| Fusarium oxysporum,as a worldwide soil-borne disease pathogen,mainly infects Solanaceae,legumes,melons and cash crops.Panax notoginseng(Burk.)F.H.Chen is a kind of precious Chinese medicinal material,and F.oxysporum is the pathogen of its root rot.As a pathogen,F.oxysporum has a huge impact on the cultivation of these crops causing serious economic losses.However,the current research on the pathogenic mechanism of F.oxysporum is still quite limited,especially the mechanism of root rot caused by F.oxysporum.Studies have shown that the pathogenicity of pathogenic fungi is related to the process of cell polar growth,which leads to the simultaneous formation of new plasma membrane and cell wall in the growth area.In the process of cell polar growth,Rho GTPases regulates the activity of cell wall biosynthetic enzymes,actin cytoskeleton and polar secretion.This is essential to maintain fungal growth and cell wall integrity.The research on Rho GTPase and its regulation mechanism mainly focuses on yeast and animal cells,and there is less research on filamentous fungi.In order to reveal the function of Rho GTPase,this study used F.oxysporum as the material.In this study,the expression level of Rho GTPase in F.oxysporum under different abiotic stress treatments was analyzed;The role of Rho2 gene in F.oxysporum sporulation,spore germination,and cell wall synthesis gene expression of F.oxysporum was studied by constructing ?Rho2 gene knockout mutant;In addition,by constructing a GFP-labeled F.oxysporum strain,the process of its infecting the roots of P.notoginseng was observed.The main results are as follows:1.The wild-type strain of F.oxysporum was treated with cell wall,cell membrane,heavy metal and osmotic stress,and the expression levels of Rho1-3 and Cdc42 in F.oxysporum were detected by fluorescence quantification PCR.The results showed that Rho1,Rho2,and Cdc42 were all down-regulated to varying degrees,and Rho3 was significantly up-regulated under cell membrane stress;The constructed gene knockout expression cassette was transformed into the protoplasts of the wild-type strain of F.oxysporum,and the ?Rho2 mutant was screened and verified by ordinary PCR and fluorescent quantitative PCR.2.Character analysis of ?Rho2 mutant strain: The sporulation rate of ?Rho2 did not change significantly,but the spore germination rate was significantly decreased.Under different abiotic stress treatments,?Rho2 was more sensitive to cell wall synthesis inhibitors and high temperature stress,and colony growth was obviously inhibited;Chitin synthase encoding gene CHS-2,CHS-4,CHS3 and ?-1,3-glucan synthase encoding gene FKS2 in ?Rho2 were down-regulated to varying degrees;The phosphorylation level of Mpk1 in ?Rho2 was significantly up-regulated.3.The GFP-labeled F.oxysporum strain was used to infect the roots of P.notoginseng by the wounded root inoculation method,and the infection process of F.oxysporum in the roots of one-year and two-year plant was observed and compared by laser confocal and scanning electron microscopy.The results showed that the infection process of F.oxysporum to annual seedlings was faster than that of two-year plant.The results showed that,compared with two-year-old P.notoginseng,F.oxysporum infects annual P.notoginseng plant faster.F.oxysporum can directly penetrate the epidermis of the roots of P.notoginseng or enter the roots from natural orifices.These results indicated that Rho2 GTPase is involved in regulating the spore germination of F.oxysporum,responding to cell wall stress and high temperature stress,regulating the expression of genes related to cell wall biosynthesis,and negatively regulateing the activation of Mpk1 in the cell wall integrity signaling pathway.During F.oxysporum infection of P.notoginseng,mycelium formed by spore germination adheres to the root surface,and then enters the root tissue through the natural orifice or directly penetrates the epidermal cells,and diffuses in the intercellular space,thus reaching the purpose of colonization. |
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