Mechanism Of OsGRDP1 Regulating Rice Lesion Mimic Formation And Disease Resistance

The lesion mimic mutants(LMM)of plants,exhibit hypersensitive response(HR)-like lesions as well as enhance plant pathogens resistance in most cases.Thus,identification of novel genes involved in controlling the plant lesion phenotype may provide candidate genetic resources for resistant cultivars development.It is also important for revealing new mechanism involved in PCD and/or plant immunity.A novel T-DNA insertion lesion mimic mutant was obtained from the genetic transformation of the Japonica rice cultivar 'Aichiasahi',which was referred to as spl-D.There was no significant difference in growth between spl-D mutants and wild-types growing under the long day condition(14h light/10h dark)or normal field conditions at the seedling stage.Later brown lesions appeared beginning at the lower leaves and gradually expanded to the sheaths and upper leaves after the heading stage,and ultimately leads to death and wilt of the whole plant.Under the short day condition(10h light/14h dark),no lesions appeared until the five-leaf stage on the lower leaves which continued to the mature stage.Shading experiment on spl-D leaves showed that darkness could lead to the lesion-mimic phenotype.High accumulation of H2O2 was detected by DAB staining in and around the lesions of spl-D leaves.Moreover,spl-D showed significantly increased resistance to rice blast and bacterial blight.Further qRT-PCR analysis on expression of defense-related genes showed that the expressions of PR1a,PR1b,PR2,PR3,PR5,PR8,PR9,PR10,NH1,WRKY45,WRKY62,and AOS2 were all up-regulated in spl-D.These results suggest that the mutated gene is involved in the SA-and JA-mediated defense response.Genetic analysis showed that spl-D was a dominant lesion mimic mutant with a single-copy T-DNA insertion,which was co-segregated with the lesion phenotype.The T-DNA flanking sequence in spl-D was amplified by TAIL-PCR and sequenced.Sequence analysis revealed that the T-DNA was localized upstream region of LOC_Os11g40590.qRT-PCR analysis indicated that the expression of LOC_Os11g40590 increased approximately 5-fold higher than that of wide type.The C-terminus of LOC_Os11g40590 encodes a protein that contains seven glycine-rich repeats,thus referred to as OsGRDP1(Glycin-Rich Domain Protein 1).Overexpression of OsGRDP1 showed similar lesion-mimic phenotype as shown in spl-D,which confirmed that OsGRDP1 is the candidate gene to spl-D mutant.OsGRDP1 is a constitutive gene,however,the expression of which could also be induced upon rice blast fungus infection.Transcriptome analysis revealed that OsGRDP1 was involved in the secondary biomass metabolism,stress responses,cell death and kinase activity in rice.Subcellular localization analysis indicated that OsGRDP1 was a cell membrane-associated protein.Screening for the putative interacting proteins of OsGRDPl in rice cDNA library through Y2H identified OsAP25,which was an aspartic protease,as a candidate interactor.Specific regions necessary for interaction identification assay using Y2H revealed that OsAP25 only interacted with the full-length OsGRDP1.Further analysis showed that the total aspartic protease activity of the mutant spl-D was higher than that of the wild-type 'Aichiasahi'.Using Pepstain A,an aspartic protease inhibitor,we found a slowdown in lesions expansion on the spl-D leaves.Therefore,it is speculated that the up-regulation of OsAP25 may increase the total aspartic protease activity,which further contributes to the lesion phynotype of spl-D.To better understand the role of OsGRDP1 in disease resistance,we developed OsGRDP1-RNAi line and knocked out OsAP25 in spl-D line using CRISPR/cas9 system.Screening and identification of the transgenic lines are currently in progress.In summary,OsGRDP1 was a novel regulator of lesion formation.Overexpression of OsGRDP1 induced cell death and activated defense signaling pathways in rice,which further enhanced resistance to rice blast and bacterial blight pathogens.These results provide a new evidence for the mechanism of rice lesion mimic formation.In addition,further study on the relationship between OsGRDPl gene expression and rice resistance will provide theoretical basis and technical support for breeding resistant varieties.