MAPKs trigger antiviral immunity by directly phosphorylating a rhabdovirus nucleoprotein in plants and insect vectors.

Signaling by the evolutionarily conserved mitogen-activated protein kinase or extracellular signal-regulated kinase (MAPK/ERK) plays critical roles in converting extracellular stimuli into immune responses. However, whether MAPK/ERK signaling induces virus immunity by directly phosphorylating viral effectors remains largely unknown. Barley yellow striate mosaic virus (BYSMV) is an economically important plant cytorhabdovirus that is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. Here, we found that the barley (Hordeum vulgare) MAPK MPK3 (HvMPK3) and the planthopper ERK (LsERK) proteins interact with the BYSMV nucleoprotein (N) and directly phosphorylate N protein primarily on serine 290. The overexpression of HvMPK3 inhibited BYSMV infection, whereas barley plants treated with the MAPK pathway inhibitor U0126 displayed greater susceptibility to BYSMV. Moreover, knockdown of LsERK promoted virus infection in SBPHs. A phosphomimetic mutant of the N Ser290 (S290D) completely abolished virus infection because of impaired self-interaction of BYSMV N and formation of unstable N-RNA complexes. Altogether, our results demonstrate that the conserved MAPK and ERK directly phosphorylate the viral nucleoprotein to trigger immunity against cross-kingdom infection of BYSMV in host plants and its insect vectors.

A rhabdovirus accessory protein inhibits jasmonic acid signaling in plants to attract insect vectors.

Plant rhabdoviruses heavily rely on insect vectors for transmission between sessile plants. However, little is known about the underlying mechanisms of insect attraction and transmission of plant rhabdoviruses. In this study, we used an arthropod-borne cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), to demonstrate the molecular mechanisms of a rhabdovirus accessory protein in improving plant attractiveness to insect vectors. Here, we found that BYSMV-infected barley (Hordeum vulgare L.) plants attracted more insect vectors than mock-treated plants. Interestingly, overexpression of BYSMV P6, an accessory protein, in transgenic wheat (Triticum aestivum L.) plants substantially increased host attractiveness to insect vectors through inhibiting the jasmonic acid (JA) signaling pathway. The BYSMV P6 protein interacted with the constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5) of barley plants in vivo and in vitro, and negatively affected CSN5-mediated deRUBylation of cullin1 (CUL1). Consequently, the defective CUL1-based Skp1/Cullin1/F-box ubiquitin E3 ligases could not mediate degradation of jasmonate ZIM-domain proteins, resulting in compromised JA signaling and increased insect attraction. Overexpression of BYSMV P6 also inhibited JA signaling in transgenic Arabidopsis (Arabidopsis thaliana) plants to attract insects. Our results provide insight into how a plant cytorhabdovirus subverts plant JA signaling to attract insect vectors.

Casein Kinase 1 Regulates Cytorhabdovirus Replication and Transcription by Phosphorylating a Phosphoprotein Serine-Rich Motif.

Casein kinase 1 (CK1) family members are conserved Ser/Thr protein kinases that regulate important developmental processes in all eukaryotic organisms. However, the functions of CK1 in plant immunity remain largely unknown. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, infects cereal crops and is obligately transmitted by the small brown planthopper (SBPH; Laodelphax striatellus). The BYSMV phosphoprotein (P) exists as two forms with different mobilities corresponding to 42 kD (P42) and 44 kD (P44) in SDS-PAGE gels. Mass spectrometric analyses revealed a highly phosphorylated serine-rich (SR) motif at the C-terminal intrinsically disordered region of the P protein. The Ala-substitution mutant (PS5A) in the SR motif stimulated virus replication, whereas the phosphorylation-mimic mutant (PS5D) facilitated virus transcription. Furthermore, PS5A and PS5D associated preferentially with nucleocapsid protein-RNA templates and the large polymerase protein to provide optimal replication and transcription complexes, respectively. Biochemistry assays demonstrated that plant and insect CK1 protein kinases could phosphorylate the SR motif and induce conformational changes from P42 to P44. Moreover, overexpression of CK1 or a dominant-negative mutant impaired the balance between P42 and P44, thereby compromising virus infections. Our results demonstrate that BYSMV recruits the conserved CK1 kinases to achieve its cross-kingdom infection in host plants and insect vectors.

Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies.

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid ß-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.

A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43-55) and the remaining region (aa 56-295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

A cytorhabdovirus-based expression vector in Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera.

The brown planthopper (BPH, Nilaparvata lugens), the small brown planthopper (SBPH, Laodelphax striatellus), and the white-backed planthopper (WBPH, Sogatella furcifera) are problematic insect pests and cause severe yield losses through phloem sap-sucking and virus transmission. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, has been developed as versatile expression platforms in SBPHs and cereal plants. However, bio-safe overexpression vectors based on recombinant BYSMV (rBYSMV) remain to be developed and applied to the three kinds of planthoppers. Here, we found that rBYSMV was able to infect SBPHs, BPHs and WBPHs through microinjection with crude extracts from rBYSMV-infected barley leaves. To ensure bio-safety of the rBYSMV vectors, we generated an rBYSMV mutant by deleting the accessory protein P3, a putative viral movement protein. As expected, the resulting mutant abolished viral systemic infection in barley plants but had no effects on BYSMV infectivity in insect vectors. Subsequently, we used the modified rBYSMV vector to overexpress iron transport peptide (ITP) in the three kinds of planthoppers and revealed the potential functions of ITP. Overall, our results provide bio-safe overexpression platforms to facilitate functional genomics studies of planthoppers.

CCR4, a RNA decay factor, is hijacked by a plant cytorhabdovirus phosphoprotein to facilitate virus replication.

Carbon catabolite repression 4 (CCR4) is a conserved mRNA deadenylase regulating posttranscriptional gene expression. However, regulation of CCR4 in virus infections is less understood. Here, we characterized a pro-viral role of CCR4 in replication of a plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV). The barley (Hordeum vulgare) CCR4 protein (HvCCR4) was identified to interact with the BYSMV phosphoprotein (P). The BYSMV P protein recruited HvCCR4 from processing bodies (PBs) into viroplasm-like bodies. Overexpression of HvCCR4 promoted BYSMV replication in plants. Conversely, knockdown of the small brown planthopper CCR4 inhibited viral accumulation in the insect vector. Biochemistry experiments revealed that HvCCR4 was recruited into N-RNA complexes by the BYSMV P protein and triggered turnover of N-bound cellular mRNAs, thereby releasing RNA-free N protein to bind viral genomic RNA for optimal viral replication. Our results demonstrate that the co-opted CCR4-mediated RNA decay facilitates cytorhabdovirus replication in plants and insects.