Functional Analysis Of Two Putative G-protein Coupled Receptors In Magnaporthe Grisea

The rice blast fungus, Magnaporthe grisea, infects many economically important cereal crops, particularly rice. The interaction of M. grisea and rice is also taken as a model for the study of fungus-plant interaction. When the fungal conidium lands on and attaches to a suitable surface, such as a rice leaf, a germ tube arises. Subsequently, germ elongation ceases and the tip of the germ tube swells to form an appressorium surrounded by a thick melanized cell wall. Turgor pressure builds up inside the appressorium, forcing a penetration peg into the plant tissues; this is then followed by invasive growth of the fungus. Thus the life cycle of M. grisea involeds many complexed cell differentiation, singal transduction and biochemical processes. G-protein coupled receptors (GPCRs), as a large family of cell surface receptors, can be activated by a diversity of extracellular stimuli including biogenic amines, odorants, amino acids, lipids, nucleotides, peptides and photons, etc. Consequently, GPCRs play an important role in perceiving different excellular suimuli.Pth11, a GPCR-like gene which encodes by transmembrane protein and localizes at the cell membrane regulating the fungal development and pathogenicity and possibly acting upstream of the cAMP-dependent pathway, is first reported to be related to the fungal surface recognition. Then 76 GPCR-like proteins were identified based on homologs searching in rice blast fungus genome by Kulkar, which include 14 GPCR-like proteins that contain homologs of characterized fungal GPCRs (GPR1,STM1,and the STE2-and STE3-like pheromone receptors), and 61 GPCR-like proteins related to PTH11．Therefore, GPCRs or GPCR-like proteins are proposed to be involved in the recognition of various plant signals during the different infection stages of M. grisea and play an important role in the regulation of interaction between the fungus and rice.In this study, MGG₀2855．5 and MGG₀4698．5 encoding GPCR-like proteins were taken as candidate genes .We constructed the loss-of-function mutants based on the knockout strategy to analyze their phenotypes such as the hyphal growth, mating, conidiation, appressorium formation and development, infection growth and so on .The results showed that the knockout mutant mgg4698 was delayed on its germination and appressorium formation, and leaded to the pathogenicity enhanced when inoculated on rice CO39 and barely leaves at the same concentration of conidia suspension. However, the gene knockout had no impact on conidiation, hyphal growth, and colony color.MGG₀2855．5 knock out mutant mgg2855 was decreased in the conidiation and the hyphal growth. The colony color of the mutant stain was darker than that of wildtype GUY11．However, there were no differences on the growth speed, mating, germination and appressoria formation. While the gene and its native promoter were reinduced into the knockout mutant mgg2855, the conidiation was recovered and reached to the same level of wildtype GUY11, which could lead to a conclusion that gene MGG₀2855．5 was related to the conidiation of M. grisea.In summary, our results would indicate that these two putative GPCRs were involved in regulating hyphal growth and the pathogenicity in M. grisea. However, different GPCR has different function. Thus, dissecting the function of other GPCRs furthermore will help to realize more about the molecular mechanism regulating the fungal pathogenicity and facilitate the rice breeding for durable resistance and the disease integrated management.