Research On Structure, Function And The Interacting Protein Of Protein Elicitor MoHrip2 From Magnaporche Oryzae

Plants have been evolved an effective system of immunity response to the environmental stress.The system of plant immunity is based on the effective detection and recognition of different inducers from the environment. As one of the main inducers, elicitors are becoming a research hotspot in the plant immunology field. The protein elicitor MoHrip2, extracted from Magnaporthe oryzae as an exocrine protein, triggers the tobacco immune system and enhances blast resistance in rice. However, detailed mechanisms by which MoHrip2 acts as an elicitor remain unclear. Here, we intensively study the crystal structure, the structure and function relationship, the interaction proteins screening and confirmation of MoHrip2, and the biological functions. Accordingly, the research attains the following achievements:1. The three-dimensional structure of MoHrip2 was determinedMoHrip2 protein and the protein ramification containing Se atom were expressed and purified using E.coli expression system. The conditions for crystal growth were screened and optimized and single crystals with high quanlity of MoHrip2 and its ramification were obtained. The diffraction data of native protein and its ramification were collected using X-ray diffraction technique at 2? and 1.8 ?. The phase position of MoHrip2 crystal was solved based on the anomalous dispersion from Se atom; the crystals contain two protein molecules per asymmetric unit. After the model constructing and refinement performed by COOT software package, the three-dimensional structure of MoHrip2 was determined; the overall architecture is a β-barrel, composed of 11β- sheets and a very short α-helix, containing 3 intramolecular disulfide bridges. A DALI search based on similarity to the three-dimensional structure of MoHrip2 revealed that it showed significant similarities to TL-XI protein.2. The functional domain was determinedBased on the secondary structure of MoHrip2 amino acid sequence, eight truncated mutants were designed and each of the mutants was expressed and purified using E.coli expression system. The abilities of the truncation mutants to induce HR and defense responses in tobacco were investigated; the results showed that the short peptide composed of 14 amino acid residues located in the middle region from 76th-89 th site forms the functional domain that is responsible for the ability of the MoHrip2 as an elicitor.3. Mo2BP1 protein was obtained as an interacting protein with MoHrip2 in ArabidopsisThe c DNA library of Arabidopsis was screened using yeast two hybrid system with the MoHrip2 as the bait protein; four candidate proteins were shown to interact with MoHrip2 in yeast. The Bi FC experiment was performed to confirm the interation of the four candidate proteins with MoHrip2 in vivo; the results showed that one protein named as Mo2BP1 can interact with MoHrip2 in tobacco live celles. Mo2BP1 belongs to thaumatin-like pathogenesis-related protein and was involved in responsing to exogenous stimulus. The codons of Mo2BP1 were optimized to express in E.coli expression system; the fusion protein GST-Mo2BP1 was expressed and purified. MoHrip2 and Mo2BP1 can interact in vitro confirmedby GST pull-down assay.4. The function of MoHrip2 was affected in the Mo2BP1 deletion ArabidopsisThe fusion protein Mo2BP1-GFP was transiently expressed in tobacco leaves cells using agrobacteriummediated method; the excited fluorescence was observed under the confocal laser scan microscope and Mo2BP1 was confirmed to locate in the cell membrane in tobacco cells. The mutant plant seeds of Mo2BP1 deletion were bought and the homozygous mutants were obtained after cultivation and screening. The abilities of MoHrip2 to induce HR and defense responses in wide type and mutant Arabidopsis were investigated; the different effects occurred in different genotype plants indicated that Mo2BP1 plays an important role in MoHrip2 inducing HR and improving disease resistance in Arabidopsis.5. MoHrip2 overexpression enhances Arabidopsis disease resistanceThe agrobacterium-mediated method was used to perform the Arabidopsis transformation and transgenic Arabidopsis with MoHrip2 overexpression stable and genetic were ibatained. Disease resistance analysis of transgenic plants showed that MoHrip2 overexpression could enhance Arabidopsis resistance to Botrytis cinerea.