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Studies the effect of the RNA5 component on pathogenicity of beet necrotic yellow vein virus. Preparation of transcripts of beet necrotic yellow virus RNA5 from the cloned cDNA in a bacteriophage T7 in vitro run-off transcription system; Co-inoculation of RNA5 transcript and RNA extracted from BNYVY mutants; Improvement in virus infections and accumulation in the hosts.

Offers a functional analysis of beet necrotic yellow vein virus (BNYVV) RNA4 in fungal transmission. Insertion of full-length RNA4 of BNYVV and its mutants into a transcription plasmid; Coinoculation of the plasmid transcripts and RNAs extracted from a BNYVV isolate to Tetragona expansa; Inoculation of the recombinant isolates to sugar beet plants grown in a sand culture system containing Polymyxa betae; Finding that the efficiency of BNYVV transmission decreased with different deletions in the coding region of RNA4 and not affected by the frame-shift mutant with four nucleotides; Conclusion that some of the RNA4 sequence in the deleted region of nucleotide acids are needed to transmit the virus by fungus.

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]

In the present study, sixteen novel RNA mycoviruses co-infecting a single strain of Rhizoctonia zeae (strain D40) were identified and molecularly characterized using metatranscriptome sequencing combined with a method for rapid amplification of cDNA ends. The fungal strain was isolated from diseased seedlings of sugar beet with damping-off symptoms. Based on genome analysis and phylogenetic analysis of amino acid sequences of RNA-dependent RNA polymerase, the sixteen mycoviruses associated with strain D40 contained three genome types with nine distinct lineages, including positive single-stranded RNA (Hypoviridae, Yadokariviridae, Botourmiaviridae, and Gammaflexiviridae), double-stranded RNA (Phlegiviridae, Megabirnaviridae, Megatotiviridae, and Yadonushiviridae), and negative single-stranded RNA (Tulasviridae), suggesting a complex composition of a mycoviral community in this single strain of R. zeae (strain D40). Full genome sequences of six novel mycoviruses and the nearly full-length sequences of the remaining ten novel mycoviruses were obtained. Furthermore, seven of these sixteen mycoviruses were confirmed to assemble virus particles present in the R. zeae strain D40. To the best of our knowledge, this is the first detailed study of mycoviruses infecting R. zeae. [ABSTRACT FROM AUTHOR]

In 2021, Plumbago indica plants with necrotic spots on their leaves were observed in Beijing, China. Through high-throughput sequencing, we discovered a putative novel member of the genus Cytorhabdovirus, which was provisionally named "plumbago necrotic spot-associated virus" (PNSaV). The full-length negative-sense single-stranded RNA genome of this virus is 13,180 nucleotides in length and contains eight putative open reading frames (ORFs), in the order 3′ leader-N-(P′)-P-P3-M-G-P6-L-5′ trailer. Phylogenetic analysis and pairwise comparisons suggested that PNSaV is most closely related to pastinaca cytorhabdovirus 1, with 59.2% nucleotide sequence identity in the complete genome and 56.4% amino acid sequence identity in the L protein. These findings suggest that PNSaV should be considered a new member of the genus Cytorhabdovirus. [ABSTRACT FROM AUTHOR]

Simple Summary: A plant's C2H2-type zinc finger proteins (C2H2-ZFPs) play crucial roles in the process of plant growth and development, as well as various stress responses. The Q-type ZFP family, which contains a conserved "QALGGH", has been reported in many plants. Sugar beet is an important crop for sugar production. Salt stress and viral infection significantly reduce both sugar yield and processing quality of sugar beet. So far, the genome-wide analysis of Q-type C2H2 ZPFs and their expression pattern in sugar beet have not been analyzed yet. This study analyzed 35 Q-type ZFPs in sugar beet and their expression patterns under salt stress and virus. These results will provide theoretical evidence for understanding the functions of Q-type ZFPs. A plant's Q-type C2H2-type ZFP plays key roles in plant growth and development and responses to biotic and abiotic stresses. Sugar beet (Beta vulgaris L.) is an important crop for sugar production. Salt stress and viral infection significantly reduce the root yield and sugar content of sugar beet. However, there is a lack of comprehensive genome-wide analyses of Q-type C2H2 ZFPs and their expression patterns in sugar beet under stress. In this study, 35 sugar beet Q-type C2H2 ZFPs (BvZFPs) containing at least one conserved "QALGGH" motif were identified via bioinformatics techniques using TBtools software. According to their evolutionary relationship, the BvZFPs were classified into five subclasses. Within each subclass, the physicochemical properties and motif compositions showed strong similarities. A Ka/Ks analysis indicated that the BvZFPs were conserved during evolution. Promoter cis-element analysis revealed that most BvZFPs are associated with elements related to phytohormone, biotic or abiotic stress, and plant development. The expression data showed that the BvZFPs in sugar beet are predominantly expressed in the root. In addition, BvZFPs are involved in the response to abiotic and biotic stresses, including salt stress and viral infection. Overall, these results will extend our understanding of the Q-type C2H2 gene family and provide valuable information for the biological breeding of sugar beet against abiotic and biotic stresses in the future. [ABSTRACT FROM AUTHOR]

