A potyvirus provides an efficient viral vector for gene expression and functional studies in Asteraceae plants.

Plant virus-derived vectors are rapid and cost-effective for protein expression and gene functional studies in plants, particularly for species that are difficult to genetically transform. However, few efficient viral vectors are available for functional studies in Asteraceae plants. Here, we identified a potyvirus named zinnia mild mottle virus (ZiMMV) from common zinnia (Zinnia elegans Jacq.) through next-generation sequencing. Using a yeast homologous recombination strategy, we established a full-length infectious cDNA clone of ZiMMV under the control of the cauliflower mosaic virus 35S promoter. Furthermore, we developed an efficient expression vector based on ZiMMV for the persistent and abundant expression of foreign proteins in the leaf, stem, root, and flower tissues with mild symptoms during viral infection in common zinnia. We showed that the ZiMMV-based vector can express ZeMYB9, which encodes a transcript factor inducing dark red speckles in leaves and flowers. Additionally, the expression of a gibberellic acid (GA) biosynthesis gene from the ZiMMV vector substantially accelerated plant height growth, offering a rapid and cost-effective method. In summary, our work provides a powerful tool for gene expression, functional studies, and genetic improvement of horticultural traits in Asteraceae plant hosts.

The plant rhabdovirus viroporin P9 facilitates insect-mediated virus transmission in barley.

Potassium (K+) plays crucial roles in both plant development and immunity. However, the function of K+ in plant-virus interactions remains largely unknown. Here, we utilized Barley yellow striate mosaic virus (BYSMV), an insect-transmitted plant cytorhabdovirus, to investigate the interplay between viral infection and plant K+ homeostasis. The BYSMV accessory P9 protein exhibits viroporin activity by enhancing membrane permeability in Escherichia coli. Additionally, P9 increases K+ uptake in yeast (Saccharomyces cerevisiae) cells, which is disrupted by a point mutation of Glycine 14 to Threonine (P9G14T). Furthermore, BYSMV P9 forms oligomers and targets to both the viral envelope and the plant membrane. Based on the recombinant BYSMV-green fluorescent protein (BYGFP) virus, a P9-deleted mutant (BYGFPΔP9) was rescued and demonstrated infectivity within individual plant cells of Nicotiana benthamiana and insect vectors. However, BYGFPΔP9 failed to infect barley plants after transmission by insect vectors. Furthermore, infection of barley plants was severely impaired for BYGFP-P9G14T lacking P9 K+ channel activity. In vitro assays demonstrate that K+ facilitates virion disassembly and the release of genome RNA for viral mRNA transcription. Altogether, our results show that the K+ channel activity of viroporins is conserved in plant cytorhabdoviruses and plays crucial roles in insect-mediated virus transmission.

A plant cytorhabdovirus modulates locomotor activity of insect vectors to enhance virus transmission.

Transmission of many plant viruses relies on phloem-feeding insect vectors. However, how plant viruses directly modulate insect behavior is largely unknown. Barley yellow striate mosaic virus (BYSMV) is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus). Here, we show that BYSMV infects the central nervous system (CNS) of SBPHs, induces insect hyperactivity, and prolongs phloem feeding duration. The BYSMV accessory protein P6 interacts with the COP9 signalosome subunit 5 (LsCSN5) of SBPHs and suppresses LsCSN5-regulated de-neddylation from the Cullin 1 (CUL1), hereby inhibiting CUL1-based E3 ligases-mediated degradation of the circadian clock protein Timeless (TIM). Thus, virus infection or knockdown of LsCSN5 compromises TIM oscillation and induces high insect locomotor activity for transmission. Additionally, expression of BYSMV P6 in the CNS of transgenic Drosophila melanogaster disturbs circadian rhythm and induces high locomotor activity. Together, our results suggest the molecular mechanisms whereby BYSMV modulates locomotor activity of insect vectors for transmission.

The small peptide VISP1 acts as a selective autophagy receptor regulating plant-virus interactions.

Selective macroautophagy/autophagy is tightly regulated by cargo receptors that recruit specific substrates to the ATG8-family proteins for autophagic degradation. Therefore, identification of selective receptors and their new cargoes will improve our understanding of selective autophagy functions in plant development and stress responses. We have recently demonstrated that the small peptide VISP1 acts as a selective autophagy receptor to mediate degradation of suppressors of RNA silencing (VSRs) of several RNA and DNA viruses. Moreover, VISP1 induces symptom recovery through fine-tuning the balance of plant immunity and virus pathogenicity. Our findings provide new insights into the double-edged sword roles of selective autophagy in plant-virus interactions.

A selective autophagy receptor VISP1 induces symptom recovery by targeting viral silencing suppressors.

