The Fungal Elicitor Hrip-induced Disease Resistance And Identification Of Interacting Protein In Rice

Fungal Fungal protein elicitor Hrip1 isolated from the Alternaria alternate is a hypersensitivity-induced protein with a molecular weight of 17.6KD.Hrip1 triggers a series of defense response including cell necrosis,medium alkalization,Ca²⁺inf lux,ROS accumulation and improves the resistance of tobacco against TMV.To further investigate the function of Hrip1-induced defense response and molecular mechanism in rice.In this study,Hrip1 recombinant proteins were obtained by using Pichia pastoris expression system and the roles of Hrip1 on rice were investigated.Interacting protein of Hrip1 in rice was screened by yeast two-hybrid experiment,and further conformed by GST pull down experiment.The interacting protein mutants were obtained by CRISPR/Cas9-mediated gene editing technique in rice.The details are as follows:1.Recombinant protein Hrip1 was successfully expressed with eukaryotic expression system.The recombinant vector pPICZ?A-Hrip1 was successfully constructed and transformed into Pichia pastoris and screened using different concentration of Zeocin.The yeast mutant containing high copy Hrip1gene was obtained.A high purity and homogeneous target protein was obtained by methanol-induced expression and affinity chromatography.Western blot analysis showed that the target protein was Hrip1recombinant protein.Bioactivity exper iments showed that Hrip1 recombinant protein could cause necrosis reaction in tobacco leaf,and the lowest concentration was 10?M.2.Hrip1 could induce rice resistance against Magnaporthe grisea.The rice was treated with Hrip1at a concentration of 30?M.After 48 hours,the spores of Magnaporthe grisea were sprayed on leaves.The results showed that Hrip1 could induce the resistance of rice to Magnaporthe grisea.In order to investigate the mechanism of Hrip1–induced disease resistance in rice,the content of salicylic acid in Hr ip1-treated rice was measured at different time points.The results demonstrated that the content of salicylic acid in the treatment group signif icantly increased compared to untreated plants and reached the highest value in 48 hours.Furthermore,the activity of PAL,POD and CAT related to the defense reaction was also increased after Hrip1treatment.These results suggested that Hrip1 can induce rice resistance to Magnaporthe grisea through defense enzyme system and salicylic acid pathway.3.Hrip1 interaction protein in rice was obtained with Y2H screening.In order to explore the mechanism of Hrip1 inducing rice resistance,a Hrip1-interacting protein was obtained from yeast c DNA library by yeast two-hybrid experiment,named Hrip1-BP.The homologous protein of Hrip1-BP in Arabidopsis is CSN5,an ubiquitin-like protein,which is mainly involved in the light morphogenesis of plants and plays an important role in the interaction between plants and pathogens.4.Hrip1 interacting protein in rice was further validated in vitro with GST pull down.According to the sequence information of Hrip1-BP in rice genome,the specific primers were designed and the full length sequence of Hrip1-BP was obtained.The purified soluble Hr ip1-BP recombinant protein was successfully obtained by constructing pGEX-6p-2-Hrip1-BP recombinant vector and transforming E.coli expressing strain BL21.The GST pull down experiment further demonstrated that Hrip1-BP could interact with Hrip1 in vitro.5.The interaction protein gene knockout mutants were generated with CRISPR/Cas9-mediated gene editing technique.In order to elucidate the role of Hrip1-BP in Hr ip1-induced rice immune response,the Hrip1-BP gene knockout mutants were constructed by CRISPR/Cas9-mediated gene editing technique in r ice.In the end,we achieved directional editing at two sites in the first exon region of the target gene,resulting in 47 mutants,including double allele mutations,homozygous mutations and heterozygous mutations.