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

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

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

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

The P1 protein of wheat yellow mosaic virus exerts RNA silencing suppression activity to facilitate virus infection in wheat plants.

Wheat yellow mosaic virus (WYMV) causes severe wheat viral disease in Asia. However, the viral suppressor of RNA silencing (VSR) encoded by WYMV has not been identified. Here, the P1 protein encoded by WYMV RNA2 was shown to suppress RNA silencing in Nicotiana benthamiana. Mutagenesis assays revealed that the alanine substitution mutant G175A of P1 abolished VSR activity and mutant Y10A VSR activity remained only in younger leaves. P1, but not G175A, interacted with gene silencing-related protein, N. benthamiana calmodulin-like protein (NbCaM), and calmodulin-binding transcription activator 3 (NbCAMTA3), and Y10A interacted with NbCAMTA3 only. Competitive Bimolecular fluorescence complementation and co-immunoprecipitation assays showed that the ability of P1 disturbing the interaction between NbCaM and NbCAMTA3 was stronger than Y10A, Y10A was stronger than G175A. In vitro transcript inoculation of infectious WYMV clones further demonstrated that VSR-defective mutants G175A and Y10A reduced WYMV infection in wheat (Triticum aestivum L.), G175A had a more significant effect on virus accumulation in upper leaves of wheat than Y10A. Moreover, RNA silencing, temperature, and autophagy have significant effects on the accumulation of P1 in N. benthamiana. Taken together, WYMV P1 acts as VSR by interfering with calmodulin-associated antiviral RNAi defense to facilitate virus infection in wheat, which has provided clear insights into the function of P1 in the process of WYMV infection.

The P2 protein of wheat yellow mosaic virus acts as a viral suppressor of RNA silencing in Nicotiana benthamiana to facilitate virus infection.

Wheat yellow mosaic virus (WYMV) causes severe viral wheat disease in Asia. The WYMV P1 protein encoded by RNA2 has viral suppressor of RNA silencing (VSR) activity to facilitate virus infection, however, VSR activity has not been identified for P2 protein encoded by RNA2. In this study, P2 protein exhibited strong VSR activity in Nicotiana benthamiana at the four-leaf stage, and point mutants P70A and G230A lost VSR activity. Protein P2 interacted with calmodulin (CaM) protein, a gene-silencing associated protein, while point mutants P70A and G230A did not interact with it. Competitive bimolecular fluorescence complementation and competitive co-immunoprecipitation experiments showed that P2 interfered with the interaction between CaM and calmodulin-binding transcription activator 3 (CAMTA3), but the point mutants P70A and G230A could not. Mechanical inoculation of wheat with in vitro transcripts of WYMV infectious cDNA clone further confirmed that VSR-deficient mutants P70A and G230A decreased WYMV infection in wheat plants compared with the wild type. In addition, RNA silencing, temperature, ubiquitination and autophagy had significant effects on accumulation of P2 protein in N. benthamiana leaves. In conclusion, WYMV P2 plays a VSR role in N. benthamiana and promotes virus infection by interfering with calmodulin-related antiviral RNAi defense.

The Carboxyl Terminal Regions of P0 Protein Are Required for Systemic Infections of Poleroviruses.

P0 proteins encoded by poleroviruses Brassica yellows virus (BrYV) and Potato leafroll virus (PLRV) are viral suppressors of RNA silencing (VSR) involved in abolishing host RNA silencing to assist viral infection. However, other roles that P0 proteins play in virus infection remain unclear. Here, we found that C-terminal truncation of P0 resulted in compromised systemic infection of BrYV and PLRV. C-terminal truncation affected systemic but not local VSR activities of P0 proteins, but neither transient nor ectopic stably expressed VSR proteins could rescue the systemic infection of BrYV and PLRV mutants. Moreover, BrYV mutant failed to establish systemic infection in DCL2/4 RNAi or RDR6 RNAi plants, indicating that systemic infection might be independent of the VSR activity of P0. Partially rescued infection of BrYV mutant by the co-infected PLRV implied the functional conservation of P0 proteins within genus. However, although C-terminal truncation mutant of BrYV P0 showed weaker interaction with its movement protein (MP) when compared to wild-type P0, wild-type and mutant PLRV P0 showed similar interaction with its MP. In sum, our findings revealed the role of P0 in virus systemic infection and the requirement of P0 carboxyl terminal region for the infection.

Development of Beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing.