Selective autophagy is a double-edged sword in antiviral immunity and regulated by various autophagy receptors. However, it remains unclear how to balance the opposite roles by one autophagy receptor. We previously identified a virus-induced small peptide called VISP1 as a selective autophagy receptor that facilitates virus infections by targeting components of antiviral RNA silencing. However, we show here that VISP1 can also inhibit virus infections by mediating autophagic degradation of viral suppressors of RNA silencing (VSRs). VISP1 targets the cucumber mosaic virus (CMV) 2b protein for degradation and attenuates its suppression activity on RNA silencing. Knockout and overexpression of VISP1 exhibit compromised and enhanced resistance against late infection of CMV, respectively. Consequently, VISP1 induces symptom recovery from CMV infection by triggering 2b turnover. VISP1 also targets the C2/AC2 VSRs of two geminiviruses and enhances antiviral immunity. Together, VISP1 induces symptom recovery from severe infections of plant viruses through controlling VSR accumulation.

Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement.

Viral replication and movement are intimately linked; however, the molecular mechanisms regulating the transition between replication and subsequent movement remain largely unknown. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein promotes viral replication and movement by interacting with the αa replicase and TGB1 movement proteins. Here, we found that γb is palmitoylated at Cys-10, Cys-19, and Cys-60 in Nicotiana benthamiana, which supports BSMV infection. Intriguingly, non-palmitoylated γb is anchored to chloroplast replication sites and enhances BSMV replication, whereas palmitoylated γb protein recruits TGB1 to the chloroplasts and forms viral replication-movement intermediate complexes. At the late stages of replication, γb interacts with NbPAT15 and NbPAT21 and is palmitoylated at the chloroplast periphery, thereby shifting viral replication to intracellular and intercellular movement. We also show that palmitoylated γb promotes virus cell-to-cell movement by interacting with NbREM1 to inhibit callose deposition at the plasmodesmata. Altogether, our experiments reveal a model whereby palmitoylation of γb directs a dynamic switch between BSMV replication and movement events during infection.

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.

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.

Barley stripe mosaic virus γb protein targets thioredoxin h-type 1 to dampen salicylic acid-mediated defenses.

Salicylic acid (SA) acts as a signaling molecule to perceive and defend against pathogen infections. Accordingly, pathogens evolve versatile strategies to disrupt the SA-mediated signal transduction, and how plant viruses manipulate the SA-dependent defense responses requires further characterization. Here, we show that barley stripe mosaic virus (BSMV) infection activates the SA-mediated defense signaling pathway and upregulates the expression of Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1). The γb protein interacts directly with NbTRXh1 in vivo and in vitro. The overexpression of NbTRXh1, but not a reductase-defective mutant, impedes BSMV infection, whereas low NbTRXh1 expression level results in increased viral accumulation. Similar with its orthologs in Arabidopsis (Arabidopsis thaliana), NbTRXh1 also plays an essential role in SA signaling transduction in N. benthamiana. To counteract NbTRXh1-mediated defenses, the BSMV γb protein targets NbTRXh1 to dampen its reductase activity, thereby impairing downstream SA defense gene expression to optimize viral cell-to-cell movement. We also found that NbTRXh1-mediated resistance defends against lychnis ringspot virus, beet black scorch virus, and beet necrotic yellow vein virus. Taken together, our results reveal a role for the multifunctional γb protein in counteracting plant defense responses and an expanded broad-spectrum antibiotic role of the SA signaling pathway.

Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection.

Liquid-liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infection is largely unknown. Here, we used barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 (CK1) in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and the 5' trailer of genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host CK1-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted viroplasm formation and virus replication. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state for virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of the viral P protein and virus infection.

Developing reverse genetics systems of northern cereal mosaic virus to reveal superinfection exclusion of two cytorhabdoviruses in barley plants.

Recently, reverse genetics systems of plant negative-stranded RNA (NSR) viruses have been developed to study virus-host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV-RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV-RFP and green fluorescent protein (GFP)-tagged barley yellow striate mosaic virus (rBYSMV-GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV-GFP and rNCMV-RFP was observed in barley leaves. Our results demonstrate that two insect-transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.

A Versatile Expression Platform in Insects and Cereals Based on a Cytorhabdovirus.

In recent years, plant virus-based vectors have been widely applied to express heterologous proteins for genomic studies and commercial production. Among these versatile RNA viral vectors, the barley yellow striate mosaic virus (BYSMV)-based expression vector system has outstanding capability to express large and multiple heterologous proteins. Here we describe a detailed protocol for expression of heterologous proteins using BYSMV expression systems in monocot plants and insects.

RNA In Situ Hybridization of Detecting Cucumber Mosaic Virus in Shoots of Nicotiana benthamiana Plants.

RNA in situ hybridization, a histological technique derived from Southern blotting and northern blotting, has been an important approach in biology studies for many years. In the studies of virus-plant interactions, RNA in situ hybridization provides a direct visualization of viral RNA in host plants. Here, we provide a detailed protocol for viral RNA in situ hybridization that has been successfully used to detect Cucumber mosaic virus genome (CMV) RNAs in shoots of N. benthamiana plants.

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.

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication.

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH-dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC-mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2-Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC-mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.

A small peptide inhibits siRNA amplification in plants by mediating autophagic degradation of SGS3/RDR6 bodies.