Anastomosis groups (AGs) or subgroups of 244 Rhizoctonia isolates recovered from sugar beet roots with symptoms of root and crown rot were characterized to be AG-A, AG-K, AG-2-2IIIB, AG-2-2IV, AG-3 PT, AG-4HGI, AG-4HGII, and AG-4HGIII, with AG-4HGI (108 isolates, 44.26%) and AG-2-2IIIB (107 isolates, 43.85%) being predominate. Four unclassified mycoviruses and one hundred and one putative mycoviruses belonging to six families, namely Mitoviridae (60.00%), Narnaviridae (18.10%), Partitiviridae (7.62%), Benyviridae (4.76%), Hypoviridae (3.81%), and Botourmiaviridae (1.90%), were found to be present in these 244 Rhizoctonia isolates, most of which (88.57%) contained positive single-stranded RNA genome. The 244 Rhizoctonia isolates were all sensitive to flutolanil and thifluzamide, with average median effective concentration (EC50) value of 0.3199 ± 0.0149 μg·mL−1 and 0.1081 ± 0.0044 μg·mL−1, respectively. Among the 244 isolates, except for 20 Rhizoctonia isolates (seven isolates of AG-A and AG-K, one isolate of AG-4HGI, and 12 isolates of AG-4HGII), 117 isolates of AG-2-2IIIB, AG-2-2IV, AG-3 PT, and AG-4HGIII, 107 isolates of AG-4HGI, and six isolates of AG-4HGII were sensitive to pencycuron, with average EC50 value of 0.0339 ± 0.0012 μg·mL−1. Correlation index (ρ) of cross-resistance level between flutolanil and thifluzamide, flutolanil and pencycuron, and thifluzamide and pencycuron was 0.398, 0.315, and 0.125, respectively. This is the first detailed study on AG identification, mycovirome analysis, and sensitivity to flutolanil, thifluzamide, and pencycuron of Rhizoctonia isolates associated with sugar beet root and crown rot. [ABSTRACT FROM AUTHOR]

Cercospora leaf spot (CLS) caused by Cercospora beticola is the most destructive foliar disease of sugar beet worldwide and is mainly controlled by timely fungicide applications. Recently, CLS control by pyraclostrobin exhibited significantly reduced efficiency in some fields of the Chifeng area in Inner Mongolia Autonomous Region of China. Purification of fungi by single spore isolation is essential for the study of fungal and fungicide resistance. However, the conventional single spore isolation and fungicide sensitivity assay for C. beticola are time‐consuming with a risk of contamination. We developed a new method for single spore isolation of C. beticola, which facilitated monitoring fungicide sensitivity of infected field samples. First, the composite spore suspension was prepared from typical lesions by vortex in the sterilized centrifuge tube. Then, the diluted spore suspension was spread on the streptomycin‐amended potato dextrose agar (PDA) plate or the PDA plate contained a threshold dosage of specific fungicide. The C. beticola colony derived from a single spore could be observed with naked eye and easily isolated by a sterilized scalpel knife after 36 h of incubation at 25°C. Meanwhile, the percentage of fungicide‐resistant or ‐tolerant isolates could be analysed based on the growth of different PDA media within 3 days. Based on this method, we identified that 22.88% of the C. beticola isolates were resistant to pyraclostrobin in the Chifeng area, indicating a high risk of emergence of C. beticola‐resistant strains to QoI fungicides in this area. Furthermore, the G143A or F129L mutation of the cytochrome b (Cyt‐b) gene was found in these resistant isolates. To our knowledge, this is the first report of C. beticola QoI‐resistant isolates with G143A or F129L mutation of CbCyt‐b gene in China. Our approach provides considerable potential for studying the biology, fitness and fungicide resistance of C. beticola. [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]

