Salicylic acid (SA) is a key phytohormone that mediates a broad spectrum of resistance against a diverse range of viruses; however, the downstream pathway of SA governed antiviral immune response remains largely to be explored. Here, we identified an orchid protein containing A20 and AN1 zinc finger domains, designated Pha13. Pha13 is up-regulated upon virus infection, and the transgenic monocot orchid and dicot Arabidopsis overexpressing orchid Pha13 conferred greater resistance to different viruses. In addition, our data showed that Arabidopsis homolog of Pha13, AtSAP5, is also involved in virus resistance. Pha13 and AtSAP5 are early induced by exogenous SA treatment, and participate in the expression of SA-mediated immune responsive genes, including the master regulator gene of plant immunity, NPR1, as well as NPR1-independent virus defense genes. SA also induced the proteasome degradation of Pha13. Functional domain analysis revealed that AN1 domain of Pha13 is involved in expression of orchid NPR1 through its AN1 domain, whereas dual A20/AN1 domains orchestrated the overall virus resistance. Subcellular localization analysis suggested that Pha13 can be found localized in the nucleus. Self-ubiquitination assay revealed that Pha13 confer E3 ligase activity, and the main E3 ligase activity was mapped to the A20 domain. Identification of Pha13 interacting proteins and substrate by yeast two-hybrid screening revealed mainly ubiquitin proteins. Further detailed biochemical analysis revealed that A20 domain of Pha13 binds to various polyubiquitin chains, suggesting that Pha13 may interact with multiple ubiquitinated proteins. Our findings revealed that Pha13 serves as an important regulatory hub in plant antiviral immunity, and uncover a delicate mode of immune regulation through the coordination of A20 and/or AN1 domains, as well as through the modulation of E3 ligase and ubiquitin chain binding activity of Pha13.

22Salicylic acid (SA)-mediated immunity plays important roles in combating virus in 23 plants. Two plant stress associated protein (SAPs) containing dual A20/AN1 zinc-24 finger domain were found to play important roles in SA-mediated immunity; however, 25 detailed mechanisms remain elusive. In this study, another orchid homolog gene of 26 Pha13, Pha21, was analyzed. Pha21 confers antiviral immunity in both transgenic 27 orchid and Arabidopsis overexpressing Pha21. Expression of Pha21 is early-induced 28 by SA treatment, and is involved in the expression of the orchid homolog of the master 29 regulator NPR1. Pha21 but not Pha13 is involved in the expression of key RNAi-related 30 genes, Dicer-like nuclease 4 (DCL4) and Argonaut 1 (AGO1) in orchids. The 31 involvement of SAPs in expression of orchid DCL4 and AGO1 is not limited to orchid, 32 as AtSAP5 also plays essential role in the expression of Arabidopsis DCL4 and AGO1. 33 However, unlike Pha13 and AtSAP5, Pha21 does not play positive role in the 34 expression of orchid homolog gene of RNA-dependent RNA polymerase 1 (RdR1), an 35 important gene in RNAi pathway. Pha21 can be found localized in the nucleus, and 36 confers self-E3 ligase and ubiquitin binding activities. Functional domain analysis 37 revealed that both A20 and AN1 domains of Pha21 are required for decreasing virus 38 accumulation, and the AN1 domain plays a more important role in the expression of 39 orchid DCL4. Collectively, our data suggests SA regulated SAPs play important roles 40 in antiviral immunity and is involved in delicate regulation of key genes in RNAi-41 mediated pathway. 42 43 IMPORTANCE 44 3