Selective autophagy mediates specific degradation of unwanted cytoplasmic components to maintain cellular homeostasis. The suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6)-formed bodies (SGS3/RDR6 bodies) are essential for siRNA amplification in planta. However, whether autophagy receptors regulate selective turnover of SGS3/RDR6 bodies is unknown. By analyzing the transcriptomic response to virus infection in Arabidopsis, we identified a virus-induced small peptide 1 (VISP1) composed of 71 amino acids, which harbor a ubiquitin-interacting motif that mediates interaction with autophagy-related protein 8. Overexpression of VISP1 induced selective autophagy and compromised antiviral immunity by inhibiting SGS3/RDR6-dependent viral siRNA amplification, whereas visp1 mutants exhibited opposite effects. Biochemistry assays demonstrate that VISP1 interacted with SGS3 and mediated autophagic degradation of SGS3/RDR6 bodies. Further analyses revealed that overexpression of VISP1, mimicking the sgs3 mutant, impaired biogenesis of endogenous trans-acting siRNAs and up-regulated their targets. Collectively, we propose that VISP1 is a small peptide receptor functioning in the crosstalk between selective autophagy and RNA silencing.

The serine/threonine/tyrosine kinase STY46 defends against hordeivirus infection by phosphorylating γb protein.

Protein phosphorylation is a common post-translational modification that frequently occurs during plant-virus interaction. Host protein kinases often regulate virus infectivity and pathogenicity by phosphorylating viral proteins. The Barley stripe mosaic virus (BSMV) γb protein plays versatile roles in virus infection and the coevolutionary arms race between plant defense and viral counter-defense. Here, we identified that the autophosphorylated cytosolic serine/threonine/tyrosine (STY) protein kinase 46 of Nicotiana benthamiana (NbSTY46) phosphorylates and directly interacts with the basic motif domain (aa 19-47) of γb in vitro and in vivo. Overexpression of wild-type NbSTY46, either transiently or transgenically, suppresses BSMV replication and ameliorates viral symptoms, whereas silencing of NbSTY46 leads to increased viral replication and exacerbated symptom. Moreover, the antiviral role of NbSTY46 requires its kinase activity, as the NbSTY46T436A mutant, lacking kinase activity, not only loses the ability to phosphorylate and interact with γb but also fails to impair BSMV infection when expressed in plants. NbSTY46 could also inhibit the replication of Lychnis ringspot virus, another chloroplast-replicating hordeivirus. In summary, we report a function of the cytosolic kinase STY46 in defending against plant viral infection by phosphorylating a viral protein in addition to its basal function in plant growth, development, and abiotic stress responses.

Three-dimensional reconstruction and comparison of vacuolar membranes in response to viral infection.

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

Tobacco Necrosis Virus-AC Single Coat Protein Amino Acid Substitutions Determine Host-Specific Systemic Infections of Nicotiana benthamiana and Soybean.

Plant viruses often infect several distinct host species. Sometimes, viruses can systemically infect a specific host whereas, in other cases, only local infections occur in other species. How viral and host factors interact to determine systemic infections among different hosts is largely unknown, particularly for icosahedral positive-stranded RNA viruses. The Tobacco necrosis virus-A Chinese isolate belongs to the genus Alphanecrovirus in the family Tombusviridae. In this study, we investigated variations in systemic infections of tobacco necrosis virus-AC (TNV-AC) in Nicotiana benthamiana and Glycine max (soybean) by alanine-scanning mutagenesis of the viral coat protein (CP), which is essential for systemic movement of TNV-AC. We found that three amino acids, R169, K177, and Q233, are key residues that mediate varying degrees of systemic infections of N. benthamiana and soybean. Further analysis revealed that variations in systemic trafficking of TNV-AC CP mutants in N. benthamiana and soybean are associated with virion assembly and stability. The CP amino acids K177 and Q233 are highly conserved among all TNV-A isolates and are replaced by Q and K in the TNV-D isolates. We demonstrated that systemic infectivity of either TNV-AC K177A and Q233A or K177Q and Q233K mutants are correlated with the binding affinity of the mutated CPs to the host-specific Hsc70-2 protein. These results expand our understanding of host-dependent long-distance movement of icosahedral viruses in plants.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A putative nuclear copper chaperone promotes plant immunity in Arabidopsis.

Copper is essential for many metabolic processes but must be sequestrated by copper chaperones. It is well known that plant copper chaperones regulate various physiological processes. However, the functions of copper chaperones in the plant nucleus remain largely unknown. Here, we identified a putative copper chaperone induced by pathogens (CCP) in Arabidopsis thaliana. CCP harbors a classical MXCXXC copper-binding site (CBS) at its N-terminus and a nuclear localization signal (NLS) at its C-terminus. CCP mainly formed nuclear speckles in the plant nucleus, which requires the NLS and CBS domains. Overexpression of CCP induced PR1 expression and enhanced resistance against Pseudomonas syringae pv. tomato DC3000 compared with Col-0 plants. Conversely, two CRISPR/Cas9-mediated ccp mutants were impaired in plant immunity. Further biochemical analyses revealed that CCP interacted with the transcription factor TGA2 in vivo and in vitro. Moreover, CCP recruits TGA2 to the PR1 promoter sequences in vivo, which induces defense gene expression and plant immunity. Collectively, our results have identified a putative nuclear copper chaperone required for plant immunity and provided evidence for a potential function of copper in the salicylic pathway.