Oligogalacturonides (OGs) are a bioactive carbohydrate derived from homogalacturonan. The OGs synthesized in this study significantly inhibited the mycelial growth of Rhizoctonia solani AG-4HGI in vitro, even at a low concentration (10 mg/L). The seed vigor test demonstrated that the application of 50 mg/L OGs to sugar beet seeds significantly increased average germination percentage, germination energy, germination index, and seedling vigor index. The same concentration of OGs also improved the seedling emergence percentage of sugar beet when seeds were sown in soil inoculated with D2 and D31 isolates, respectively. The lesion diameter on mature sugar beet roots caused by R. solani AG-4HGI isolates D2 and D31 also decreased by 40.60% and 39.86%, respectively, in sugar beets roots first treated with 50 mg/mL OGs in the wound site, relative to lesion size in untreated/pathogen inoculated wounds. Sugar beet roots treated with 50 mg/mL OGs prior to inoculation with the D2 isolate exhibited up-regulation of the defense-related genes glutathione peroxidase (GPX) and superoxide dismutase (SOD) by 2.4- and 1.6-fold, respectively, relative to control roots. Sugar beet roots treated with 50 mg/mL OGs prior to inoculation with D31 exhibited a 2.0- and 1.6-fold up-regulation of GPX and SOD, respectively, relative to the control. Our results indicate that OGs have the potential to be used for the protection of sugar beet against R. solani AG-4HGI. [ABSTRACT FROM AUTHOR]

Strawberry crinkle virus (SCV) has always been one of the major viruses affecting the strawberry production in China. Developing a quick and accurate method of detection is a crucial step for controlling the spread of SCV. In this study, a reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay was developed for the detection of SCV. RNA was extracted from SCV‐infected strawberry leaves using a commercial extraction kit. According to the sequence of 1553 L protein (RdRp), four primers were developed for RT‐LAMP and a one‐step reaction was carried out at 65℃ for 30 min. Conventional reverse transcription Polymerase Chain Reaction (RT‐PCR) was performed as a comparison. The amplified RT‐LAMP products were separated on agarose gel by running electrophoresis and could be detected by visual inspection using Green‐DNA‐staining dye too. The detection sensitivity of the RT‐LAMP assay was 1000 times higher than that of the conventional RT‐PCR method, and no cross reaction was found with other strawberry viruses. Field samples with the positive rate of about 60% were collected to demonstrate the applicability of the RT‐LAMP for field detection and its high consistency with the traditional RT‐PCR method. The LAMP method described in this report is a sensitive, simple, economy and reliable assay for the rapid detection of SCV infection in strawberry plants. [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]

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. Synopsis: Genomic analyses have predicted numerous potentially expressed small peptides in plants, but their functions remain largely unknown. Here, a virus‐induced peptide is identified as a new autophagy receptor triggering degradation of SGS3/RDR6‐bodies and negatively regulating siRNA amplification. VISP1 is a novel virus‐induced small peptide composed of 71 amino acids in Arabidopsis.VISP1 harbors a ubiquitin‐interacting motif (UIM), which mediates the interaction with ATG8.VISP1 serves as a new autophagy receptor and triggers autophagic degradation of SGS3/RDR6‐bodies.VISP1 negatively regulates SGS3/RDR6‐dependent siRNA amplification. [ABSTRACT FROM AUTHOR]

Reports research on transgenic wheat and gene silencing. Use of particle bombardment to insert the plasmid containing the promoter Act1, the coat protein (cp) gene of wheat yellow mosaic virus (WYMV) and the selectable bar gene, into immature embryos of a wheat cultivars; PCR and PCR-RFLP used to screen for the cp gene in TO and T1 generations; How seeds from the positive T1 plants were sowed in WYMV-contaminated soil to determine their resistance; Finding that one of the transgenic wheat lines, PS-T2, showed high disease-resistance; Western blot and RT-PCR analysis indicating the expression level of cp gene in the resistant transgenic line was reduced significantly; Conclusion that the mechanism of the virus induced gene silencing stimulated resistance.

