Discussion
As obligate parasites with limited genome, plant viruses interact extensively with their hosts to hijack the plant intracellular machinery for self survival and infection, which usually leads to disordered physiological responses and develops to visible diseases that compromising plants growth and development. For counteraction, plants employ multiple strategies to restrict viral infection, including gene silencing, hormone-mediated defense, immune receptor signaling, protein modification and degradation (Alcaide-Loridan and Jupin, 2012; Incarbone and Dunoyer, 2013; Korner et al., 2013; Mandadi and Scholthof, 2013; Calil and Fontes, 2017). The UPS is a highly conserved protein degradation pathway among eukaryotes and is involved in regulating many cellular biological mechanisms, including defense responses against viruses (Alcaide-Loridan and Jupin, 2012; Zhou and Zeng, 2017).
To date, the UPS pathway has been reported to be involved in disrupting different stages in viral infection, such as viral genome replication and movement. For instance, UPS restricted the replication of turnip yellow mosaic virus (TYMV) by degrading and eliminating its RdRp accumulation during viral infection in Arabidopsis (Prod’homme et al., 2001; Camborde et al., 2010). A recent finding proved that an E3 ligase NbUbE3R1 (ubiquitin E3 ligase containing RING domain 1) functioned in inhibiting the replication of bamboo mosaic virus (BaMV), probably because of its interaction with the viral replicase (Chen et al., 2019). And here we found that MdBT2 interacted with and promoted the ubiquitination and degradation of viral protein 1a, which plays an essential role in viral replication according to its homologous in BMV case (Diaz and Wang, 2014), to inhibit the replication of ApNMV in apple (Figs. 3, 4, and 6).
Upon establishment of infection in an individual cell, plant viruses spread from cell to cell to achieve a systemic infection, and MP plays a critical role in virus movement. Specifically, MP forms complexes with viral genome, and then increases the permeability of plasmodesmatal to allow the transportation of the complexes into neighboring cells (Lucas, 2006; Ueki and Citovsky, 2011). Thus, MPs are also potential substrates of UPS pathways in plant defense responses to control viral infection. For example, the MP of tobacco mosaic virus (TMV) (Reichel and Beachy, 2000), TYMV (Drugeon and Jupin, 2002), and potato leafroll virus (PLRV) (Vogel et al., 2007) have been reported to be degraded by the UPS pathways. We also tested the protein interactions between ApNMV MP and MdBT2 to see if the latter is involved in regulating virus movement, but MdBT2 only interacted with 1a, but not with any other viral proteins, including the MP (Supplementary Fig. S3). Nevertheless, UPS pathway has been developed as one of the common strategies that plants utilized to defense virus infection.
BT2 was first identified in an Y2H assay to screen calmodulin-binding proteins (Du and Poovaiah, 2004), and it was later found to be involved in assembling of E3 ligase with CUL3 and RBX1, and functional in substrate recognition in model plant Arabidopsis (Figueroa et al., 2005). Then, BT2 was proved to behave downstream of TAC1 to regulate induction of telomerase (Ren et al., 2007), and also functioned in mediating responses to nutrients (nitrogen and sugar), hormones (auxin and ABA), and stresses (cold and H2O2) (Mandadi et al., 2009). These evidences suggest that BT2 is a multifunctional protein in plant growth and development.
As a nitrate-responsive protein, BT2 coding gene expression is induced by nitrate in both Arabidopsis  (Mandadi et al., 2009) and apple (Supplementary Fig. S1A). And recent findings suggest that MdBT2 regulates accumulation of anthocyanin and malate in apple through interacting with and degrading MdMYB1 (Wang et al., 2018), MdCIbHLH1 (Zhang et al., 2020 a), and MdMYB73 (Zhang et al., 2020 b), respectively, in response to nitrate. And here, we found that nitrate treatment promoted the protein degradation of viral protein 1a in vitro through proteasome pathway (Fig. 2; Supplementary Fig. S2), and overexpressing MdBT2 inhibited the viral RNA replication by targeting and degrading viral protein 1a (Figs. 4 and 6). These findings imply that moderately increased nitrate application might help to control ApNMV infection in apple cultivation. In addition to nitrate, and our recent findings demonstrate that MdBT2 plays critical roles in regulating cellular metabolisms in response to multiple environmental factors as a subunit of E3 ligase. For example, MdBT2 integrates the signals from ABA, wounding, drought, light, and UV-B to regulate biosynthesis and accumulation of anthocyanin by targeting and degrading bZIP44 (basic leucine zipper 44) (An et al., 2018), WRKY40 (An et al., 2019 b), ERF38 (ethylene response factor 38) (An et al., 2020 c), TCP46 (teosinte branched/cycloidea/proliferating 46) (An et al., 2020 d), and BBX22 (B-box 22) (An et al., 2019 c), respectively. In addition, MdBT2 was reported to regulate leaf senescence through MdbHLH93 (An et al., 2019 a), MdMYC2, and MdJAZ2 (JAZMONATE ZIM domain 2) (An et al., 2021). Collectively, these proofs suggested that MdBT2 serves as a signal hub to regulate cellular metabolisms in response to biotic and abiotic stresses.
