Research Article: Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity

Date Published: September 13, 2018

Publisher: Public Library of Science

Author(s): Li Chang, Ho-Hsiung Chang, Jui-Che Chang, Hsiang-Chia Lu, Tan-Tung Wang, Duen-Wei Hsu, Yuh Tzean, An-Po Cheng, Yi-Shu Chiu, Hsin-Hung Yeh, Savithramma P. Dinesh-Kumar.


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.

Partial Text

The plant hormone salicylic acid (SA) plays a major role in triggering local and systemic immune response for combating a broad-spectrum of biotrophic pathogens including viruses [1–3]. SA is involved in innate immunity including pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) to ward off invaders [1]. Plants trigger PTI as the first line of defense through recognition of conserved microbe-associated molecular patterns (MAMPs) by pattern-recognition receptors [4, 5]. PTI is also involved in plant viral resistance, and virus dsRNA has been shown to serve as a MAMP [6–8]. To successfully infect plant hosts, pathogens utilize various effectors to compromise PTI [9]. However, plants have evolved resistance (R) proteins capable of detecting these effectors to trigger ETI, which is a second line of plant defense against viruses [9]. Of note, elevated SA concentration is also essential to establish systemic acquired resistance (SAR) to further protect plants from diverse pathogens [10]. In summary, SA plays an important role in the regulation of PTI, ETI, and SAR to ward off virus infection [1].

In this report, we provided evidence indicating that an orchid SAP gene, Pha13, serves pivotal roles in resistance to viruses through important but previously unidentified SA responsive transcriptional reprogramming of immune responsive gene(s). First, our analysis revealed the striking similarities between Arabidopsis and orchids in plant immune responses. Counterparts of the SA-dependent plant immune responsive genes found in Arabidopsis including PR1, NPR1, RdR1, and GRX were also identified in orchids (i.e. PhaPR1, PhaNPR1, PhaRdR1, and PhaGRX). Indeed, SA induces these orchid genes as they do in Arabidopsis counterparts (Figs 2D and 4A). The dependency on PhaNPR1 for expression of PhaPR1, PhaRdR1, and PhaGRX are also similarly reported in Arabidopsis (Fig 4C). Taking these results together we can see that this central immunity is conserved across plants. Furthermore, we also found that overexpression of Pha13 in engineered transgenic Arabidopsis conferred resistance to various viruses and bacteria (Fig 9). Similarly, a previous report indicated that overexpression of a rice SAP gene, OsSAP1, in tobacco can enhance protection of plants against bacterial pathogen infection [33]. More importantly, we also demonstrated that Arabidopsis homolog of Pha13, AtSAP5, also play similar role in virus resistance and immune regulation (Figs 10 and 11). These findings together suggest that the downstream immune responsive pathways of SAPs are conserved among plants.