Strawberry mottle virus (SMoV) is one of the most important viruses infecting strawberry all over the world, seriously affecting the yield and quality of strawberry especially in the occasion of compound infection. Loop‐mediated isothermal amplification (LAMP) as a new method of nucleic acid amplification has the advantages of rapid detection, simple operation and high sensitivity. Therefore, four specific LAMP primers using the 3′ end conservative coat protein (CP) gene were selected and designed according to the RNA2 sequence of SMoV obtained in NCBI GenBank. Results showed that the optimized RT‐LAMP reaction conditions including 1.0 µmol/L1 primers FIP/BIP, 0.1 µmol/L1 primers F3/B3, 2 mmol/L1 Mg2+, 1.2 mmol/L1 dNTPs, 1.0 mol/L1 Betaine and 2 mol/L1 DTT, with an optimal temperature set at 62°C for 45 min. The amplicons were successfully inspected by visual assessment using SYBR Green I as well as electrophoresis on agarose gel. The sensitivity of the LAMP was 100 times higher than the RT‐PCR method. Furthermore, it was highly specific for SMoV and showed no cross‐reaction with several other strawberry viruses. This assay will provide a practical method for rapid detection of large quantities of field samples and be useful in reducing the virus incidence through production of virus‐free planting material. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Sugar beet is widely grown throughout the world and represents the second largest crop used to produce sugar. Root rot in sugar beet, caused by Fusarium, significantly reduces yield, juice purity, and sugar concentration. Here, 307 Fusarium isolates were collected from sugar beet roots exhibiting typical root rot symptoms in eight provinces or autonomous regions of China from 2009 to 2012. Based on morphological characteristics and sequence data of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and the translation elongation factor 1α (EF-1α), Fusarium oxysporum (38.4%) was identified as the most prevalent species, followed by F. solani (20.9%), and F. equiseti (18.9%). These three species were widely distributed in all eight of the provinces and autonomous regions. F. tricinctum (5.9%), F. brachygibbosum (4.6%), F. redolens (3.3%), F. proliferatum (3.3%), F. graminearum (2.3%), F. verticillioides (1.6%), F. nygamai (0.7%), and F. culmorum (0.3%) were less frequently obtained. Of the 307 Fusarium isolates, 117 representing different species and geographic locations were demonstrated to cause tip rot and vascular discoloration in sugar beet roots, with disease incidence ranging from 84.2 to 100.0% and disease index ranging from 41.94 to 75.83. This is the first detailed report of Fusarium species, in particular F. tricinctum, F. brachygibbosum, F. redolens, F. proliferatum, F. nygamai, and F. culmorum, causing sugar beet root rot in China. [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]

Summary: 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 γb. 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. [ABSTRACT FROM AUTHOR]

Summary: Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA‐binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear‐cytoplasmic trafficking is required for BSMV cell‐to‐cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95–104) and a nuclear localization signal (NLS) (amino acids 227–238). NoLS mutations reduced BSMV cell‐to‐cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine–arginine‐rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV‐infected cells, Fib2 accumulation increased by about 60%–70% and co‐localized with TGB1 in the plasmodesmata. In addition, BSMV cell‐to‐cell movement in fib2 knockdown transgenic plants was reduced to less than one‐third of that of non‐transgenic plants. Fib2 also co‐localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1–Fib2 interactions play a direct role in cell‐to‐cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell‐to‐cell movement. [ABSTRACT FROM AUTHOR]

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. [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]

Key message: Spot blotch disease resistance gene Sb3 was mapped to a 0.15 centimorgan (cM) genetic interval spanning a 602 kb physical genomic region on chromosome 3BS. Abstract: Wheat spot blotch disease, caused by B. sorokiniana, is a devastating disease that can cause severe yield losses. Although inoculum levels can be reduced by planting disease-free seed, treatment of plants with fungicides and crop rotation, genetic resistance is likely to be a robust, economical and environmentally friendly tool in the control of spot blotch. The winter wheat line 621-7-1 confers immune resistance against B. sorokiniana. Genetic analysis indicates that the spot blotch resistance of 621-7-1 is controlled by a single dominant gene, provisionally designated Sb3. Bulked segregant analysis (BSA) and simple sequence repeat (SSR) mapping showed that Sb3 is located on chromosome arm 3BS linked with markers Xbarc133 and Xbarc147. Seven and twelve new polymorphic markers were developed from the Chinese Spring 3BS shotgun survey sequence contigs and 3BS reference sequences, respectively. Finally, Sb3 was mapped in a 0.15 cM genetic interval spanning a 602 kb physical genomic region of Chinese Spring chromosome 3BS. The genetic and physical maps of Sb3 provide a framework for map-based cloning and marker-assisted selection (MAS) of the spot blotch resistance. [ABSTRACT FROM AUTHOR]

