Construction And Verification Of Complementary Technology System For Phytophthora Sojae Gene Editing

Phytophthora are an important class of plant pathogens,which are classified as oomycetes rather than fungi.Oomycetes vegetative bodies are diploid,and the probability of homologous recombination in oomycetes is extremely low,so the gene knockout technology widely used in fungi cannot be used in oomycetes.For a long time,the research on the function of oomycete genes has been slow,limited by the low efficiency of genetic transformation system,and it is urgent to develop a new gene research platform.In recent years,CRISPR/Cas9 technology has developed rapidly,providing a new solution for targeted editing of oomycete genomes.Because CRISPR/Cas9 technology can stably knock out target genes from the oomycete genome,it is widely used in oomycete genome editing.However,there are very few selection markers for genetic transformation experiments of oomycetes at this stage,and the neomycin phosphotransferase（NPT II）is mainly used in the experiments to confer resistance to geneticin（G418）in oomycetes.If the NPT II-containing transformed strain is to be complemented or knocked out again,the transformed strain needs to be screened with a new selection marker.Therefore,no efficient gene homologous replacement and complementation technology similar to fungi has been developed in the research of oomycetes so far,which greatly limits the research of oomycetes.At this stage,the urgent need to study the gene function of oomycetes is to develop new screening marker genes,and realize the diversity of oomycete genetic manipulation through gene complementation.Nourseothricin（NTC）can inhibit the growth of eukaryotic organisms by inhibiting protein synthesis.At present,Nourseothricin is widely used in the study of model species such as yeast.This study found that Nourseothricin can effectively inhibit the growth of Phytophthora sojae,and we speculate that Nourseothricin can screen oomycete transformant strains.In this study,an oomycete genetic transformation vector based on the Nat1 gene was constructed,and we successfully obtained transformant strains by screening with nourseothricin.Further analysis of the transformant strain showed that Nat1 gene could confer resistance to nourseothricin in Phytophthora sojae,but it had no effect on the growth and development of the strain.RxLR-like effectors are exocrine proteins of Phytophthora,and theoretically knocking out exocrine proteins will not negatively affect the growth of the strain.Therefore,using CRISPR/Cas9 technology and Nat1 gene to edit RxLR-like effector-encoding genes can detect whether it affects the growth of Phytophthora.In this study,gene knockout and gene complementation were performed on the gene PsAvh109 encoding the RxLR effector of Phytophthora sojae.Experiments have shown that the selection marker gene Nat1 and CRISPR/Cas9 technology have no effect on the growth and development of Phytophthora.In order to verify this conclusion,this study took the RxLR effector-encoding gene PsAvh31 of Phytophthora sojae as the research object to conduct repeated experiments,and obtained the same conclusion.In this study,we successfully applied Nat1 as a screening marker gene to the oomycete genetic transformation experiment,which enriched the genetic manipulation methods of oomycetes,improved the oomycete genetic transformation system,and provided technical support for in-depth study of oomycete gene functions in the future.