AGO1 And AGO2 Are Involved In A New Pathway Of DCL4-mediated RNA Silencing In Arabidopsis

RNA silencing is a ubiquitous RNA-induced regulatory mechanism in eukaryotes which not only regulates the growth and development of plant,but also responds to biotic and abiotic stresses and is an important antiviral defense mechanism as well.RNA silencing pathway involves multiple proteins such as DCLs(Dicer-like.DCL),DRBs(Double strand RNA binding protein,DRB),AGOs(Argonaute)and so on in which AGOs are the major effectors.It was reported that there is a DCL4 initiate antiviral RNA silencing pathway which is incomplete DRB4-dependent in Arabidopsis theliana,while the molecular mechanism is still unclear.In this study we employ molecular biology and genetic approaches to determine whether AGO1 and AG02 involve in the new discovered antiviral RNA silencing pathway and achieve following results:(1)CPB1B and CPB1F which are TCV-based vectors,harbouring 44 bp PDS(phytoene desaturase,PDS)gene sequence from Arabidopsis theliana inserted in the antisense and sense orientation respectively could trigger silencing of the target gene efficiently indicated by the photobleching of leaves.(2)The maximum insert of the VIGS vector CPB-CC which is derived from TCV is no more than 357 bp.CPB-CC based vectors,harbouring less than 187 bp foreign inserts in the antisense orientation could trigger silencing of the target genes in Arabidopsis theliana efficiently.(3)VIGS vectors CPB1B-DCL4A and CPB1B-DCL4B which are CPB1B derived,harbouring 100 bp DCL4 gene fragments from Arabidopsis theliana respectively,could silence PDS and DCL4 gene simultaneously.And the replication of TCV increase significantly when the DCL4 gene in Arabidopsis theliana dcl2 mutant was silenced which indicate we have establish an efficient system for studying whether target genes are involved in anti-TCV silencing pathways by employing TCV-derived VIGS vector CPBI B and Arabidopsis theliana.(4)Using the established research systems reveal that the expression level of AGO1 and AG02 in Arabidopsis theliana dcl2drh4 mutant are up-regulated by infecting with CPBIBGUSA which is TCV-derived VIGS vector harbouring 100 bp GUS gene fragment which indicate both of them are involved in the new discovered antiviral RNA silencing pathway.While the expression level of AGO1 and AGO2 in Arabidopsis theliana dcl2drb4 mutant are down-regulated by infecting with CPB1B-AGO1B,CPB1B-AGO1C and CPB1B-AG02A,CPB1B-AG02B which harbouring 100 bp AGO1 and AG02 gene fragment respectively.Furthermore the replication of TCV increase significantly after knock-down AGO1 or AGO2 in dcl2drb4 mutant.The present data indicate both of them are involved in the new discovered antiviral RNA silencing pathway.(5)To confirm the above result we obtain agoldcl2drb4,ago2dcl2drb4 and agol/2dcl2drb4 Arabidopsis theliana mutants in which AGO1,AG02 were knocked out alone or simultaneously by genetic cross agol,ago2 single mutant or agolago2 double mutant with dcl2drb4 double mutant respectively.The above resulting mutants and dcl2drb4 double mutant as well show varying degrees of PDS silencing after inoculted with CPB1B.The single of photobleach dcl2drb4 mutant in which AGOl and AG02 knockouted simultaneously is much weaker than those mutants in which knockout AGOl or AG02 alone while show most serious viral symptoms.And the replication of TCV increase significantly in ago1dcl2drb4,crgo2dcl2drb4 or agol/2dcl2drb4 mutants comparing with dcl2drb4 mutant.The presented data confirm that both of AGO1 and AG02 involve in DCL4-mediated while incomplete DRB4-dependent antiviral defense and besides the two AGO proteins there is some other AGO protein implicated in the pathway.This study will contribute to elucidate the molecular mechanism of antiviral silencing pathway initiated by DCL4 while independent of DRB4 protein and deepen understanding of the complex molecular mechanisms of virus-host interactions,and explore new and more effective viral control strategies.