Phospholipid-binding Protein AVI6 Regulates Antiviral Innate Immunity In Arabidopsis

RNA interference(RNAi)is a highly conserved homologous RNA silencing induced by double-stranded RNA in the evolutionary process,which is involved in the regulation of various physiological processes in eukaryotic organism.Meanwhile,small RNA mediated gene silencing is the main natural mechanism against viral infection in plants.With the discovery of some important regulatory factors,the basic pathway of antiviral RNAi immunity has been preliminarily revealed.However,few new regulatory factors have been discovered for many years,and the mechanism of antiviral RNAi needs to be further explored.We use Viral Suppressor of RNA Silencing(VSR)2b protein deficiency of Cucumber Mosaic virus(CMV-Δ2b),through the efficient forward genetic screen system,to isolate antiviral RNAi-defective(avi)mutant in Arabidopsis by finding new antiviral immune regulatory factors.In this study,we isolated the avi6 mutant through genetic screening.Compared with wild type Col-0,avi6 showed severe disease symptoms,such as growth retardation,the yellow mature leaves and curly young leaves and cell death,two weeks after being infected with CMV-Δ2b.Through genome resequencing and bioinformatics analysis,we found that the single base A insertion mutation on the 13 th exon of the AVI6(At2g15900)gene encoding phospholipid-binding protein in the avi6 mutant.Moreover,through complementary analysis and CRISPR knockout analysis,AVI6 was confirmed as the true gene for the mutant.Surprisingly,although avi6 mutants showed severe disease after the infection of CMV-Δ2b,CMV-2aTΔ2b or wild-type CMV,the accumulation of viral RNAs did not show much change,but the accumulation of viral siRNA accumulation was decreased significantly.It indicated that the mutation of AVI6 destroyed antiviral RNAi immunity in Arabidopsis.Further,through BiFC interaction experiments,we found that AVI6 and two other candidate antiviral regulators,GLTP1 and LPEAT1,interact strongly on the membranous system of cell,respectively.AVI6 is a phospholipid binding protein with PX domain which specifically interact with PI3 P phospholipid.Interestingly,we found that PI3P-deficiency mutant also showed antiviral defects.Combined with the above results,it is further indicated that specific phospholipids or phospholipid-interacting proteins regulate small RNA mediated antiviral innate immunity in plant.