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Autophagy is a conserved defense strategy against viral infection. However, little is known about the counterdefense strategies of plant viruses involving interference with autophagy. Here, we show that γb protein from Barley stripe mosaic virus (BSMV), a positive single-stranded RNA virus, directly interacts with AUTOPHAGY PROTEIN7 (ATG7). BSMV infection suppresses autophagy, and overexpression of γb protein is sufficient to inhibit autophagy. Furthermore, silencing of autophagy-related gene ATG5 and ATG7 in Nicotiana benthamiana plants enhanced BSMV accumulation and viral symptoms, indicating that autophagy plays an antiviral role in BSMV infection. Molecular analyses indicated that γb interferes with the interaction of ATG7 with ATG8 in a competitive manner, whereas a single point mutation in γb, Tyr29Ala (Y29A), made this protein deficient in the interaction with ATG7, which was correlated with the abolishment of autophagy inhibition. Consistently, the mutant BSMVY29A virus showed reduced symptom severity and viral accumulation. Taken together, our findings reveal that BSMV γb protein subverts autophagy-mediated antiviral defense by disrupting the ATG7-ATG8 interaction to promote plant RNA virus infection, and they provide evidence that ATG7 is a target of pathogen effectors that functions in the ongoing arms race of plant defense and viral counterdefense.

Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.

A double-stranded RNA (dsRNA) segment was identified in Rhizoctonia solani anastomosis group (AG)-2-2IIIB, the primary causal agent of Rhizoctonia crown and root rot of sugar beet. The dsRNA segment represented the genome replication intermediate of a new mitovirus that was tentatively designated as \"Rhizoctonia solani mitovirus 39\" (RsMV-39). The complete sequence of the dsRNA was 2805 bp in length with 61.9% A+U content. Using either the fungal mitochondrial or universal genetic code, a protein of 840 amino acids containing an RNA-dependent RNA polymerase (RdRp) domain was predicted with a molecular mass of 94.46 kDa. BLASTp analysis revealed that the RdRp domain of RsMV-39 had 43.55% to 72.96% sequence identity to viruses in the genus Mitovirus, and was the most similar (72.96% identical) to that of Ceratobasidium mitovirus A (CbMV-A). Phylogenetic analysis based on RdRp domains clearly showed that RsMV-39 is a member of a distinct species in the genus Mitovirus of the family Mitoviridae. This is the first full genome sequence of a mycovirus associated with R. solani AG-2-2IIIB.

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. Symptom recovery is induced by a balanced “arms race” between viruses and plants. Here, the authors show that a small peptide mediates autophagic degradation of viral silencing suppressors to reach the balance of virus pathogenicity and plant immunity.

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. Most plant viruses are transmitted by insect vectors in finely regulatory mechanisms. Here, the authors show that a plant rhabdovirus can modify circadian rhythm of its insect vectors and enhances locomotor activity for efficient transmission.

RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes.

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

P0 protein of some polerovirus members can target ARGONAUTE1 (AGO1) to suppress RNA silencing. Although P0 harbors an F-box-like motif reported to be essential for interaction with S phase kinase-associated protein 1 (SKP1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO1 degradation. Therefore, the role of P0–SKP1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0Br). We found that several residues within P0Br are required for local and systemic silencing suppression activities. Remarkably, the F-box-like motif mutant of P0Br, which failed to interact with SKP1, is destabilized in vivo. Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP1 ortholog leads to the destabilization of P0Br. Genetic analyses indicated that the P0Br–SKP1 interaction is not directly required for silencing suppression activity of P0Br, but it facilitates stability of P0Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0Br. Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

The Barley stripe mosaic virus (BSMV) γb protein is a viral suppressor of RNA silencing (VSR) and symptom determinant. However, it is unclear how post-translational modification affects the different functions of cb. Here, we demonstrate that γb is phosphorylated at Ser-96 by a PKA-like kinase in vivo and in vitro. Mutant viruses containing a nonphosphorylatable substitution (BSMVS96A or BSMVS96R) exhibited reduced viral accumulation in Nicotiana benthamiana due to transient induction of the cell death response that constrained the virus to necrotic areas. By contrast, a BSMVS96D mutant virus that mimics γb phosphorylation spread similarly to the wild-type virus. Furthermore, the S96A mutant had reduced local and systemic γb VSR activity due to having compromised its binding activity to 21-bp dsRNA. However, overexpression of other VSRs in trans or in cis failed to rescue the necrosis induced by BSMVS96A, demonstrating that suppression of cell death by γb phosphorylation is functionally distinct from its RNA silencing suppressor activities. These results provide new insights into the function of γb phosphorylation in regulating RNA silencing and the BSMV-induced host cell death response, and contribute to our understanding of how the virus optimizes the balance between viral replication and virus survival in the host plants during virus infection.

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.