The Interaction Between The Mycoviruses AfPV1 And RNAi-related Genes Of The Host Aspergillus Flavus

Objective:Knockdown of Aspergillus flavus（A.flavus）RNA interference（RNAi）-related genes was validated for growth and development using the mycoviruses Af PV1infection.To explore the connection between mycoviruses Af PV1 and RNAi-related genes in A.flavus hosts.Methods:（1）Confirmation of the genes associated with A.flavus RNAi by search for three classes of genes related to Dicer,Argonaut,and RDRP in NCBI,then constructing phylogenetic trees with MEGA7.0 and predicting conserved structural domains in NCBI;（2）Infection of A.flavus strain CA14 with mycoviruses Af PV1 and RT-PCR detection of the infection results.To detection of the promoted RNAi-related genes by q RT-PCR.（3）Knockdown of RNAi-associated genes promoted by mycoviruses Af PV1 using homologous recombination and identification of CA14 RNAi-associated gene mutant strains using PCR and sourthern blot;（4）Infection of A.flavus RNAi-associated gene mutant strains with mycoviruses Af PV1 and detection of infection results using RT-PCR.（5）The following tests were performed on the RNAi-related gene mutant strains and those infected by mycoviruses Af PV1:（1）Phenotypic observation:Inoculate 10⁶CFU/m L spore suspension in PDA+UU medium and observe the phenotype after 8 days of incubation at 30℃in the dark.（2）Growth rate and spores production measurement:Inoculate 10⁶CFU/m L spore suspension in PDA+UU medium and measure the growth diameter after 5 days of incubation at 30℃in the dark using the crossover method.Conidia on the surface were eluted after 7 days of incubation and counted by hematocrit plate.（3）Sclerotia production determination:Inoculate 10⁶CFU/m L spore suspension in WKM+UU medium,to incubate at 30℃for 14 days,then washing off the mycelium with 75%ethanol and manually counting the number of Sclerotia.（4）Stress response:Inoculate10⁶CFU/m L spore suspension in YGM+UU medium containing cell wall disruptor Congo red（CR）,oxidizer hydrogen peroxide（H₂O₂）,osmotic stress reagent sodium chloride（Na Cl）,and genomic damage reagent methyl methanesulfonate（MMS）,and detect mycoviruses infection with RNAi-related gene mutant strains by measuring the growth diameter after 5 days of incubation continuously with cross hair associated deletion mutant strains.（5）q RT-PCR to detect the expression of mycoviruses Af PV1in RNAi-associated mutant strains.（6）Sequencing of s RNA using A.flavus strains LDF1,LDF1-b.Differential expression of mi RNA enrichment assays predicts the targeting pathways and involved functions of virus Af PV1-mediated mi RNA interference.Results:The phylogenetic tree of Dicer,Argonaut,and RDRP of A.flavus and the prediction of conserved structural domains showed that the three classes of genes of A.flavus RNAi-related proteins had the highest homology and were in the same cluster with Aspergillus oryzae,and also had high homology with other Aspergillus,reaching more than 96%.The structural domains were also highly conserved among Aspergillus.Infection of CA14 with mycoviruses was found to promote the expression of Dcl1,Dcl2,Ago1,Ago2,RDRP1,RDRP2,RDRP3,and individual knockdown of the above genes.The corresponding RNAi-related gene mutant strains were obtained by Sourthern blot identification.Phenotypic experiments on A.flavus RNAi-related genes mutant strains and mycoviruses Af PV1-infected RNAi-related genes mutant strains demonstrated that compared to CA14,?Dcl1,?Dcl2,?Ago1,?Ago2,?RDRP1,?RDRP2,and?RDRP3 mutant strains did not differences in performance on phenotypically,grow rate,asexual reproduction,and Sclerotia development.The CA14-b strain was obtained by infecting CA14 with Mycoviruses Af PV1 and showed aberrant phenotype,significantly reduced growth rate,spore production,and no Sclerotia production.In contrast,compared to CA14-b,those strains?Dcl1-b,?Dcl2-b,?Ago1-b,?Ago2-b,?RDRP1-b,?RDRP2-b,and?RDRP3-b infected with mycoviruses Af PV1 showed a significant increase in growth rate and spore production,but the Sclerotia was still not produced.Stress response assay:cell wall stress assay,RNAi-related gene mutant strains carrying mycoviruses Af PV1 compared to virus-free RNAi mutant strains,the deletion of Dcl1,Dcl2,Ago1,and Ago2 genes all reflect reduced sensitivity to CR.Virus-free RNAi-related gene mutant strains,Dcl1,Dcl2,Ago1 and Ago2 gene deletion reflects reduced sensitivity to CR;In oxidative stress experiments:before the mycoviruses Af PV1 was infected with the RNAi-related mutant strains,the RNAi genes significantly involved in the regulation of oxidative stress were Dcl2,Ago1,RDRP1,RDRP3;However,when the RNAi-related gene mutant strains were infected with the mycoviruses Af PV1,Dcl1and Ago2 were involved in the regulation of oxidative stress.Osmotic stress experiment:RNAi-related gene mutant strains presented Dcl2,Ago1,Ago2,RDRP1genes involved in the stressful role of regulating osmotic stress.In contrast,the mycoviruses Af PV1 infected with RNAi-related genes mutant strains showed RDRP1,RDRP2 and RDRP3 involved in the regulatory role;Genomic stress experiments:RNAi-related gene mutant strains carrying the mycoviruses Af PV1produced mainly the effect of Dcl1,Dcl2,RDRP3 genes compared to virus-free mutant strains.And the virus-free mutant strains exhibited Dcl2,Ago1,Ago2,and RDRP1 genes involved in the regulation of genomic stress.Expression of mycoviruses Af PV1 in RNAi-related genes mutant strains was significantly reduced by q RT-PCR compared to CA14-b.The s RNA transcriptome data of mycoviruses Af PV1 infected A.flavus demonstrated that the conserved Know mi RNA and Novel mi RNA,r RNA,sno RNA,sn RNA,t RNA,unknown RNA expressions have been annotated to LDF1-b were reduced compared to LDF1.The length of mi RNAs was mainly concentrated in the range of 18-22 bp,with the most abundant expression in the size of 20 bp.A total of 40 mi RNAs,including aof-mi R395a,bra-mi R5711,cre-mi R1153-5p.1,gma-mi R10438,gma-mi R1520j,mtr-mi R5272f,were induced to be significantly upregulated at LDF1.mi RNAs were significantly up-regulated,and438 mi RNAs such as aly-mi R163-3p.1,aly-mi R3441-5p.1,aly-mi R3444a-5p,aly-mi R3446-5p,aly-mi R399h-5p,and aly-mi R4229 were significantly down-regulated,and these mi RNAs were mainly involved in interfering with organelle membrane formation and chromosome assembly.Conclusions:Interaction of mycoviruses Af PV1 with Dcl1,Dcl2,Ago1,Ago2,RDRP1,RDRP2,RDRP3 genes in A.flavus RNAi inhibits host growth rate and asexual reproduction.Different degrees of mediated expression of Dcl1,Dcl2,Ago1,Ago2,RDRP1,RDRP2,RDRP3 genes in response to cell wall stress,oxidative stress,osmotic stress and genomic stress.Deletion of Dcl1,Dcl2,Ago1,Ago2,RDRP1,RDRP2,and RDRP3 genes resulted in significant down-regulation of fungal virus Af PV1 expression.Mycoviruses Af PV1 interacted with A.flavus LDF1 RNAi to upregulate the expression of 40 mi RNAs and downregulate the expression of 438mi RNAs to interfere with host growth and development.