Study On The Role Of Ustilago Esculenta Auxin In The Formation Of Culm Gall In Zizania Latifolia

Ustilago esculenta is a biotrophic fungus that exclusively parasitizes in Zizania latifolia.Unlike most diseases caused by pathogens,a special symbiotic relationship was formed between smut fungi and Z.latifolia,which inhibits the heading and flowering of stalks,and stimulates the base of the host plant stem to expand and form a fresh and delicious vegetable——"Jiaobai"."jiaobai" was a common aquatic vegetables in Southeast Asia,and had the largest cultivation area in China,especially in Taihu Lake Basin of Jiangsu and Zhejiang provinces.Previous studies shown that auxin played an important regulatory role in plant-pathogen interaction,and both microorganisms and host plants have the ability to synthesize Indole acetic acid（IAA）.The content of IAA changed significantly during the expansion of the stem gall,and IAA biosynthesis was also found in the culture medium of U.esculenta in vitro.However,whether IAA biosynthesis by U.esculenta plays a role in the growth and development of "Jiaobai" and functions of the related enzymes in the auxin biosynthesis pathway in hyphal formation and stem gall expansion are not clear.In this study,10 enzymes related to auxin biosynthesis were identified in the U.esculenta genome（INSDC:JAAKGJ010000000）by bioinformatics methods,and their physicochemical properties,evolutionary relationship,gene structure and motif of the proteins were analyzed.At the same time,the expression difference of these gene during the hyphal formation in sex compatible strain and in the process of formation and expansion of Z.latifolia were also studied.Furthermore,the gene knockout vector was constructed,and the mutant strain was obtained through homologous recombination by PEG mediated protoplast transformation method,which laid a foundation for further study on the function of UeIAD1.The main results were as follows:1.A total of 10 genes encoding enzymes related to the IAA biosynthetic pathway were identified from the U.esculenta genome database.The molecular weight of the proteins were 33.48～66.24 kDa,and the gene structures were relatively conservative,and all these encoding genes were closely related to the related genes in Ustilago maydis.In the IPA pathway,there were two Trp aminotransferase（TAM）,one Indole-3-pyruvate decarboxylase（IPDC）and four Indole-3-acetaldehyde dehydrogenase（IAD）were found,and the domain structures of Aminotran₁₂,Thiamine pyrophosphatase（TPP）and Aldedh were contained respectively.In the IAM pathway,there were two L-amino acid oxidase（IaaM）gene and one N-acylethanolamine Amidohydrolase（IaaH）were found,and the domain structures of Amino oxidase and Amidase,respectively.2.Three haploid MT-type strains of U.esculenta:UeYD-3 MT10,UeYD-3 MT12 and UeYD-3 MT24 were isolated through single spore separation from "Jiaobai".Further research found that UeYD-3 MT10 and UeYD-3 MT12 combinations（UeYD-3 MT10-12）,UeYD-3 MT12 and UeYD-3 MT24（UeYD-3 MT12-24）combinations could produce the fusion of sexual affinity strains,and the length of the mycelial of UeYD-3 MT10-12 was about twice than UeYD-3 MT12-24.The expression of enzyme related to IAA biosynthetic pathway during the mycelial growth showed that the expression of the UeIADl was significantly increased in the combination with faster mycelial growth.It indicated that auxin probably played an important role in the growth of mycelial,and UelAD1 maybe involved in regulating the growth of mycelial in vitro culture conditions.3.The content of endogenous hormones and gene expression of auxin biosynthesis related enzymes of U.esculenta in different stages of "Jiaobai" were also analyzed.And the result showed that the IAA content increased significantly during the initial formation stage of the culm gall,then gradually decreased but still maintained a high content level during the rapid enlargement stage,and the content of IAA in the late stage of enlargement of "Jiaobai"was the lowest.The expression of most number of the TAM,IPDC,IAD,IaaM and IaaH gene family members in U.esculenta did not change or slightly decreased,then gradually increased thereafter to reach a peak and then showed a decreasing trend during the enlargement of "Jiaobai",and the expression of UeIAD1 increased at the rapid expansion stage of "Jiaobai" and maintained for the longest time.This suggested that IAA may played a critical role in the initiation of stem expansion and IAA synthesized by U.esculenta may not directly induce the initiation of host tumour formation but rather related to the maintenance of auxin levels after stem expansion in Z.latlfolia.4.The genetic transformation system of U.esculenta was established:the strains were grown at 28℃ with shaking for 36-48 h,the highest yield of protoplasts was prepared at 30℃,85 rpm for 3-4h by 2%Driselase+1.5%Lysing enzyme as enzymatic solution and 0.8 M sorbitol as stabilizing agent.The gene knockout vector was constructed,and the mutant strain was obtained through homologous recombination by PEG mediated protoplast transformation method.