Background: Rhizomania is one of the most devastating diseases of sugar beet. It is caused by Beet necrotic yellow vein virus (BNYVV) transmitted by the obligate root-infecting parasite Polymyxa betae. Beta macrocarpa, a wild beet species widely used as a systemic host in the laboratory, can be rub-inoculated with BNYVV to avoid variation associated with the presence of the vector P. betae. To better understand disease and resistance between beets and BNYVV, we characterized the transcriptome of B. macrocarpa and analyzed global gene expression of B. macrocarpa in response to BNYVV infection using the Illumina sequencing platform. Results: The overall de novo assembly of cDNA sequence data generated 75,917 unigenes, with an average length of 1054 bp. Based on a BLASTX search (E-value ≤ 10−5) against the non-redundant (NR, NCBI) protein, Swiss-Prot, the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, there were 39,372 unigenes annotated. In addition, 4,834 simple sequence repeats (SSRs) were also predicted, which could serve as a foundation for various applications in beet breeding. Furthermore, comparative analysis of the two transcriptomes revealed that 261 genes were differentially expressed in infected compared to control plants, including 128 up- and 133 down-regulated genes. GO analysis showed that the changes in the differently expressed genes were mainly enrichment in response to biotic stimulus and primary metabolic process. Conclusion: Our results not only provide a rich genomic resource for beets, but also benefit research into the molecular mechanisms of beet- BNYV Vinteraction. [ABSTRACT FROM AUTHOR]

Background: Beet necrotic yellow vein virus (BNYVV), encodes either four or five plus-sense single stranded RNAs and is the causal agent of sugar beet rhizomania disease, which is widely distributed in most regions of the world. BNYVV can also infect Nicotiana benthamiana systemically, and causes severe curling and stunting symptoms in the presence of RNA4 or mild symptoms in the absence of RNA4. Results: Confocal laser scanning microscopy (CLSM) analyses showed that the RNA4-encoded p31 protein fused to the red fluorescent protein (RFP) accumulated mainly in the nuclei of N. benthamiana epidermal cells. This suggested that severe RNA4-induced symptoms might result from p31-dependent modifications of the transcriptome. Therefore, we used next-generation sequencing technologies to analyze the transcriptome profile of N. benthamiana in response to infection with different isolates of BNYVV. Comparisons of the transcriptomes of mock, BN3 (RNAs 1+2+3), and BN34 (RNAs 1+2+3+4) infected plants identified 3,016 differentially expressed transcripts, which provided a list of candidate genes that potentially are elicited in response to virus infection. Our data indicate that modifications in the expression of genes involved in RNA silencing, ubiquitin-proteasome pathway, cellulose synthesis, and metabolism of the plant hormone gibberellin may contribute to the severe symptoms induced by RNA4 from BNYVV. Conclusions: These results expand our understanding of the genetic architecture of N. benthamiana as well as provide valuable clues to identify genes potentially involved in resistance to BNYVV infection. Our global survey of gene expression changes in infected plants reveals new insights into the complicated molecular mechanisms underlying symptom development, and aids research into new strategies to protect crops against viruses. [ABSTRACT FROM AUTHOR]

Highlights: [•] We examined the reference gene stability in five different monocots. [•] Four kinds of viruses were used to produce biotic stresses in plants. [•] The most suitable reference genes for accurate normalisation in virus-infected monocot plants were determined by three different software programs. [•] The suitability of selecting appropriate reference genes was further validated by normalising the expression levels of PR-1 in virus-infected monocots. [•] The diversity of PR-1 expression pattern mirrors the complexity of gene regulation during virus–plant interaction. [Copyright &y& Elsevier]

