Barley GRIK1‐SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection

Abstract

Viruses often usurp host machineries for their amplification, but it remains unclear if hosts may subvert virus proteins to regulate viral proliferation. Here, we show that the 17K protein, an important virulence factor conserved in barley yellow dwarf viruses (BYDVs) and related poleroviruses, is phosphorylated by host GRIK1-SnRK1 kinases, with the phosphorylated 17K (P17K) capable of enhancing the abundance of virus-derived small interfering RNAs (vsiRNAs) and thus antiviral RNAi. Furthermore, P17K interacts with barley small RNA-degrading nuclease 1 (HvSDN1) and impedes HvSDN1catalyzed vsiRNA degradation. Additionally, P17K weakens the HvSDN1-HvAGO1 interaction, thus hindering HvSDN1 from accessing and degrading HvAGO1-carried vsiRNAs. Importantly, transgenic expression of 17K phosphomimetics (17K 5D ), or genome editing of SDN1, generates stable resistance to BYDV through elevating vsiRNA abundance. These data validate a novel mechanism that enhances antiviral RNAi through host subversion of a viral virulence protein to inhibit SDN1-catalyzed vsiRNA degradation and suggest new ways for engineering BYDV-resistant crops.

Appropriate reduction of importin‐α gene expression enhances yellow dwarf disease resistance in common wheat

“…To examine the role of the 33 aa segment in BYDV 17K nuclear import in more detail, we expressed 17K 33del -YFP and 17K 4A -YFP fusions in N. benthamiana using the pea early browning virus (PEBV) vector, with PEBV-mediated expression of YFP or 17K-YFP as controls (Figure 5a). In agreement with our previous finding (Jin et al, 2022b), the plants with 17K-YFP expression exhibited more severe disease symptoms and had increased viral proliferation compared with those expressing YFP (Figure 5b-d). Enhancement of disease symptoms and viral proliferation was also noted for the plants expressing 17K 33del -YFP or 17K 4A -YFP, but the magnitude of the enhancement was much weaker (Figure 5b-d).…”

“…As anticipated, HvIMP‐α1 and ‐α2 were found located in the nucleus when expressed as YFP fusions (HvIMP‐α1‐YFP and HvIMP‐α2‐YFP) in wheat protoplasts (Figure 3d ). In the same set of assays, 17K‐YFP was observed in both the cytoplasm and nucleus of wheat protoplasts (Figure 3d ), which is consistent with previous findings on the subcellular distribution pattern of BYDV 17K (Jin et al ., 2020 , 2022b ; Nass et al ., 1998 ; Xia et al ., 2008 ). Similar results were obtained when HvIMP‐α1‐GFP, HvIMP‐α2‐GFP and 17K‐GFP were transiently expressed in N. benthamiana leaf cells (Figure S2 ).…”

“…Because BYDVs usually exist in very low amounts in their host plants (Miller and Lozier, 2022 ; Nass et al ., 1998 ), we used separate sets of samples for analysing the levels of 17K accumulation in the infected barley and wheat plants and their nuclear and cytoplasmic fractions. Briefly, a comparison of 17K levels in the infected plants by immunoblotting was conducted as described in our previous studies (Jin et al ., 2020 , 2022b ). For preparing the cytosolic fraction, each starting sample was 2 g. But for preparing the nuclear fraction, each starting sample was increased to 5 g in order to get a reliable result.…”

“…Bacterial expression of histidine-tagged recombinant HvIMP-a2 was executed as described before (Jin et al, 2020(Jin et al, , 2022b. Briefly, the coding sequences of HvIMP-a2 were PCR-amplified and cloned into the bacterial expression vector pET30a.…”

Plant virology in the 21st century in China: Recent advances and future directions

“…Moreover, phosphorylation of the cysteine‐rich protein (CRP) of CWMV by serine/threonine‐protein kinase SAPK7 promotes virus infection (Li et al, 2022c). In contrast, phosphorylation of the satBaMV‐encoded P20 protein by cellular kinases inhibits the formation of the satBaMV‐P20 RNP (Vijayapalani et al, 2011), and GRIK1‐SnRK1 kinases phosphorylate barley yellow dwarf virus (BYDV) 17 K protein to upregulate antiviral RNAi and inhibit infection (Jin et al, 2022). In addition, the conserved casein kinase 1 (CK1) in host plants and insect vectors can directly target an intrinsically disordered region of P and inhibit LLPS of P, driving the transition from viral replication to transcription (Gao et al, 2020b; Fang et al, 2022a).…”