Introduction
Apple mosaic disease is one the major and widely distributed viral diseases affecting apple growth and production all over the world. The causal agent of the disease was traditionally believed to be apple mosaic virus (ApMV), which belongs to the genus Ilarvirus , familyBromoviridae  (Bujarski et al., 2012). However, recent studies revealed that apple necrotic mosaic virus (ApNMV), other than ApMV, is highly associated with the occurrence of apple mosaic disease in China (Noda et al., 2017; Xing et al., 2018), whose apple production accounts for half of the world.
ApNMV is in the same genus with ApMV, and both of them share the same genomic structure (Noda et al., 2017). They have three positive single-stranded genomic RNAs (RNA1, RNA2, and RNA3) and an encapsidated subgenomic RNA4 derived from RNA3 (Noda et al., 2017). RNA1 encodes the 1a protein, which is characterized with an N-terminal methyltransferase (MET) domain and a C-terminal NTP-binding helicase (HEL) domain. RNA2 encodes the viral RNA-dependent RNA polymerase (RdRp, 2apol). The movement protein (MP) is encoded by the RNA3, while the coat protein (CP) is encoded the subgenomic RNA4 (Bujarski et al., 2012). Based on a well-characterized model virus brome mosaic virus (BMV), which belongs to the same family and shares similar genomic structure with ApNMV, 1a is a multifunctional protein playing essential roles in virus replication, including inducing the formation of viral replication complex (VRC), recruiting 2apol and template RNAs into these VRCs, and facilitating the viral genomic RNA replication (Diaz and Wang, 2014).
In natural environments, plants face continuous biotic and abiotic stresses that compromise their survival. To counteract these environmental challenges, plants have evolved various complex and efficient mechanisms of resistance, including the ubiquitin proteasome system (UPS) that is highly conserved among eukaryotes (Zhou and Zeng, 2017; Adams and Spoel, 2018). The UPS is an enzymatic process in which ubiquitin moieties are covalently conjugated to substrate proteins for degradation by proteasome, and this process has been demonstrated to play key roles in many intracellular biological processes of plants (Bachmair et al., 2001; Vierstra 2009; Alcaide-Loridan and Jupin, 2012). The ubiquitination process is mediated by a series of enzymes including an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and an ubiquitin E3 ligase (E3). Among them, E3 is a key component for targeting specificity by interacting with target substrates and transferring ubiquitins from E2 to the targets, and thus, generating the ubiquitin modification (Metzger et al., 2014). Based on the composition and activation mechanisms, four types of E3 ligases are mainly found in plants, including HECT (homologous to E3 associated protein C-terminus), RING (really interesting new gene), U-box, and CRLs (cullin-RING ligases) (Mazzucotelli et al., 2006; Vierstra, 2009). CRLs are the most abundant E3 ligases in plants and they exist as complexes with a cullin (CUL) subunit serving as molecular scaffold, and three types of CUL (CUL1, CUL3, and CUL4) have been reported in various plants (Hotton and Callis, 2008).
BTB (bric-a-brac, tramtrack and broad complex) type E3 ligase is one of the CRL subfamilies (Vierstra, 2009). In the CUL3-RING E3 ligase (CRL3) of model plant Arabidopsis thaliana , BTB/POZ (poxvirus and zinc finger) domain-containing proteins directly interact with both the CUL3 and substrate target, and thus serve as the substrate receptor to select proteins for degradation via the UPS (Hua and Vierstra, 2011; Genschik et al., 2013). A body of evidences has been reported to reveal the critical roles of BTB/POZ domain-containing proteins in multiple intracellular processes. For instance, BTB-BACK domain protein POB1 regulated plant immunity by interacting with and targeting PUB (Plant U-box) 17 and PUB29 for degradation in Nicotiana benthamiana  (Orosa et al., 2017) and apple (Malus domestica ) (Han et al., 2019), respectively. In Arabidopsis, AtBT2 contains an N-terminal BTB/POZ domain, a central TAZ (transcriptional adaptor zinc finger) domain, and a C-terminal calmodulin-binding domain (Ren et al., 2007). AtBT2 has been reported to be involved in regulation of multiple responses, such as responding to circadian, light, stresses, and nutrients; suppressing the sugar signaling; modulating plant hormone responses by suppressing abscisic acid (ABA) signaling while enhancing auxin signaling; and regulating telomerase activity by acting downstream of TAC1 (TELOMERASE ACTIVATOR1) (Ren et al., 2007; Mandadi et al., 2009; Kunz et al., 2015; Misra et al., 2018). MdBT2, a homologue of AtBT2, shares similar protein structure with AtBT2, and has also been demonstrated to function as a signal hub to regulate anthocyanin biosynthesis, leaf senescence, iron homeostasis, and malate accumulation in response to multiple hormonal and environmental signals (Zhao et al., 2016; An et al., 2019a; An et al., 2020 a; Zhang et al., 2020 a, b). For example, MdBT2 interacts with and promotes the ubiquitination and degradation of MdMYB1 and MdCIbHLH1 to inhibit accumulation of anthocyanin (Wang et al., 2018) and malate (Zhang et al., 2020 a, b), respectively, in response to nitrate. In addition, MdBT2 functions in delaying the leaf senescence by interacting with and promoting the ubiquitination and degradation of MdbHLH93 and MdMYC2 in apple (An et al., 2019 a; An et al., 2021).
Nitrogen (N) is a major nutrient for plant growth and productivity, and it has been reported to play key roles in plant immunity by regulating plant resistance against various pathogens (Dordas, 2008). To date, a well known defense-related N-derivant is nitric oxide (NO), which is partially generated through nitrate reductase (NR), a key enzyme in nitrate assimilation. Multiple evidences have demonstrated the roles of NO in transcriptional regulation of defense genes encoding pathogen-related (PR) proteins or proteins involved in phytoalexin synthesis, post-translational protein modifications, and salicylic acid (SA) accumulation (reviewed in Wendehenne et al., 2014). For example, NO was functional in brassinosteroid (BR)-mediated resistance against virus infection in N. benthamiana (tobacco mosaic virus, TMV) (Deng et al., 2016) and Arabidopsis (cucumber mosaic virus, CMV) (Zou et al., 2018). Additionally, nitrate, an inorganic nitrogen that is usually taken up by roots from aerobic soil, was also proved to be involved in disease resistance. For example, application of NO3- efflux inhibitor delayed and reduced the hypersensitive cell death triggered by cryptogein in tobacco, which was accompanied by the suppression of induction of some defense-related genes (Wendehenne et al., 2002). Moreover, feeding the tobacco plants with NO3- enhanced the accumulation of SA and expression of PR1 gene, as well as the speed of cell death upon infection of Pseudomonas syringae pv.Phaseolicola (Gupta et al., 2013). All these reports revealed the potential role of nitrogen in resistance against pathogens.
In this study, we found that a nitrate-responsive protein MdBT2 interacted with ApNMV protein 1a, and promoted its ubiquitination and degradation through 26S proteasome pathways in a MdCUL3A-independent manner. ApNMV genomic RNA accumulation was inhibited in MdBT2overexpression (MdBT2-OE ) transgenic apple leaves but enhanced inMdBT2 antisense (MdBT2-anti ) compared to that in the wild-type (WT). In addition, MdBT2 interfered with the interaction between 1a and 2apol through competitive interacting with 1a.