Functional Analysis Of GTPase-activating Protein Gene Gyp3 In Metarhizium Acridum

Metarhizium anisopliae is one of the most important general of entomopathogenic fungi, it has a great potential as biocontrol agents due to its environmental safety and low likelihood of the development of insect resistance. In addition, the wide occurrence and its broad spectrum of action make it has been become a very important model organisms for study infection process in arthropods. Nevertheless, some shortcomings have hinder it widespread application, such as sensitivity to various adversites and low insecticidal speed. Better understanding on gene which influence virulence and cellular stresses could make improve strain come true and also provides an important theoretical basis of fungal pesticides.GYP3 is a principal Rab5 GAP(GTPase-activating Protein), it could slow GTP hydrolysis rate and inactivates the Rab5. It has been shown that Rab GTPases are required for filamentous growth and virulence, deletion of Rab5 led to mild sensitivities to some cellular stresses, it is a general requirement for Rab5 signaling during ionic stress to identify GYP3 as the principal in vivo GAP of Rab5.We investigated whether Gyp3 deletion influences some cellular stresses, filamentous growth and virulence by inactivates the Rab5, there is no studies and reports about this. To elucidate the role of Rab5 GTPase-activating Protein in entomopathogenic fungi and explore the mechanisms involved, the Rab5 GTPase-activating Protein gene of M. acridum( Gyp3), was functionally studied by the construction of disrupted transformant and complementary transformant.The main results are as follows:1、Gyp3 affect the sensitive to Na+ stress,regulates the thermo- and UV- tolerances of conidia in M. acridum: Under hyperosmotic stress conditions(1/4 SDA containing Na Cl), the growth of ΔGyp3 was dramatically reduced compared to that of WT and CP. The stress tolerance levels of conidial germination to UV-B and heat shock were analyzed to clarify the function of Gyp3. Every strain was immediately exposed to irradiances and 45 heat shock for ℃ 0 h, 2 h, 4 h, 6 h and 8 h, then incubated in darkness at 28 °C. The results revealed that ΔGyp3 had a significant reduction compared with WT, while the CP was similar to that of the WT.2、Gyp3 does not affect sporulation, appressorium formation on locust hind wings, cell wall integrity, antioxidant capacity, the non ion tolerance to osmotic pressure:ΔGyp3 exhibited remarkable resistance to the cell wall disturbing agents CR、SDS and CFW. The conidial yielda and appressorium formation on locust hind wings of the ΔGyp3 was no difference among WT and CP.3、Gyp3 affects conidial germinationon on locust hind wings, affects pressure of appressorium on locust hind wings, affects fungal growth in the hemolymph, affect the transcriptional level of of hydrolytic enzyme,deletion exhibited a reduced level of pathogenicity:deletion of Gyp3 has a declined fungal growth in the hemolymph of the host insect in vivo and in vitro; the half germination time(GT50) of the ΔGyp3 was delayed; the transcriptional level of of hydrolytic enzyme and pressure of appressorium of ΔGyp3 had a significant reduction compared with WT、CP.4、Gyp3 does affect the transcriptional level of Rab5, and inactivates the Rab5 in Gyp3 deletion stain:...