Maize rough dwarf disease (MRDD), caused by several Fijiviruses in the family Reoviridae, is a global disease that is responsible for substantial yield losses in maize. Although some maize germplasm have low levels of polygenic resistance to MRDD, highly resistant cultivated varieties are not available for agronomic field production in China. In this work, we have generated transgenic maize lines that constitutively express rnc70, a mutant E. coli dsRNA-specific endoribonuclease gene. Transgenic lines were propagated and screened under field conditions for 12 generations. During three years of evaluations, two transgenic lines and their progeny were challenged with Rice black-streaked dwarf virus (RBSDV), the causal agent of MRDD in China, and these plants exhibited reduced levels of disease severity. In two normal years of MRDD abundance, both lines were more resistant than non-transgenic plants. Even in the most serious MRDD year, six out of seven progeny from one line were resistant, whereas non-transgenic plants were highly susceptible. Molecular approaches in the T12 generation revealed that the rnc70 transgene was integrated and expressed stably in transgenic lines. Under artificial conditions permitting heavy virus inoculation, the T12 progeny of two highly resistant lines had a reduced incidence of MRDD and accumulation of RBSDV in infected plants. In addition, we confirmed that the RNC70 protein could bind directly to RBSDV dsRNA in vitro. Overall, our data show that RNC70-mediated resistance in transgenic maize can provide efficient protection against dsRNA virus infection. [ABSTRACT FROM AUTHOR]

Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the Barley yellow dwarf virus (BYDV) – like translational enhancer (BTE) is present in Tobacco necrosis virus A (TNV-A), a Necrovirus member in the Tombusviridae family. In this paper, an RNA stretch flanking the 5′ proximal end of the TNV-AC coat protein (CP) gene was shown to be essential for viral replication in Chenopodium amaranticolor plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5′ or 3′ sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (−3 to +6 of the CP initiation site) within TNV-AC RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from in vitro synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-AC, ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses. [ABSTRACT FROM AUTHOR]

Abstract: Beet black scorch virus (BBSV) encodes three movement proteins (P7a, P7b and P5′) that facilitate its cell-to-cell movement. An arginine-rich motif of P7a N-terminus was found to determine nuclear and nucleolar localization. Amino acids substitution or deletion of the R-rich motif interfered with P7a nuclear and nucleolar localization. Bimolecular fluorescence complementation (BiFC) assays revealed that P7a protein interacted with Nicotiana benthamiana nuclear import factor importin α, suggesting that P7a is translocated into the nucleus by the classical importin α/β-dependent pathway. Moreover, P7a also interacted with the nucleolar protein fibrillarin. Mutations in the R-rich motif of P7a diminished P7a interactions with importin α and fibrillarin, influenced viral replication in Nicotiana benthamiana protoplasts and altered the symptom phenotype and viral RNA accumulation in Chenopodium amaranticolor plants. These results demonstrate that the R-rich motif of P7a is correlated with nuclear and nucleolar localization, viral replication and virus infection. [Copyright &y& Elsevier]

Abstract: Beet black scorch virus (BBSV) is a positive-sense, single-stranded RNA virus belonging to Necrovirus genus. In order to better understand the life cycle of BBSV, we have investigated the subcellular localization of BBSV capsid protein (CP) by its fusion with green fluorescent protein (GFP) agroinfiltrated into Nicotiana benthamiana leaves and by particle bombardment into onion (Allium cepa) epidermal cells. Confocal laser scanning microscopy (CLSM) showed that BBSV CP fused to GFP displayed enhanced fluorescence in nuclei and nuclear import of the CP was confirmed in BBSV-infected N. benthamiana leaves. Mutational analysis revealed that the N-terminal basic amino acid cluster 4KRNKGGKKSR13 of the CP is essential for nuclear localization. Bimolecular fluorescence complementation (BiFC) assays indicated that the CP could interact with the nuclear import factor importin α, suggesting that the CP is possibly imported into the nucleus via an importin α-dependent pathway. This is the first report of the nuclear localization of the CP encoded by a necrovirus. [ABSTRACT FROM AUTHOR]

Abstract: Plant viruses show significant potential as expression vectors for producing foreign proteins in plants. In this study, codon-optimized VP6 gene (sVP6) of human rotavirus was engineered as a replacement to the coat protein (CP) open reading frame of Beet black scorch virus (BBSV). In vitro-generated RNA transcripts corresponding to the engineered virus were infectious when inoculated onto the leaves of Chenopodium amaranticolor. Molecular analysis revealed that sVP6 was efficiently expressed and accounted for 0.25% of the total soluble protein (TSP) in plant leaves on 7dpi. On average, a high level 1.54μg of sVP6 was expressed in each gram of infected leaves. Oral immunization of female BALB/c mice with the plant-based sVP6 protein induced high titers of anti-VP6 mucosal IgA and serum IgG. 60% of suckling pups born from immunized dams were protected against the virulent rotavirus challenge and those infected pups developed less severe diarrhea. These results suggested that it is feasible to induce lactogenic immunity against an enteric pathogen through oral vaccination, by using the antigen produced in a new BBSV-based plant protein expression system. [ABSTRACT FROM AUTHOR]

