Study On The Virulence Factors Of Alternaria Alternata Croftonweed Pathotype Infection Of Its Host

Alternaria alternata(Fr.)Keissler was recognized as a natural filamentous pathogen to croftonweed.TeA toxin is one of its metabolites which has been evaluated as a potential bio-based herbicide for croftonweed control,so as other monocotyledon and dicotyledon weeds.Our previous studies shows that TeA is a novel natural photosystem ? inhibitor functioned by blocking electron transport beyond QA by competing with QB for QB-site in D1 protein.It was furthermore verified that oxidative damage resulting from TeA-induced ROS generation in chloroplasts of mesophyll cells was responsible for TeA-triggered cell destruction and leaf necrosis.However,the content of toxin produced by A.alternata is so low that could not-produce-by fermentation commercially.In order to use the TeA as a novel bioherbicide more efficiently,it is necessary to improve its toxin production capability,so studying the biosynthesis metabolic process and genes in relation to toxin production.A toxin-deficient mutant strain had already been obtained by restriction enzyme-mediated integration(REMI).The physiological and pathogenicitic comparison studies between wild type and mutant strains showed the mutant has a 70%reduction in sporulation and did not cause disease spots after inoculated with mycelium onto croftonweed leaves.Based on the disrupted gene in the mutant by plasmid pSH75,the disrupted gene HP001 were cloned.This study is mainly on the virulence factor of TeA toxin for infection by A.alternate to the host,and the function analysis of the histidine phosphatese gene HP001 during fungi development and pathogenicity of A.alternata.The main study results are given as followings:1.TeA is a key virulence factor for A.alternata when infecting its hostUsing the wild type and HP001 mutant as materials,the role of TeA in the process of the infection of the host was studied.It was found that the TeA toxin could damage the host leaves before the hyphae infection,and the PSII reaction center was blocked,which inhibited the formation of cross membrane proton gradient,and caused the damage of the photosynthetic system.The mutant hyphae could not cause disease spots in the host,but when the wild-type cultured filtrate or a certain concentration of TeAwas added,the mutant could also cause the damage in the host leaves.In addition,it was found that the ROS content of the mutant in the growth stage was higher than that in the wild type.And ROS content in the infection stage was deteced,and the results were the same as in the growth stage.At the same time,the mutant by adding TeA could reduce its ROS content.These results indicated that lack of TeA toxin seems to responsible for the loss of pathogenicity of the HP001 mutant.As a key virulence factor,TeA toxin not only damages the host plant but also involved in maintaining ROS content,host recognition andinducing appressoria to infect the host and for allowing completion of the infection process.2.HP001 is the key pathogenic factor in the development and pathogenicity of A.alternataA suppression subtractive hybridization(SSH)forward substraction was constructed,using the wild type strain as tester and mutant as driver,to study the function of HP001 gene.1210 colonies were selected from forward substraction,845 colonies which had inserts after colony PCR validation were used for dot blot hybridization,and 73 difference significant expression genes were obtained by blasting in NCBI.Gene was classified by COGEME Phytopathogenic Fungi and Oomycete EST database.The results showed that most genes were related to the fungi growth and metabolism,while others were ribosomal proteins and unknown genes.These genes were selected to regulate the downstream regulation of HP001 gene,also associated with the production of toxin.To further verify the accuracy of these genes,real-time PC R was used to detect the expression differences between wild type and mutant.It was found that the expression of 9 genes which had been reported associated with pathogenicity were significantly reduced in the mutant.In addition,the mitogen activated protein kinase(MAPK)gene was also found in those differential genes.And the MAPK signaling pathway could be regulated by the G protein signaling pathway.Previous studies had indicated that G? could be the substrate for histidine phosphatase.The yeast two-hybrid results showed that the yeast contained HP001 and G? could get blue colors on SD-Trp-Leu-Ade-His.The His pull-down was used to verify the results.The results showed that the two proteins could be eluted from the Ni2+column together by 50mM imidazole buffer.These results indicated that HP001 is involved in the G signaling pathway.To proving the role of HP001 gene function in the fungi pathogenicity,the complemented strain of HP001 was obtained through protoplast transformation.Stell,Ste7,Fus3 and Ste12 genes in the MAPK signaling pathway were cloned,expression levels of G? and these genes were determined by real-time PCR to further verify the function of HP001 gene.And the results showed that the expression of these genes are all reduced in the mutant and had recoveried in the complemented strain Further analysis of the physiological function of the wild type,HP001 mutant and the complemented strain was showed that HP001 disruption led to a reduction in the aerial hypha growth and conidiation.The spores of three strains could germinate normally on the host surfaces,but the mutant could not infect the host.The pathogenicity of the mutant was significantly decreased,and the complemented strain was partially restored.The cell wall thickness of the mutant was only half compared to the wild type,and the sensitivity of the mycelium cell wall to cell wall degrading enzymes was increased,while resistant to the salt stress.The content of melanin in the mutant was also significantly lower than that of the wild type.Meanwhile,the ROS content in the mutant was significantly higher than that of the wild type,and the extracellular peroxidase activity was lower.These results indicated that HP001 is an important pathogenic factor in the regulation of fungal growth,infection and pathogenicity.In summary,TeA and HP001 are the key virulence factors of A.alternata infected to the host.TeA toxin which could be regulated by HP001 gene is the direct virulence factor of A.alternata.Exton TeA could recover the pathogensis of the mutant.Thus,TeA and HP001 gene are inseparably interconnected and responsible for the regulation of the pathogenic process of A.alternata.