Gene network mediated by WRKY13 to regulate resistance against sheath infecting fungi in rice (Oryza sativa L.)

OsWRKY13 TF gene is known to play a regulatory role of signaling in physiological pathways related to either development or disease resistance in rice plants. Rice cultivars IR 50 and TRY 3, resistant and susceptible respectively to sheath blight, TRY 3 and CO 43 resistant and susceptible respectively to sheath rot were challenged with fungal pathogens and disease scoring was carried out. Percent Disease Index (PDI) was significantly higher in susceptible varieties than resistant varieties. RT-PCR and qPCR analyses of WRKY13 using RNA extracted from the plant tissues revealed higher WRKY13 expression in resistant varieties (both diseases) upon pathogen challenge compared to uninfected control and also the susceptible varieties. To compute and evaluate the possible molecular mechanism for observed resistance correlated to WRKY13 gene expression, rice gene expression profiles against bacterial leaf blight and leaf blast disease from ROAD database were used to prioritize the locus IDs that were used as input in RiceNet v2 tool. The expression of WRKY13-regulated TIFY9 gene was predicted and validated using RT-PCR and qRT-PCR along with WRKY12 and PR2. All three genes showed induced expression in R. solani challenged sheath blight resistant variety. WRKY12 and PR2 expression in S. oryzae challenged sheath rot resistant variety was higher. Agrobacterium mediated transformation was carried out in rice plants using overexpression construct of WRKY13 (agroinfection in seeds of varieties susceptible to sheath blight and sheath rot, followed by selection in antibiotic media, germinating and hardening of putative transgenic lines). Based on qPCR analysis, the expression level of WRKY13 and the co-expression levels of WRKY12, TIFY9 and PR2 were found higher in PCR-positive T-1 plants compared to wild-type. Infection bioassays in the transgenic plants of both varieties revealed enhanced resistance to the pathogens. A mechanism by which WRKY13 would influence the MAPK cascade with TIFY9 acting as a mediator, is proposed.