Functional Analysis Of Genes Involved In The Small RNA Pathways In Valsa Mali

Valsa canker of apple tree,caused by the fungus Valsa mali,is one of the most destructive diseases on apple in China and eastern Asia.Small-RNA-guided gene regulation has emerged as one of the fundamental principles in cell function.Dicer and Argonaute are the two major protein players in this process.The function of the genes involved in small RNA pathway have been reported in many species,including some phytopathogenic fungus,while the roles of dicer-like(DCL)and Argonaute(AGO)in Vinali are not clear.In this study,the function of DCL and AGO were reseached through comparing the growth,phenotype and pathogenicity between the wild strain and DCL,AGO gene deletion mutants.Our study will lay a theoretical basis for revealing the molecular genetic mechanisms of the small RNAs generation in Vmali.The results were as follows:1.Two genes,dcl-1 and dcl-2,encoding Dicer-like proteins and three genes,encoding Argonaute protein,were identified in Vmali through bioinformatics analysis.2.Four replacement mutants of VMDCL1,five replacement mutants of VMDCL2,six replacement mutants of VMAGO2 and one replacement mutants of VMAGO3 were obtained.All deletion mutants had been verified through PCR and Southern blot.3.DCL2 may be involved in the mycelia growth.Compared with the wild strain,the mycelium production of DCL2 deletion mutant increased significantly.The results of infecting Fuji blade experiments showed that DCL1 may be related to the pathogenicity of Vmali.The pathogenicity of DCL1 knockout mutant increased while the deletion of DCL2 had no obvious effect on the pathogenicity of Vmali.4.AGO2 may be related to the growth of mycelium.Compared with the wild strain,AGO2 deletion mutant had less branches and the mycelia was thicker;AGO3 may be related to the formation of propagules.Compared with wild strain 03-8,the propagules production of AGO3 knockout mutant increased significantly;AGO2 and AGO3 may be associated with virulence of Vmali.The deletion of AGO2 led to the reduction of pathogenicity and the reduction was significant while the pathogenicity of AGO3 deletion mutant enhanced significantly.5.DCL1、DCL2 and AGO2 may be involved in reponse to stress.A test of mutants under hydrogen peroxide stress was conducted.Deletion of DCL1 resulted in becoming more resistant to hydrogen peroxide stress while the DCL2 deletion mutant was more sensitive to the hydrogen peroxide stress.The colony morphology of AGO2 mutant was very different from other mutants and the wild strain 03-8;The deletion of DCL1 made the strain more sensitive under high osmotic pressure in PDA while the other mutants made no difference from wild strain 03-8 under under this pressure.