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive-stranded RNAs. Here, we have established a BNYVV full-length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV-based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co-localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV-based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV-based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

Brassica yellows virus P0 protein impairs the antiviral activity of NbRAF2 in Nicotiana benthamiana.

In interactions between poleroviruses and their hosts, few cellular proteins have been identified that directly interact with the multifunctional virus P0 protein. To help explore the functions of P0, we identified a Brassica yellows virus genotype A (BrYV-A) P0BrA-interacting protein from Nicotiana benthamiana, Rubisco assembly factor 2 (NbRAF2), which localizes in the nucleus, cell periphery, chloroplasts, and stromules. We found that its C-terminal domain (amino acids 183-211) is required for self-interaction. A split ubiquitin membrane-bound yeast two-hybrid system and co-immunoprecipitation assays showed that NbRAF2 interacted with P0BrA, and co-localized in the nucleus and at the cell periphery. Interestingly, the nuclear pool of NbRAF2 decreased in the presence of P0BrA and during BrYV-A infection, and the P0BrA-mediated reduction of nuclear NbRAF2 required dual localization of NbRAF2 in the chloroplasts and nucleus. Tobacco rattle virus-based virus-induced gene silencing of NbRAF2 promoted BrYV-A infection in N. benthamiana, and the overexpression of nuclear NbRAF2 inhibited BrYV-A accumulation. Potato leafroll virus P0PL also interacted with NbRAF2 and decreased its nuclear accumulation, indicating that NbRAF2 may be a common target of poleroviruses. These results suggest that nuclear NbRAF2 possesses antiviral activity against BrYV-A infection, and that BrYV-A P0BrA interacts with NbRAF2 and alters its localization pattern to facilitate virus infection.

Amino acid sequence motifs essential for P0-mediated suppression of RNA silencing in an isolate of potato leafroll virus from Inner Mongolia.

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.

Infection of Beet necrotic yellow vein virus with RNA4-encoded P31 specifically up-regulates pathogenesis-related protein 10 in Nicotiana benthamiana.

BACKGROUND: Beet necrotic yellow vein virus (BNYVV) is the infectious agent of sugar beet rhizomania, which consists of four or five plus-sense RNAs. RNA4 of BNYVV is not essential for virus propagation in Nicotiana benthamiana but has a major effect on symptom expression. Early reports showed that RNA4-encoded P31 was associated with severe symptoms, such as curling and dwarfing, in N. benthamiana. RESULTS: We discovered that the pathogenesis-related protein 10 (PR-10) gene can be up-regulated in BNYVV-infected N. benthamiana in the presence of RNA4 and that it had a close link with symptom development. Our frame-shift, deletion and substitution analysis showed that only the entire P31 could induce PR-10 up-regulation during BNYVV infection and that all the tryptophans and six cysteines (C174, C183, C186, C190, C197 and C199) in the cysteine-rich P31 had significant effects on PR-10 expression. However, P31 could not interact directly with PR-10 in yeast. CONCLUSIONS: Our data demonstrated that only integrated P31 specifically induced PR-10 transcription, which coincided closely with the appearance of severe symptoms in BNYVV-infected N. benthamiana, although they could not interact directly with each other in yeast.

Complete genome sequence analysis identifies a new genotype of brassica yellows virus that infects cabbage and radish in China.

For brassica yellows virus (BrYV), proposed to be a member of a new polerovirus species, two clearly distinct genotypes (BrYV-A and BrYV-B) have been described. In this study, the complete nucleotide sequences of two BrYV isolates from radish and Chinese cabbage were determined. Sequence analysis suggested that these isolates represent a new genotype, referred to here as BrYV-C. The full-length sequences of the two BrYV-C isolates shared 93.4-94.8 % identity with BrYV-A and BrYV-B. Further phylogenetic analysis showed that the BrYV-C isolates formed a subgroup that was distinct from the BrYV-A and BrYV-B isolates based on all of the proteins except P5.

Development of full-length infectious cDNA clones and host range identification of an echinacea strain of tobacco streak virus.

Tobacco streak virus induces severe diseases on a wide range of plants and becomes an emerging threat to crop yields. However, the infectious clones of TSV remain to be developed for reverse genetics studies. Here, we obtained the full genome sequence of a TSV-CNB isolate and analyzed the phylogenetic characteristics. Subsequently, we developed the full-length infectious cDNA clones of TSV-CNB driven by 35 S promoter using yeast homologous recombination. Furthermore, the host range of TSV-CNB isolate was determined by Agrobacterium infiltration and mechanical inoculation. The results reveal that TSV-CNB can infect 10 plant species in 5 families including Glycine max, Vigna radiate, Lactuca sativa var. Ramosa, Dahlia pinnate, E. purpurea, Calendula officinalis, Helianthus annuus, Nicotiana. Benthamiana, Nicotiana tabacum and Chenopodium quinoa. Taken together, the TSV infectious clones will be a useful tool for future studies on viral pathogenesis and host-virus interactions.

