Antiviral RNAi defective 4 (AVI4) encodes a lysophospholipid acyltransferase coopting with AVI2 and ATG8a to promote antiviral immunity in Arabidopsis.

RNA interference (RNAi)-mediated antiviral immunity is an evolutionarily conserved mechanism preventing virus infection in eukaryotes. In recent studies, we developed a sensitized genetic screen to identify antiviral RNAi-defective (avi) mutants for uncovering components of the antiviral pathway in Arabidopsis. We revealed that AVI2 is a novel regulator that enhances the biogenesis of viral small interfering RNAs (vsiRNAs) to promote small-RNA-based antiviral immunity. However, the molecular mechanisms that modulate the efficiency of RNAi-mediated antiviral immunity remain largely unclear. Here, we identified the avi4 mutant using the same genetic screen. Mapping by sequencing identified a T-DNA insertion in LPEAT2 as the causal mutation for avi4, and this result was confirmed through analysis of an allelic lpeat2 mutant and complementation analysis. LPEAT2, a lysophospholipid acyltransferase in Arabidopsis, functions in the biosynthesis of phospholipids (especially phosphatidylethanolamine [PE]) and modulation of the autophagy pathway. We revealed that LPEAT2, but not its homolog LPEAT1, interacts with AVI2 to enhance RDR1/6-dependent vsiRNA biogenesis. Interestingly, LPEAT2 also interacts with and stabilizes the core autophagy component ATG8a and promotes autophagy before or after viral infection. Notably, overexpression of ATG8a enhances vsiRNA production and antiviral resistance, which is dependent on LPEAT2 function and can be repressed by VSR 2b. Therefore, our research demonstrates that AVI4 (LPEAT2) is a novel regulator of RNAi-mediated antiviral immunity, it cooperates with AVI2 and ATG8a to enhance vsiRNA biogenesis for robust antiviral defense in Arabidopsis.