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.

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.

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.

Beet necrotic yellow vein virus (BNYVV) is an economically important pathogen of sugar beet and has been found worldwide, probably as the result of recent worldwide spread. The BNYVV genome consists of four or five RNA components. Here, we report analysis of sequence variation in the RNA3-p25, RNA4-p31, RNA2-CP, and RNA5-p26 genes of 73 worldwide isolates. The RNA3-p25 gene encodes virulence and avirulence factors. These four sets of gene sequences each fell into two to four groups, of which the three groups of p25 formed eight subgroups with different geographical distributions. Each of these subgroup isolates (strains) could have arisen from four original BNYVV population and their mixed infections. The genetic diversity for BNYVV was relatively small. Selection pressure varied greatly depending on the BNYVV gene and geographical location. Isolates of the Italy strain, in which p25 was subject to the strongest positive selection, were able to overcome the Rz1-host resistance gene to differing degrees, whereas other geographically limited strains could not. Resistance-breaking variants were generated by p25 amino acid changes at positions 67 and 68. Our studies suggest that BNYVV originally evolved in East Asia and has recently become a pathogen of cultivated sugar beet followed by the emergence of new resistance-breaking variants.

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 (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 and in 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 BSMV 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.

Polerovirus P0 suppressors of host gene silencing contain a consensus F-box-like motif with Leu/Pro (L/P) requirements for suppressor activity. The Inner Mongolian Potato leafroll virus (PLRV) P0 protein (P0(PL-IM)) has an unusual F-box-like motif that contains a Trp/Gly (W/G) sequence and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other P0 proteins. We used Agrobacterium infiltration-mediated RNA silencing assays to establish that P0(PL-IM) has a strong suppressor activity. Mutagenesis experiments demonstrated that the P0(PL-IM) F-box-like motif encompasses amino acids 76-LPRHLHYECLEWGLLCG THP-95, and that the suppressor activity is abolished by L76A, W87A, or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141 region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of P0(PL-IM). As has been observed with other P0 proteins, P0(PL-IM) suppression is correlated with reduced accumulation of the host AGO1-silencing complex protein. However, P0(PL-IM) fails to bind SKP1, which functions in a proteasome pathway that may be involved in AGO1 degradation. These results suggest that P0(PL-IM) may suppress RNA silencing by using an alternative pathway to target AGO1 for degradation. Our results help improve our understanding of the molecular mechanisms involved in PLRV infection.

Summary P0 protein of some polerovirus members can target ARGONAUTE 1 ( AGO 1) 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 ( SKP 1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO 1 degradation. Therefore, the role of P0– SKP 1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0 Br ). We found that several residues within P0 Br are required for local and systemic silencing suppression activities. Remarkably, the F‐box‐like motif mutant of P0 Br , which failed to interact with SKP 1, 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 SKP 1 ortholog leads to the destabilization of P0 Br . Genetic analyses indicated that the P0 Br – SKP 1 interaction is not directly required for silencing suppression activity of P0 Br , but it facilitates stability of P0 Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of Br YV requires P0 Br . Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

Replication of viral RNA genomes in fruit flies and mosquitoes induces the production of virus-derived small interfering RNAs (siRNAs) to specifically reduce virus accumulation by RNA interference (RNAi). However, it is unknown whether the RNA-based antiviral immunity (RVI) is sufficiently potent to terminate infection in adult insects as occurs in cell culture. We show here that, in contrast to robust infection by Flock house virus (FHV), infection with an FHV mutant (FHV⌬B2) unable to express its RNAi suppressor protein B2 was rapidly terminated in adult flies. FHV⌬B2 replicated to high levels and induced high mortality rates in dicer-2 and argonaute-2 mutant flies that are RNAi defective, demonstrating that successful infection of adult Drosophila requires a virus-encoded activity to suppress RVI. Drosophila RVI may depend on the RNAi activity of viral siRNAs since efficient FHV⌬B2 infection occurred in argonaute-2 and r2d2 mutant flies despite massive production of viral siRNAs. However, RVI appears to be insensitive to the relative abundance of viral siRNAs since FHV⌬B2 infection was terminated in flies carrying a partial loss-of-function mutation in loquacious required for viral siRNA biogenesis. Deep sequencing revealed a low-abundance population of Dicer-2-dependent viral siRNAs accompanying FHV⌬B2 infection arrest in RVI-competent flies that included an approximately equal ratio of positive and negative strands. Surprisingly, viral small RNAs became strongly biased for positive strands at later stages of infection in RVI-compromised flies due to genetic or viral suppression of RNAi. We propose that degradation of the asymmetrically produced viral positive-strand RNAs associated with abundant virus accumulation contributes to the positive-strand bias of viral small RNAs.Innate immunity includes distinct mechanisms that provide immediate and broad-spectrum protection against microbial infection and often are conserved across different kingdoms (56). The fruit fly Drosophila melanogaster is a powerful model for studying innate immunity (21). Drosophila innate immunity against bacteria and fungi involves recognition of microbial molecular patterns by germ line-encoded pattern recognition receptors and the production of potent antimicrobial peptides via closely related Toll and IMD signaling pathways (21). In contrast, protection of Drosophila against viruses is mainly mediated by the RNA interference (RNAi) pathway (24,40,67,72,74) as found in fungi and plants (13,18,32).RNAi and related RNA silencing pathways operate in diverse eukaryotes, including fungi, plants, invertebrates, and mammals, and recruit small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) to guide specific silencing of genes by an Argonaute protein in an RNA-induced silencing complex (RISC) or related effector complexes (2, 10, 34). Available data indicate that in Drosophila, doublestranded RNA (dsRNA) replicative intermediates of viruses with an RNA genome are recognized by Dicer-2 (Dcr-2) and further processed into v...