Studies on the temporal,structural,and interacting features of the clubroot resistance gene Rcr1 using CRISPR/Cas9-based systems  

作　　者：Hao Hu Fengqun Yu 

机构地区：[1]Agriculture and Agri-Food Canada,Saskatoon Research and Development Centre,107 Science Place,Saskatoon,SK,S7N OX2,Canada

出　　处：《Horticultural Plant Journal》2024年第4期1035-1048,共14页园艺学报（英文版）

基　　金：supported by the Genomics Initiative of Agriculture and Agri-Food Canada。

摘　　要：Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.

关 键 词：Clubroot resistance Brassica crops CANOLA Rcr1 CRISPR/Cas9 system Gene knock-out Timing control Non-synonymous mutation Protein-protein interaction 

分 类 号：S432[农业科学—植物病理学]