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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 North Dakota 18 (ND18), Beijing (BJ), Xinjiang (XJ), Type (TY) and CV21 strains are unable to infect the Brachypodium distachyon Bd3-1 inbred line, which harbours a resistance gene designated Bsr1, but the Norwich (NW) strain is virulent on Bd3-1. Analysis of ND18 and NW genomic RNA reassortants and RNAβ mutants demonstrates that two amino acids within the helicase motif of the triple gene block 1 (TGB1) movement protein have major effects on their Bd3-1 phenotypes. Resistance to ND18 correlates with an arginine residue at TGB1 position 390 (R(390)) and a threonine at position 392 (T(392)), whereas the virulent NW strain contains lysines (K) at both positions. ND18 TGB1 R390K ((ND)TGB1(R390K)) and (ND)TGB1(T392K) single substitutions, and an (ND)TGB1(R390K,T392K) double mutation resulted in systemic infections of Bd3-1. Reciprocal (ND)TGB1 substitutions into (NW)TGB1 ((NW)TGB1(K390R) and (NW)TGB1(K392T)) failed to affect virulence, implying that K(390) and K(392) compensate for each other. In contrast, an (NW)TGB1(K390R,K392T) double mutant exhibited limited vascular movement in Bd3-1, but developed prominent necrotic streaks that spread from secondary leaf veins. This phenotype, combined with the appearance of necrotic spots in certain ND18 mutants, and necrosis and rapid wilting of Bd3-1 plants after BJ strain ((BJ)TGB1(K390,T392)) inoculations, show that Bd3-1 Bsr1 resistance is elicited by the TGB1 protein and suggest that it involves a hypersensitive response.

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.

Vicinal oxygen chelate (VOC) proteins are members of an enzyme superfamily with dioxygenase or non‐dioxygenase activities. However, the biological functions of VOC proteins in plants are poorly understood. Here, we show that a VOC in Nicotiana benthamiana (NbVOC1) facilitates viral infection. NbVOC1 was significantly induced by infection by beet necrotic yellow vein virus (BNYVV). Transient overexpression of NbVOC1 or its homolog from Beta vulgaris (BvVOC1) enhanced BNYVV infection in N. benthamiana, which required the nuclear localization of VOC1. Consistent with this result, overexpressing NbVOC1 facilitated BNYVV infection, whereas, knockdown and knockout of NbVOC1 inhibited BNYVV infection in transgenic N. benthamiana plants. NbVOC1 interacts with the basic leucine zipper transcription factors bZIP17/28, which enhances their self‐interaction and DNA binding to the promoters of unfolded protein response (UPR)‐related genes. We propose that bZIP17/28 directly binds to the NbVOC1 promoter and induces its transcription, forming a positive feedback loop to induce the UPR and facilitating BNYVV infection. Collectively, our results demonstrate that NbVOC1 positively regulates the UPR that enhances viral infection in plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Synopsis: Viral replication and subsequent intercellular movement are two critical steps for efficient plant infection. This work reveals induction of a replication‐to‐movement switch by palmitoylation of the viral γb protein during Barley stripe mosaic virus (BSMV) infection. Non‐palmitoylated γb is anchored to the chloroplasts and promotes BSMV replication.During the late stages of replication, S‐acyltransferases NbPAT15 and NbPAT21 palmitoylate γb at the periphery of chloroplasts.Palmitoylated γb recruits the TGB1 movement protein to chloroplasts, thereby inducing a shift between viral replication and movement phases.Palmitoylated γb promotes viral cell‐to‐cell movement by inhibiting NbREM1‐dependent callose deposition at plasmodesmata. [ABSTRACT FROM AUTHOR]

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. SYNOPSIS: The Barley stripe mosaic virus (BSMV) γb protein promotes viral infection in plants via various mechanisms. This work reveals an additional role in disrupting chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication. BSMV infection disturbs chloroplast redox homeostasis and induces oxidative stress.The BSMV γb protein directly interacts with host NADPH‐dependent thioredoxin reductase C (NTRC) at chloroplasts.NTRC expression suppresses BSMV replication in Nicotiana benthamiana.γb protein competitively interferes with NTRC binding to 2‐Cys peroxiredoxins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Wheat ( Triticum aestivum L.) is an important crop, and wheat yellow mosaic virus (WYMV) can cause a severe loss in wheat yield. A genetically modified (GM) wheat carrying a WYMV 72kD coding gene (Wheat 72kD) with resistance to WYMV has been constructed in a previous study. However, neither the influence of genetic modification on wild-type wheat (WT) metabolism nor the effect of WYMV-infection on the metabolic profiling in 72kD is clear. Gas chromatography-mass spectrometry (GC-MS) was used to detect the metabolic profiling in GM, WT, GM with WYMV-inoculation (GMV), WT with WYMV-inoculation (WTV) wheat, respectively. As a result, GM and WTV samples were close to each other on the principal component analysis (PCA) plot, indicating genetic modification and WYMV-infection might cause similar changes in wheat metabolism. Only 54 metabolites were annotated, and 16, 12, 17, and 14 metabolites were significantly different between GMV and GM, GMV and WTV, GM and WT, as well as between WTV and WT, respectively. Furthermore, overlapped metabolites were identified, including 3-chloro-4-hydroxybenzoic acid (CHBA), 3-sulfocatechol, S-mercaptocysteine, 1-chloro-2-nitrobenzene (2-chloronitrobenzene) and melarsoprol. In conclusion, genetic modification or/and WYMV-infection significantly affected the metabolism in wheat. This is the first report investigating the effects of WYMV-infection on metabolic profiling in GM wheat. [ABSTRACT FROM AUTHOR]