Functional Study Of NRPS2 And NRPS6 Genes In Rice False Smut

Rice false smut is a fungal disease that damages the late stage of rice panicle.Since its discovery in 1878,it has been spread throughout the world,in 40 countries in Europe,Americas,and Africa,and most seriously in Asia.The intensity and incidence of rice false smut disease increased seriousely with the large-scale popularization of high-yielding rice varieties cultivated by Oryza indica and Oryza japonica hybrid,excessive planting density,large amounts of top dressing nitrogen fertilizer at booting stage,and global climate change.Due to the increased disease incidence and serious serious yield losses,rice false smut has been risen as the main rice diseases from the secondary disease.The disease is caused by Ustilaginoidea virens(Cooke)Takah,an ascomycetous fungus.Rice false smut not only causes a serious yield reduction and affects the quality of rice,but also produces Ustiloxins which causes human and animal poison,and seriously threatens the health of humans and livestock.Nonribosomal peptide synthetase(NRPS)is a class of peptide synthetase that is composed of multiple domains.Each domain synergistically catalyzes the synthesis of many important peptide secondary metabolites.Among them,NRPS2 and NRPS6 have been shown to be related to ferritin synthesis and iron metabolism in Fusarium graminearum.Iron is a trace element necessary for the growth of plants and microorganisms.Ferrocarriers can absorb iron ions that are not directly available from the environment,transport them into the cell and convert them into iron ions that can be absorbed and released.This is an important way for plants and microorganisms to obtain iron.The research on pathogenic mechanism of Ustilaginoidea virens can provide scientific basis for prevention and control of rice smut disease.However,the genetic transformation of U.virens is very difficult due to the characteristics of this fungus,which seriously hampers the progress of its molecular mechanism research.In recent years,CRISPR-Cas9 gene editing technology become the hottest research tool in the field of life science with its features of easy-to-operate and high efficiency.In this thesis,the deletion mutants of NRPS2 and NRPS6 genes of U.virens were successfully obtained using the constructed CRISPR-Cas9 knockout system.The results showed that the deletion mutants of UvNRPS2 and UvNRPS6 in U.virens attenuated its sensitivity to oxidative stress and other stresses(such as NaCl,Sorbitol,SDS,and Congo Red),and the loss of UvNRPS2 and UvNRPS6 led to slower growth of the fungus on YTD medium.At the same time,the formation and germination of mutants’ conidia were inhibited,some of the conidial morphology variated,and the inhibitory effect on the germination of rice root buds was weakened.Based on this study,the virulence of U.virens was reduced which with NRPS2 and NRPS6 gene deletion.