Preliminary Study On The Relationship Between Oryza Sativa Auxin Repressed Gene OsARP1 And The Pid3-mediated Resistance To Magnaporthe Oryzae In Rice

Auxin repressed protein(ARP)genes are widely distributed in plants and has been confirmed to play an important role in growth and defense and seed dormancy in several plants.However,the structure and expression characteristics,biological functions,and especially the relationship with rice blast disease of rice ARP gene Os ARP1 have not yet been reported.In this study,bioinformatics methods were used to analyze the structure characteristics and the promoter cis-acting elements of Os ARP1 and its functional related genes,and the structure characteristics of their encoding proteins.To understand the relationship between the Os ARP1 and Pid3-mediated rice blast resistance,we analyzed expression levels of OsARP1 and its functional related genes in blast susceptible rice TP309 and blast resistant transgenic rice TP309-Pid3.CRISPR/Cas9 knockout and RNAi vectors for OsARP1 and vectors over expressing Os ARP1 were further generated,transgenic rice were obtained,and their levels of resistance to rice blast fungus were identified.The main findings are as follows:1.Bioinformatic analysis on OsARP1 and its five family genes detected Auxin-related domains in amino acid sequences of four proteins encoded by OsARP1 and LOC_Os03g22270,LOC_Os08g35190,and LOC_Os09g26620.Their 2 kb promoter regions contain cis-acting elements for plant defense hormone such as salicylic acid,abscisic acid,and jasmonic acid.No signal peptides and transmembrane domain,but many Threonine(T),serine(S)and tyrosine(Y)phosphorylation sites were identified on these four gene encoding protein sequences.Os ARP1 encoded protein is predicted to be located in the cytoplasm(Certainty= 0.450),LOC_Os03g22270 and LOC_Os09g26620 encoded proteins in the cytoplasm(Certainty= 0.650 and 0.600,respectively),and LOC_Os08g35190 encoded protein in the nucleus(Certainty= 0.650).2.Blast susceptible rice TP309 and blast resistant transgenic rice TP309-Pid3 were inoculated with rice blast Zhong 10-8-14 and the expression levels of OsARP1,LOC_Os03g22270,LOC_Os08g35190 and LOC_Os09g26620 were quantified by RT-PCR.Expression of these genes were induced following rice blast fungus infection,with the highest expression level detected in Os ARP1.Faster induction rate and higher level was shown in susceptible rice cultivars.These results indicate that Os ARP1 might play an important role in rice blast resistance signal pathway.3.No regenerated plant was obtained after transformation of the Os ARP1 CRISPR/Cas9 knockout and RNAi vector into TP309 and TP309-Pid3.Slow growth of calli,severe tissue browning and yellowing and eventually death of the seedling without roots were observed.After transforming the OsARP1 overexpression vector into TP309,most transgenic plants were not viable due to poor growth and only 7 transgenic rice were obtained.Upregulation of OsARP1 was only detected in 1 transgenice rice and downregulation in the rest 6 plants.Overexpression of OsARP1 was found to enhance rice resistance to Magnaporthe oryzae after inoculation of rice blast fungus into the transgenic rice with up-regulated OsARP1.This study discusses the relationship between OsARP1 and the rice blast resistance,and premilinarily estimate a close association between Os ARP1 and the resistance to Magnaporthe oryzae in rice.Difficulites in obtaining OsARP1 knockout and RNAi transgenic plants were also dissussed.