Plant virus-based expression systems provide attractive alternatives for production of animal virus-originated antigenic peptides. In the present study, an infectious cDNA clone of Tobacco necrosis virus A Chinese isolate (TNV-AC) was used for expression of different peptides derived from Foot and mouth disease virus (FMDV) serotype O VP1 fused downstream of the coat protein (CP) open reading frame (ORF). Chenopodium amaranticolor inoculated with in vitro transcripts of the chimaeras developed symptoms similar to those caused by wild-type TNV-AC. Western blot and RT-PCR detection of the infected leaves demonstrated that the chimaeras were infective, and a large number of self-assembled virions could be purified and observed under electron microscopy. Immunogold labelling revealed that highly expressed FMDV VP1 peptides could be displayed on the surfaces of virus particles. Additional immunoblotting and DNA sequence analyses showed that most of the chimaeras contained unmodified foreign peptides even after six successive passages in C. amaranticolor and three passages in Nicotiana benthamiana. Our results also suggest that the amino acid sequence and peptide length have a substantial influence on viral morphogenesis and systemic infections. Finally, animal experiments showed that purified chimaeric virus particles (CVPs) could induce a strong immune response against FMDV structural protein VP1 via an intramuscular route. And when inoculated nasally, CVPs could induce systemic and mucosal immune responses in mice. [ABSTRACT FROM AUTHOR]

Cercospora leaf spot (CLS) caused by Cercospora beticola is a devastating foliar disease of sugar beet (Beta vulgaris), resulting in high yield losses worldwide. Mycoviruses are widespread fungi viruses and can be used as a potential biocontrol agent for fugal disease management. To determine the presence of mycoviruses in C. beticola, high-throughput sequencing analysis was used to determine the diversity of mycoviruses in 139 C. beticola isolates collected from major sugar beet production areas in China. The high-throughput sequencing reads were assembled and searched against the NCBI database using BLASTn and BLASTx. The results showed that the obtained 93 contigs were derived from eight novel mycoviruses, which were grouped into 3 distinct lineages, belonging to the families Hypoviridae, Narnaviridae and Botourmiaviridae, as well as some unclassified (−)ssRNA viruses in the order Bunyavirales and Mononegavirales. To the best of our knowledge, this is the first identification of highly diverse mycoviruses in C. beticola. The novel mycoviruses explored in this study will provide new viral materials to biocontrol Cercospora diseases. Future studies of these mycoviruses will aim to assess the roles of each mycovirus in biological function of C. beticola in the future. [ABSTRACT FROM AUTHOR]

Based on a full-length infectious cDNA clone, gene modifications of Tobacco necrosis virus A Chinese isolate (TNV-Ac) were made by site-directed mutagenesis or nucleotide deletions for in vitro transcription of mutant viral RNAs. Mechanical inoculations of Chenopodium amaranticolor with in vitro transcripts, containing a single nucleic acid substitution at the presumed transcriptional start sites for the two subgenomic (sg) RNAs, showed that the sgRNA1 and sgRNA2 of TNV-Ac were initiated at G2184 or C2460, respectively. Mutagenesis of the translational initiation-codons for the open-reading frame (ORF) P8 or P6 encoded by sgRNA1 indicated that each of the two genes was essential for formation of local lesions on C. amaranticolor leaves, perhaps by blocking virus cell-to-cell movement, but were not necessary for viral RNA replication in the protoplast of tobacco cell BY-2. Results of prokaryotic expression showed that the ORF coding for coat protein on TNV-Ac sgRNA2 was initiatively translated by the first AUG codon at nucleotides 2612-261 4. Site-directed mutation of translational start codons, and deletion of the entire coding region, showed that the intact TNV-Ac coat protein was dispensable for establishment of TNV-Ac infection in C. amaranticolor, otherwise the numbers of local lesions and the viral RNA accumulation level were reduced, or the time to symptom appearance significantly delayed. These results suggested that the nucleotide sequence around the translational start codon coding for TNV-Ac coat protein gene may play an important role in the local symptoms. Aspects of the involvement of the coat protein in the TNV-Ac life cycle were discussed. [ABSTRACT FROM AUTHOR]

