Functional Analysis Of Several Cell Death-inducing Effector Proteins In Magnaporthe Oryzae

Magnaporthe oryzae effectors play important roles in the interactions between M. oryzae and rice. Functional analysis of the effectors would lead to deep understanding of M. oryzae pathogenic mechanisms, and provide theoretical basis for the prevention and controlling of the rice blast disease.In previous study, we have identified nine cell death inducing effector proteins from 101 in-planta expressed M. oryzae secreted proteins. The nine effectors, named MoCdiEP1-9, contain a predicted signal peptide sequence at the N terminal except for MoCdiEP3. Transient expression of non-signal peptide version of MoCdiEPs in plant cells did not result in cell death, suggesting that these proteins function in the plant apoplastic space.To further study the function of MoCdiEPs genes, the overexpression and knockout vectors have been constructed and transformed into M. oryzae isolate Guyll. Overexpression transformants of the nine MoCdiEPs and knockout mutants of five out of the nine MoCdiEPs were developed. The overexpression and knockout transformants were characterized for their growth rate, colony morphology, sporulation and pathogenicity. Compared with the wild type, MoCdiEP1-OE showed weaker vegetative growth rate; â–³MoCdiEP3 mutants showed enhanced pathogenicity towards barley in some distance; and MoCdiEP7-OE and MoCdiEP9-OE showed accelerated pathogenic speed. However, the pathogenic phenotypes of MoCdiEP7-OE and MoCdiEP9-OE at late infection stage were similar to that of wild type Guy11.We further constructed prokaryotic expression vectors of eight out the nine MoCdiEPs harboring a maltose binding protein (MBP) tag, and successfully expressed and purified soluble recombinant proteins from three effectors MoCdiEP1, MoCdiEP2 and MoCdiEP6. Spray inoculation results showed that high concentration recombinant protein of MoCdiEP1 or MoCdiEP2 induced rice leaves withering death. When treated the rice seedlings with low concentration recombinant protein of MoCdiEP1 or MoCdiEP2, the remarkable up-regulation of rice pathogenesis-related genes of OsChtl, OsCht3, OsNAC4, OsPR1b and OsPBZl was detected by qRT-PCR. Moreover, the rice seedlings pretreated with low concentration MoCdiEP1 or MoCdiEP2 exhibited enhanced resistance to M. oryzae, suggesting that these effector proteins can be recognized by rice plants and stimulate the immune defense response of rice. These results establish a certain foundation for deeply understanding the roles of the MoCdiEPs in the interactions between M. rovzae and rice.