Effect Of Magnaporthe Oryzae Effector PWL2/PWL2^D90N In Plant Innate Immunity

Rice blast was caused by Magnaporthe oryzae and lead to tremendous impact on rice production and food security around the world. Besides rice, M. oryzae can also infect other gramineous crops and grasses, such as barley, wheat, maize, weeping lovegrass, crabgrass. Recently, it is reported that M. oryzae could compatibly interact with the model plant Arabidosis thaliana.Effectors, which were secreted by pathogens, play important roles during invasion and disease development. Based on the genome sequencing of M. oryzae, more and more effector genes were identified. One of these effectors PWL2is a member of PWL family which plays a important role in pathogenicity on weeping lovegrass. However, PWL2has no effect in pathogenicity on rice and barley. PWL2protein belongs to cytoplasmic effector, but it is still unclear that how PWL2functions in plant. In order to illuminate the function of PWL2protein, we firstly analysed the sequence variation of PWL2in different strains, and then investigated the effect of PWL2and its mutation version PWL2D90N on plant innate immunity using biochemistry and molecular biology approaches. The main results were as follows:1. The PWL2effector displays sequence diversity in different M. oryzae strains. We blasted the whole genome database of seven published M oryzae strain (70-15, P131, Y34,4091-5-8, FJ81278, HN19311, KJ201). Except4091-5-8and P131, the others contain the PWL2gene. However, PWL2proteins in these five strains have two versions, one with the aspartic acid (D) at the90th amino acid residue, which is named as PWL2(70-15); in the other one, aspartic acid (D) is replaced with asparagine (N) at the90th amino acid residue, which is named as PWL2D90N (Y34, FJ81278. HN19311、KJ201).2. PWL2D90N could be N-glycosylated in plant cell. Based on the prediction from bioinformation analysis using bioinformatics, PWL2D90N contains two possible ubiquitylation sites, one possible SUMOylation site and one glycosylation site. Transient expression in protoplasts showed that ubiquitylation and SUMOylation could not be detected on PWL2D90N, while the glycosylation of PWL2D90N was confirmed.3. To investigate the effect of PWL2and PWL2D90N on PTI (PAMPs-triggered immunity) and the reactive oxygen species (ROS) production, dual luciferase reporter system and DCFH2-DA staining were used to detect the expression of PTI marker gene FRK1and ROS accumulation, respectively. The results showed that PWL2D90N, but not PWL2, can trigger plant innate immunity and accumulate reactive oxygen species (ROS) in Arabidopsis cell.4. Subcellular localization indicates that PWL2and PWL2D90N both located in plasma membrane of A. thaliana, but mainly targeted to nucleus in E. curvula. These results implied that glycosylation of PWL2and PWL2D90N are not corelated with their subcellular localization and the function of PWL2and PWL2D90N. The function of PWL2and PWL2D90N might be distinct in A. thaliana and weeping lovegrass (Eragrostis curvula).