Function Analysis Of The Transcription Factor PsMAD1 And Effector PsAvh238 In Phytophthora Sojae

Phytophthora sojae is one of the most destructive threats to soybean production in the world.Morphologically and physiologically,P.sojae resemble filamentous fungi,but evolutionally they are classified in kingdom Stramenopila,which includes diatoms and brown algae.Therefore,most fungicides show no control effect to plant diseases caused by P.sojae.Knockdown of genes in P.sojae by RNAi is incomplete.Also the level of silencing is unpredictable and often unsatisfactory,and some genes have required a major effort to find a few silenced lines.Which caused selective silencing of closely related genes is difficult.The CRISPR/Cas9 system operated easily,which simply uses a guide RNA and Cas9 nuclease to identify and cut target DNA sequences.Tyler report that the efficient disruption and replacement an effector gene in the Oomycete Phytophthora sojae using CRISPR/Cas9.Transcription factor is a key factor in the regulation of gene expression and play an important role in biological processes as pathogenic.A preliminary study found a MADS-box transcription factor PsMAD1.PsMAD1-silenced affected the number of zoospores,pathogenicity and abiotic stresses response regulation.Gene silencing is unstable so that we was knocking out PsMAD1 by CRISPR/Cas9 system to futher validate its biological function.We has obtained three PsMAD1 knocking out mutants.Mutants results in phenotypes:Compared with wide-type,PsMAD1 mutants no change in growth rate and abiotic stress response regulation;PsMAD1 mutants no cleavage of the cytoplasm into uninucleate zoospores or release zoospores,direct germination of sporangia,pathogenicity decrease when mycelia infect soybean leaf and root;the effector expression was down regulated.Transcription factor can transfer signal by regulate the expression of downstream genes.However,many effector genes can be up-regulated in the infection process,which can promote the infection of pathogens.Because PsMAD1 is related to the pathogenicity of P.sojae,it is speculated that the transcription of some effector genes may be regulated by them.This study screening an RXLR effector gene PsAvh238,which expression was change when infect soybean by the transcription analysis of RXLR effectors during infection of P.sojae P6497 or PsMAD1 mutants based on real-time PCR.PsAvh238 introduction of Non-Homologous End Joining mutation by CRISPR/Cas9 system,which cause insertons or deletions that could alter open reading frames and insert premature stop signals resulting in a gene knockout.Analysis for PsAvh238 mutants phenotypes found:Compared with wide-type,PsAvh238 mutants no change in growth rate;However,the pathogenicity of PsAvh238 mutants is decrease.PsAvh238 is an effector associate with pathogenesis.The question whether MADS-box transcription factor PsMAD1 regulates the transcription of effector PsAvh238 remains to be studied in the following studies.