Identification and Functional Analyses of Host Proteins Interacting with the P3a Protein of Brassica Yellows Virus.

Viruses are obligate parasites that only undergo genomic replication in their host organisms. ORF3a, a newly identified non-AUG-initiated ORF encoded by members of the genus Polerovirus, is required for long-distance movement in plants. However, its interactions with host proteins still remain unclear. Here, we used Brassica yellows virus (BrYV)-P3a as bait to screen a plant split-ubiquitin-based membrane yeast two-hybrid (MYTH) cDNA library to explain the functional role of P3a in viral infections. In total, 138 genes with annotations were obtained. Bioinformatics analyses revealed that the genes from carbon fixation in photosynthetic, photosynthesis pathways, and MAPK signaling were affected. Furthermore, Arabidopsis thaliana purine permease 14 (AtPUP14), glucosinolate transporter 1 (AtGTR1), and nitrate transporter 1.7 (AtNRT1.7) were verified to interact with P3a in vivo. P3a and these three interacting proteins mainly co-localized in the cytoplasm. Expression levels of AtPUP14, AtGTR1, and AtNRT1.7 were significantly reduced in response to BrYV during the late stages of viral infection. In addition, we characterized the roles of AtPUP14, AtGTR1, and AtNRT1.7 in BrYV infection in A. thaliana using T-DNA insertion mutants, and the pup14, gtr1, and nrt1.7 mutants influenced BrYV infection to different degrees.

Transcriptomic and Proteomic Analyses of Myzus persicae Carrying Brassica Yellows Virus.

Viruses in the genus Polerovirus infect a wide range of crop plants and cause severe economic crop losses. BrYV belongs to the genus Polerovirus and is transmitted by Myzus persicae. However, the changes in transcriptome and proteome profiles of M. persicae during viral infection are unclear. Here, RNA-Seq and TMT-based quantitative proteomic analysis were performed to compare the differences between viruliferous and nonviruliferous aphids. In total, 1266 DEGs were identified at the level of transcription with 980 DEGs being upregulated and 286 downregulated in viruliferous aphids. At the protein level, among the 18 DEPs identified, the number of upregulated proteins in viruliferous aphids was twice that of the downregulated DEPs. Enrichment analysis indicated that these DEGs and DEPs were mainly involved in epidermal protein synthesis, phosphorylation, and various metabolic processes. Interestingly, the expressions of a number of cuticle proteins and tubulins were upregulated in viruliferous aphids. Taken together, our study revealed the complex regulatory network between BrYV and its vector M. persicae from the perspective of omics. These findings should be of great benefit to screening key factors involved in the process of virus circulation in aphids and provide new insights for BrYV prevention via vector control in the field.

Nucleotide sequence of a chickpea chlorotic stunt virus relative that infects pea and faba bean in China.

We determined the genome sequence of a new polerovirus that infects field pea and faba bean in China. Its entire nucleotide sequence (6021 nt) was most closely related (83.3% identity) to that of an Ethiopian isolate of chickpea chlorotic stunt virus (CpCSV-Eth). With the exception of the coat protein (encoded by ORF3), amino acid sequence identities of all gene products of this virus to those of CpCSV-Eth and other poleroviruses were <90%. This suggests that it is a new member of the genus Polerovirus, and the name pea mild chlorosis virus is proposed.

Complete genomic sequence analysis reveals a novel fabavirus infecting cucurbits in China.

The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.

Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification.

For the detection of wheat yellow mosaic virus (WYMV), we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Using Primer Explorer software, four sets of primers were designed and RT-LAMP assay reaction conditions were optimized. The RT-LAMP was performed at different times by four primer sets. Agarose gel analysis showed that WYMV could be detected after 30 min with the primer set III and after 45 min with the other three primer sets, both under the 80-min reaction time. RT-LAMP had the same results with the four primer sets, thus primer set III and 65°C for 80 min reaction were selected for virus detection. There was no significant different when avian myeloblastosis virus (AMV) and moloney murine leukemia virus (M-MLV) RT-LAMP with the four primer sets and M-MLV was chosen due to its relatively cheap price. The result on specificity showed that the assay could amplify WYMV specifically, and the sensitivity comparison showed that the RT-LAMP was 100 times more sensitive than conventional reverse-transcriptase-polymerase chain reaction (RT-PCR). Overall, RT-LAMP was found to be a simple, specific, sensitive, convenient and time-saving method for WYMV detection.

