Identification And Preliminarily Functional Analysis Of New Effector Proteins From Magnaporthe Oryzae

Rice is one of the most important crops in the world. Rice blast disease, caused by the fungus Magnaporthe oryzae, is a serious threat to rice production. The effector proteins secreted by M. oryzae play important roles in the interaction of rice and M. oryzae. Screening and identification of M. oryzae effector proteins, can reveal the molecular mechanism of the interaction between rice and M. oryzae, and which were significant for understanding the mechanism of rice disease resistance.In previous works, we have cloned a total of101secreted proteins genes from M. oryzae. In the present research, by building pGD-M plant expression vectors for transient expression of M. oryzae secreted protein genes in Nicotiana benthamiana leaves, a total of9novel M. oryzae effector proteins that can cause necrosis in plant cells were successfully screened, and were named as MoCdiEP1-9(Magnaporthe oryzae Cell death-inducing Effector protein).Sequence prediction of the nine sceened effector proteins revealed that eight effector proteins contain N-terminal signal peptide sequence and the size of the sequence were about16-22amino acids. According to the prediction, plant expression constructs of non-signal peptide sequence version of eight MoCdiEPs were made by PCR method. Agroinfiltration results showed that the peptide sequences were required for cell death induction of the MoCdiEPs, suggesting that MoCdiEPs need to be secreted into the extracellular for cell death induction.Conserved domain prediction analysis found four effector proteins have conserved motifs. Deletion constructs of the four proteins were made based on the prediction, and were tested in Nicotiana benthamiana leaves. The result showed that there were two effector proteins N-terminal region and its conservative structure domain plays an important role to induce cell death in plants, while the remaining two effector proteins mutant function area is related to its C-terminal region. Using green fluorescent protein gene, we constructed the fusion expression vectors of nine effectors full-length genes and eight without signal peptide sequences with GFP. Detection of MoCdiEP1-GFP revealed that the sinal peptide plays important role for MoCdiEPl sublocalization.