gene silencing phenomenone in eukaryotes,which is a classic defense against the genetic invasion and endogenous gene regulation at the transcriptional and post-transcriptional level.Discovery on fungi and oomycete small RNAs,their roles in host gene regulation by taking advantage of host gene silencing system during infection and the prediction on RNAi core components have supported a cannonical RNAi in fungi and oomycete similar to mammals and plants.However,the secreted small RNA group and their functions during the trans-kingdom crosstalk still remain uncharacterized.Plus,RNAi would be an efficient method to analyze the essential gene to avoid the lethal effects even though CRISPR/Cas9 has turned out to be the most common gene editing method.But the study on RNAi mechanisms in fungi and oomycete was scarce.To lay the foundation to study RNAi mechanisms and biological functions mediated by AGOs,we systematically analyzed amounts,functional domains,phylogenetic relations and transcription levels of encoded AGO genes in 127 fungi and oomycete species.We selected PsAGO1 bioinformatically as our key to unraveling the uncharacterized interaction between Phytophthora sojae and the plants.The main contents are as followed:Bioinformatics was utilized for bioinformatic predicting,analyzing and identifying AGO proteins in fungi and oomycete.Fungi and oomycete shared the similar vegetative form as well as distant phylogenetic relation.Fungi were compared with oomycete to demonstrate AGO functional domains,sequence polymorphisms and transcription levels.The results suggested that most fungi and oomycete encoded two AGOs,with the exception of four AGOs in Phytophthora species and Chytridiales.AGOs in fungi and oomycete evolve independently while AGO1 is conserved and distributed separately from other AGOs.Overall look in the AGO protein functional domains indicated that most AGOs have the predicated six domains known as N terminal,Linker 1,PAZ,Linker 2,MID and PIWI.Functional domain conservation performed by WebLogo suggested 3’ and 5’ small RNA binding sites and catalytic residues on PAZ and PIWI domain,such as YF motif,were conserved.But there’ re still several sites exhibiting variation.The catalytic residues in oomycete were in the form of DEDH,while in fungi,DTDH/D was the primary form.Moreover,we analyzed the expression level and found the two pairs of homologous AGO gene in P.sojae and Pythium ultimum transcripted in similar patterns and expressed highly during infection,which could be associated with their biological functions.Overexpressing and silencing PsAGO1 by transformation system in P.sojae were applied into further exploration of PsAGO1 function.After comfirmation on transcriptional patterns and conserved domains,we found the PsAGO1 expression level was the highest in the predicted AGOs and we speculated that PsAGO1 may be involved in the gene silencing in P.sojae.We generated the PsAGO1 with N-terminal and C-terminal GFP tag and found the N terminal mutants could overexpress PsAGO1.The candidate mutants of PsAGO1 overexpression and silencing were then screened by genome PCR,qRT-PCR and Western Blot.T18,T80,T187,T213 and T262 of PsAGO1 overexpression lines and si111-T26,si892-T2,si892-T16 of PsAGO1 silenced lines were screened for further experiment.The phenotypes of the overexpressed and silenced lines indicated no significant difference in mycelium growth rate,zoospore production and pathogenecity to control.