MdBT2, a member of the BTB-TAZ subfamily, contains a N-terminal BTB domain, a BACK-like domain in the middle, and a C-terminal TAZ domain. As an adaptor protein in the CRL3 ligase complex, BT2 interacts with both CUL3 and potential substrate to mediated the target protein ubiquitination and degradation (Petroski and Deshaies, 2005). We found both the BACK-like and TAZ domains were responsible for interacting with ApNMV 1a (Fig. 3A). MdBT2 also interacted with MdCUL3A (Supplementary Fig. S5A), which has already been proved previously  (Zhao et al., 2016; Wang et al., 2018). However, as a possible component of the E3 ligase, MdCUL3A had rare effect in 1a protein degradation in vitro (Fig. 5A), and MdCUL3A even competed with ApNMV 1a to interact with MdBT2 (Fig. 5B), indicating MdBT2 promotes 1a ubiquitination and degradation in an MdCUL3A-independent pathway. Actually, this has been reported in apple that MdBT2 promotes MdMYB1 degradation in an MdCUL3A-independent pathway (Wang et al., 2018). These findings suggest that MdBT2 might recruit some other, yet unknown, E3 ligases to mediate the ubiquitination and degradation of targets like MdMYB1 and viral 1a protein.
In the well-established model virus BMV, both 1a and 2apol are required and sufficient to support viral genome replication, and 1a-2apol interactions play a critical role in this process (Diaz and Wang, 2014). Deletion of N-terminal of 2apol, which is responsible for interacting with 1a, severely inhibits BMV viral RNA replication (Traynor et al., 1991; Kao and Ahlquist, 1992). Our previous findings revealed that the C-terminal of 1a and N-terminal of 2apol are required for 1a-2apolinteraction (Zhang et al., 2020). We reported here that full-length 1a was required for interacting with MdBT2 (Fig. 3B), and increased amount of MdBT2 interfered with the interaction between 1a and 2apol (Fig. 7), which might be another possible reason that ApNMV viral replication was inhibited in MdBT2-OE transgenic apple leaves (Fig. 6C). In addition, 1a’s inter- and intra-molecular interactions also play an important role in BMV replication (Diaz et al., 2012), and those interactions of ApNMV have been verified in our previous reports (Zhang et al., 2020), thus we predicted that the MdBT2-1a interaction may also interrupt the 1a’s inter- or intra-molecular interactions, and lead to restricted viral RNA replication.
Nitrogen is a macronutrient for plant growth and development, and it is also involved in regulating interactions between the host plants and pathogens (Dordas, 2008). With the nature of available nitrogen in soil, lack or excess of nitrogen might modulate plant resistance through some yet unknown mechanisms to counteract pathogens (Huber and Wstson, 1974). Currently, NO, generated partially in nitrate assimilation by nitrate reductase, is a well-accepted compound that plays critical roles in plant immunity (Wendehenne et al., 2014). NO was first found to mediate defense reactions against bacterial in plants (Delledonne et al., 1998), and it was later demonstrated to active hypersensitive reaction to counteract pathogens in tobacco (Kumar and Klessig, 2000; Asai and Yoshioka, 2009), and enhanced NO content was proved to increase the resistance to TMV (Klessig et al., 2000). What’s more, NO also played a role in BR-mediated resistance against CMV and TMV in plants (Deng et al., 2016; Zou et al., 2018). Moreover, feeding the tobacco plants with nitrate could enhance the resistance to Pseudomonas syringae pv.Phaseolicola by increasing the accumulation of NO and SA, SA-mediated PR gene expression, as well as polyamine-mediated hypersensitive reactions (Gupta et al., 2013). And here, we found that increased nitrate favored the apple plantlets inhibit ApNMV genomic RNA replication through MdBT2-mediated ubiquitination and degradation of viral replication protein 1a. Of course, enhanced nitrate might also inhibit ApNMV genomic RNA replication through SA or NO signaling pathways, which is an interesting point and need further investigation to elucidate.
In sum, we identified a nitrate-responsive BTB domain-containing protein MdBT2 in apple, which inhibits the ApNMV viral RNA replication by mediating degradation of ApNMV 1a protein, as well as interfering with the interaction between viral replication proteins 1a and 2apol. Our work provides the theoretical foundation to control apple mosaic disease in apple cultivation, and also determines a potential target gene that may be applied in genetic breeding to control apple mosaic disease in apple.
Supplementary Fig. S1 The gene expression of MdBT2 (A) and MdNRT1.1 (B) in response to KNO3 and KCl. The apple plantlets were treated with KNO3 or KCl with the indicated time, and samples were collected for RNA extraction. Then qRT-PCR were utilized to test the expression of the two genes. **, P<0.01; ***, P<0.001.
Supplementary Fig. S2 Protein degradation of 1a-HIS and 2apol-HIS in vitro. A. 2apol-HIS protein degradation in proteins extracted from ‘GL3’ leaves treated with KNO3 or KCl. 1a-HIS protein degradation in proteins extracted from wt apple calli pretreated with KNO3 or KCl in the absence (A) or presence (B) of MG132. The charts on the right side indicated the degradation trends in (A)-(C), and the intensity of protein bands at 0 h was set as 1.00. MdACTIN served as loading control.
Supplementary Fig. S3 A Y2H assay was used to test the interactions of MdBT2 with the four viral proteins, 1a, 2apol, MP, and CP.
Supplementary Fig. S4 Using qRT-PCR to test the gene expression of MdBT2 in transgenic apple calli (A) and GL3 shoots (B). *, P<0.05; ***, P<0.001.
Supplementary Fig. S5 MdBT2 interacts with MdCUL3A in vitro. A. Pull-down assay showed the in vitro interactions between GST-MdBT2 and MdCUL3A-HIS. The anti-GST and anti-HIS antibodies were used to detect the target proteins. GST-MdBT2, GST, MdCUL3A-HIS bands are indicated by short lines on the right side. B. Using qRT-PCR to test the expression of MdCUL3A in its overexression transgenic apple calli. ***, P<0.001.