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.