Functional analysis for gene silencing suppressor of P14 geneof Beet necrotic yellow vein virus and S6 gene ofRice black streak dwarf virus was carried out by agro-infiltration with recombinant vectors ofPotato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing ofgfp transgenicNicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the suppressed plants, thegfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially thegfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors. [ABSTRACT FROM AUTHOR]

Functional analysis for gene silencing suppressor of P14 gene of Beet necrotic yellow vein virus and S6 gene filtration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors. [ABSTRACT FROM AUTHOR]

To counteract host antiviral RNA silencing, plant viruses encode numerous viral suppressors of RNA silencing (VSRs). P0 proteins have been identified as VSRs in many poleroviruses. However, their suppressor function has not been fully characterized. Here, we investigated the function of P0 from pea mild chlorosis virus (PMCV) in the suppression of local and systemic RNA silencing via green fluorescent protein (GFP) co-infiltration assays in wild-type and GFP-transgenic Nicotiana benthamiana (line 16c). Amino acid deletion analysis showed that N-terminal residues Asn 2 and Val 3, but not the C-terminus residues from 230–270 aa, were necessary for PMCV P0 (P0PM) VSR activity. P0PM acted as an F-box protein, and triple LPP mutation (62LPxx79P) at the F-box-like motif abolished its VSR activity. In addition, P0PM failed to interact with S-phase kinase-associated protein 1 (SKP1), which was consistent with previous findings of P0 from potato leafroll virus. These data further support the notion that VSR activity of P0 is independent of P0–SKP1 interaction. Furthermore, we examined the effect of P0PM on ARGONAUTE1 (AGO1) protein stability, and co-expression analysis showed that P0PM triggered AGO1 degradation. Taken together, our findings suggest that P0PM promotes degradation of AGO1 to suppress RNA silencing independent of SKP1 interaction. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) infections induce stunting and leaf curling, as well as root and floral developmental defects and leaf senescence in Nicotiana benthamiana. A microarray analysis with probes capable of detecting 1596 candidate microRNAs (miRNAs) was conducted to investigate differentially expressed miRNAs and their targets upon BNYVV infection of N. benthamiana plants. Eight species-specific miRNAs of N. benthamiana were identified. Comprehensive characterization of the N. benthamiana microRNA profile in response to the BNYVV infection revealed that 129 miRNAs were altered, including four species-specific miRNAs. The targets of the differentially expressed miRNAs were predicted accordingly. The expressions of miR164, 160, and 393 were up-regulated by BNYVV infection, and those of their target genes, NAC21/22, ARF17/18, and TIR, were down-regulated. GRF1, which is a target of miR396, was also down-regulated. Further genetic analysis of GRF1, by Tobacco rattle virus-induced gene silencing, assay confirmed the involvement of GRF1 in the symptom development during BNYVV infection. BNYVV infection also induced the up-regulation of miR168 and miR398. The miR398 was predicted to target umecyanin, and silencing of umecyanin could enhance plant resistance against viruses, suggesting the activation of primary defense response to BNYVV infection in N. benthamiana. These results provide a global profile of miRNA changes induced by BNYVV infection and enhance our understanding of the mechanisms underlying BNYVV pathogenesis. [ABSTRACT FROM AUTHOR]

Plant virus coat proteins (CPs) play a fundamental role in protection of genomic RNAs, virion assembly, and viral movement. Although phosphorylation of several CPs during virus infection have been reported, little information is available about CP phosphorylation of the spherical RNA plant viruses. Here, we demonstrate that the CP of Beet black scorch virus (BBSV), a member of the genus Necrovirus, can be phosphorylated at threonine-41 (T41) by cAMP-dependent protein kinase (PKA)-like kinase in vivo and in vitro. Mutant viruses containing a T41A non-phosphorylatable alanine substitution, and a T41E glutamic acid substitution to mimic threonine phosphorylation were able to replicate but were unable to move systemically in Nicotiana benthamiana. Interestingly, the T41A and T41E mutants generated unstable 17 nm virus-like particles that failed to package viral genomic (g) RNA, compared with wild-type BBSV with 30 nm virions during viral infection in N. benthamiana. Further analyses showed that the T41 mutations had little effect on the gRNA-binding activity of the CP. Therefore, we propose a model whereby CP phosphorylation plays an essential role in long-distance movement of BBSV that involves formation of stable virions. [ABSTRACT FROM AUTHOR]