Molecular characterization of two genotypes of a new polerovirus infecting brassicas in China.

The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.

A novel strain of Beet western yellows virus infecting sugar beet with two distinct genotypes differing in the 5'-terminal half of genome.

The complete genomic sequences of two distinct Beet western yellows virus (BWYV) genotypes infecting sugar beet in Beijing, named as BWYV-BJ(A) and BWYV-BJ(B) (GenBank accession number HM804471, HM804472, respectively), were determined by RT-PCR sub-cloning approach. BWYV-BJ(A) and BWYV-BJ(B) were 5674 and 5626nt in length, respectively. BWYV-BJ(B) was 48nt shorter than BWYV-BJ(A) in the regions 1589-1615 and 1629-1649nt. Sequence alignment analysis showed that the full length of BWYV-BJ(A) and BWYV-BJ(B) shared 93% nucleotide sequence identity, with relatively high variability within ORFs 0, 1, 2 (at the nucleotide level was 86.3-88.8%) and high conservation within ORFs 3, 4, 5 (at the nucleotide level was 99.3-99.5%). The complete nucleotide sequences of BWYV-BJ(A) and BWYV-BJ(B) were most related to BWYV-US (80.6 and 79.0%, respectively). ORFs 1, 2 of BWYV-BJ(A) and BWYV-BJ(B) shared the highest homology with BWYV-US (nucleotide identity 91.2-93.3, 86.7-89.5%, respectively) and their ORFs 3, 4 were more closely related to BWYV-IM. However, their ORF5 were more closely related to that of Cucurbit aphid-borne yellows virus China strain (CABYV-CHN), with 68.1 and 68.5% nucleotide identity, respectively. Based on the sequence and phylogenetic analysis, we proposed that BWYV-BJ was at least a novel strain of BWYV, and BWYV-BJ(A), BWYV-BJ(B) were two distinct genotypes of BWYV-BJ. In addition, phylogenetic analysis and recombination analysis suggested that BWYV-BJ(A) and BWYV-BJ(B) might be recombinant viruses.

Comparative Analysis of Biological Characteristics among P0 Proteins from Different Brassica Yellows Virus Genotypes.

Brassica yellows virus (BrYV) is a tentative species of the genus Polerovirus, which has at least three genotypes (A, B, and C) in China. The P0 protein of BrYV-A (P0BrA) has been identified as a viral suppressor of RNA silencing (VSR), which can also induce cell death in infiltrated Nicotiana benthamiana leaves. In this study, we demonstrated that the cell death induced by P0BrA was accompanied by the accumulation of reactive oxygen species (ROS) and increased Pathogenesis-related protein genes-1 (PR1) expression. Meanwhile, this cell death phenotype was delayed by salicylic acid (SA) pretreatment. Biological function comparison of the three P0 proteins showed that transiently expressed P0BrB or P0BrC induced a significantly delayed and milder cell death response compared with P0BrA. However, like P0BrA, they also suppressed local and systemic RNA silencing. Six residues of P0BrA essential for inducing cell death were identified by comparative analysis and amino acid substitution assay. We also show that all three BrYV genotypes have synergistic interactions with pea enation mosaic virus 2 (PEMV 2) in N. benthamiana. This study provides theoretical guidance for controlling the viral disease caused by poleroviruses in the future.

A Simple Method for the Acquisition and Transmission of Brassica Yellows Virus from Transgenic Plants and Frozen Infected Leaves by Aphids.

Brassica yellows virus (BrYV) is a tentative species of the genus Polerovirus, which occurs widely, and mostly damages Brassicaceae plants in East Asia. Because BrYV cannot be transmitted mechanically, an insect-based transmission method is required for further virus research. Here, a reliable and unrestricted method is described, in which non-viruliferous aphids (Myzus persicae) acquired BrYV from transgenic Arabidopsis thaliana, harboring the full-length viral genome germinated from seeds and its frozen leaves. The aphids then transmitted the virus to healthy plants. There was no significant difference in acquisition rates between fresh and frozen infected leaves, although the transmission rate from frozen infected leaves was lower compared to fresh infected leaves. This simple novel method may be used to preserve viral inocula, evaluate host varietal resistance to BrYV, and investigate interactions among BrYV, aphids, and hosts.