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.