Studies On The Genes Involving In Phytotoxin Biosynthesis In Fusarium Oxysporum F.sp.cubense4

Banana fusarium wilt,a soil-borne disease caused by F.oxysporum f.sp.cubense(Foc),has already become one of the main diseases in banana production.When Foc infects bananas,it can secrete two phytotoxins,fusaric acid and Beauverin,to contribute to its pathogenicity.However,some studies have suggested that the amount of Beauverin secreted by Foc during the infection process is very low,whether is one of phytotoxins to its pathogenicity remains uncertainty.In addition,in some researches on the pathogenesis of banana fusarium between the types of vessel obstruction type and the phytotoxin pathogenicity still have controversy.The type of vessel obstruction suggests that during the Foc infecting bananas,it colonizes in the vascular bundle of bananas,and induces callose invasion to be produced,and blocks the vessel,prevents upward transportion of water and nutrients,which consequencely causes plant wilting.However,the type of phytotoxin pathogenicity suggests that the phytotoxins damage the plasma membrane is the most important reason for disease symptom.These arguments can only be resolved through studying the phytotoxin synthesis pathway in the pathogens and interrupting the phytotoxin synthesis and testing the pathogenicity of the mutants.Brown et al.identified a Fub S gene cluster for fusaric acid biosynthesis in Fusarium and speculated a biosynthesis pathway for fusaric acid.The studies of the mutants without the key genes in the Fub gene cluster of F.oxysporum f.sp.lycopersici(tomato specialization)found that it still produced a small amount of fusaric acid and was still pathogenic to plants.Therefore,we speculate that there may be another biosynthesis pathway for fusaric acid in F.oxysporum.The Fub1 and Fub7 key genes in the Fub S gene cluster,the ACMSD and HAD genes in the kynurenine pathway for 2-picolinic acid biosynthesis,and the Beauverin synthase gene Esyn1 were respectively knocked out or interfered in the Banana fusarium wilt pathogen Foc4 using reverse genetic research methods.The phytotoxin products of these mutants were detected by HPLC;and the pathogenicity of these mutants were tested in Banana leaves and seedlings.The detail results were as follows.1.The Fub1 and Fub7 genes in Fub S cluster are the key genes for fusaric acid biosynthesis in Foc4It was predicted by bioinformatic approach that the Fub S gene cluster also exists in Foc4.The two key genes Fub1 and Fub7 in the Fub gene cluster were knocked out in Foc4,and the phytotoxin products of the knockout mutants were tested by HPLC.Similar to F.oxysporum f.sp.lycopersici,the products of fusaric acid in both knockout mutants were reduced,and the fusaric acid product in the Fub1 knockout mutant was reduced more than that in the Fub7 knockout mutant,but both of them did not completely disappear fusaric acid biosynthesis.This result indicates that the Fub gene cluster pathway is also responsible for fusaric acid biosynthesis in Foc4.The decreased fusaric acid products in the Foc4 mutants could reduced banana leaf toxicity and pathogenicity,but not completely lost the pathogenicity.Because without any extra cluster gene homologous to Fub1 to compensate its function in Foc4,thus,it is deuced that there might be the existence of another fusaric acid biosynthesis pathway in Foc4.2.The kynurenine pathway for 2-picolinic acid synthesis may be an another synthetic pathway for fusaric acid in Foc4.The molecular structure of fusaric acid is similar to 2-picolinic acid,in which the hydrogen on the 5-position carbon of 2-picolinic acid is substituted by butyl.In most organisms,2-picolinic acid is biosynthesized via the kynurenine pathway.The five ACMSD-encoding homology genes in the kynurenine pathway have been knocked out in Foc4,and found when the EMT74385 gene was knocked out(one of the five ACMSD-encoding genes),the products of both 2-picolinic acid and fusaric acid were reduced,which was similar as the expressions of the two HAD genes were interfered in Foc4.Thus,the kynurenine pathway for 2-picolinic acid synthesis may be an another synthetic pathway for fusaric acid in Foc4.3.Beauverin might not be directly contribute to the pathogenicity to banana in Foc4The deletion of the Esyn1 gene encoding beauverin synthase resulted in undetectable product of beauverin in Foc4.However,the pathogenicity to banana potted seedlings was not lesser in the double-knockout mutant of both Esyn1 and EMT74385 genes than the single-knockout mutant of EMT74385 gene.It suggests that the lack of beauverin does not affect the pathogenicity,and it might not be directly contribute to the pathogenicity to banana in Foc4.However,the increased pathogenicity resulted from the double-knockout need further investigate.4.Fusaric acid is the main phytotoxin to induce Banana fusarium wilt in Foc4Based on the knocking out the key gene Fub1,the EMT74385 in the kynurenine pathway for another biosynthetic pathway of fusaric acid was further knocked out.The results showed that the productions of fusaric acid by the double-knockout mutant dropped to an undetectable level,which indicates that the kynurenine pathway is another biosynthetic pathway for fusaric acid.At the same time,this double-knockout mutant of Foc4 almost lost their pathogenicity to banana leaves or seedlings.It suggests that only fusaric acid can be considered as phytotoxin in Foc4.Finally,the "toxin pathogenicity type" in the pathogenesis of Banana fusarium wilt was thought to be more reasonable.