The coat protein of the ilarvirus prunus necrotic ringspot virus mediates long-distance movement.

The coat protein (CP) of plant viruses generally has multiple functions involving infection, replication, movement and pathogenicity. Functions of the CP of prunus necrotic ringspot virus (PNRSV), the causal agent of several threatening diseases of Prunus fruit trees, are poorly studied. Previously, we identified a novel virus in apple, apple necrotic mosaic virus (ApNMV), which is phylogenetically related to PNRSV and probably associated with apple mosaic disease in China. Full-length cDNA clones of PNRSV and ApNMV were constructed, and both are infectious in cucumber (Cucumis sativus L.), an experimental host. PNRSV exhibited higher systemic infection efficiency with more severe symptoms than ApNMV. Reassortment analysis of genomic RNA segments 1-3 found that RNA3 of PNRSV could enhance the long-distance movement of an ApNMV chimaera in cucumber, indicating the association of RNA3 of PNRSV with viral long-distance movement. Deletion mutagenesis of the PNRSV CP showed that the basic motif from amino acids 38 to 47 was crucial for the CP to maintain the systemic movement of PNRSV. Moreover, we found that arginine residues 41, 43 and 47 codetermine viral long-distance movement. The findings demonstrate that the CP of PNRSV is required for long-distance movement in cucumber, which expands the functions of ilarvirus CPs in systemic infection. For the first time, we identified involvement of Ilarvirus CP protein during long-distance movement.

The ABI5-dependent down-regulation of mitochondrial ATP synthase OSCP subunit facilitates apple necrotic mosaic virus infection.

Apple necrotic mosaic virus (ApNMV) is associated with apple mosaic disease in China. However, the mechanisms of ApNMV infection, as well as host defence against the virus, are still poorly understood. Mitochondrial ATP synthase plays a fundamental role in the regulation of plant growth and development. However, mitochondrial ATP synthase function in response to virus infection remains to be defined. In the present study, a yeast two-hybrid (Y2H) screening revealed that the apple mitochondrial ATP synthase oligomycin sensitivity-conferring protein (OSCP) subunit (MdATPO) interacts with ApNMV coat protein (CP). It was further verified that overexpression of MdATPO in Nicotiana benthamiana inhibited viral accumulation. In contrast, silencing of NbATPO facilitated viral accumulation, indicating that ATPO plays a defensive role during ApNMV infection. Further investigation demonstrated that ApNMV infection accelerated abscisic acid (ABA) accumulation, and ABA negatively regulated ATPO transcription, which was related to the ability of ABA insensitive 5 (ABI5) to bind to the ABA-responsive elements (ABREs) of the ATPO promoter. Taken together, our results indicated that transcription factor ABI5 negatively regulated ATPO transcription by directly binding to its promoter, leading to the susceptibility of apple and N. benthamiana to ApNMV infection. The current study facilitates a comprehensive understanding of the intricate responses of the host to ApNMV infection.

A novel pathogenicity determinant hijacks maize catalase 1 to enhance viral multiplication and infection.

Pathogens have evolved various strategies to overcome host immunity for successful infection. Maize chlorotic mottle virus (MCMV) can cause lethal necrosis in maize (Zea mays) when it coinfects with a virus in the Potyviridae family. However, the MCMV pathogenicity determinant remains largely unknown. Here we show that the P31 protein of MCMV is important for viral accumulation and essential for symptom development. Ectopic expression of P31 using foxtail mosaic virus or potato virus X induced necrosis in systemically infected maize or Nicotiana benthamiana leaves. Maize catalases (CATs) were shown to interact with P31 in yeast and in planta. P31 accumulation was elevated through its interaction with ZmCAT1. P31 attenuated the expression of salicylic acid (SA)-responsive pathogenesis-related (PR) genes by inhibiting catalase activity during MCMV infection. In addition, silencing of ZmCATs using a brome mosaic virus-based gene silencing vector facilitated MCMV RNA and coat protein accumulation. This study reveals an important role for MCMV P31 in counteracting host defence and inducing systemic chlorosis and necrosis. Our results have implications for understanding the mechanisms in defence and counter-defence during infection of plants by various pathogens.

First report of peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and mume virus A (MuVA) from Mei (Prunus mume) in China.

Mei (Prunus mume Sieb. et Zucc.), widely distributed in East Asian countries for both fruiting- and flowering-purposes, is susceptible to viral infections (Marais et al. 2018). Infection by plum bark necrosis stem pitting-associated virus (PBNSPaV) or little cherry virus 2 (LChV-2) possibly caused overall yield loss in mei in Japan due to incomplete flower development, low fruit bearing rate, and interveinal chlorosis (Numaguchi et al. 2019). Virus-like disease showing mosaic, interveinal chlorosis, vein clearing, or necrotic spot on leaf was observed in mei trees in Beijing, Wuhan, Wuxi, and Nanjing in spring and early summer from 2017 to 2018. Symptomatic leaves collected from the four regions were pooled as two samples for RNA-sequencing (RNA-seq) analysis. After ribosomal RNA (rRNA)-depletion, total RNA extracted by TRNzol reagent (TIANGEN, China) was subjected to library construction using NEBNext Ultra RNA Library Prep Kit (NEB, MA, USA) and sequenced on an Illumina Hiseq 4000 (Novogene, China). Sequencing data was filtered, screened, and assembled as described previously (Zhou et al. 2020) to generate contigs, following by BLAST-x/n search in viral genomes in GenBank. We identified >300 contigs (208-10756 nt) homologous to Asian prunus virus 1 and Asian prunus virus 2 (APV1 and 2), mume virus A (MuVA), PBNSPaV, and peach leaf pitting-associated virus (PLPaV), with 71-100% of nucleotide sequence identity values. APV1 and 2 have been reported in mei in China (Wang et al. 2018), here, we focused on the other three viruses. Contigs homologous to these three viruses were further assembled into three scaffolds of 14,224 nt, 1107 nt, and 753 nt for PBNSPaV, MuVA, and PLPaV, respectively. The scaffold of PBNSPaV (MW217574) nearly covered the whole genome of the isolate VIC3 from Australia (LC523039.1) (Kinoti et al. 2020) with 92.30% of sequence identity; the scalffold of MuVA (MW217572) covered 14.50% of the genome of the isolate pm14 from Japan (NC 040568.1) (Marais et al.2018) with 98.47% sequence identity; the scaffold of PLPaV (MW217573) covered 15.26% of the genome of the isolate XJ-6 from peach (KY867750.1) (He et al. 2017) with 85.23% sequence identity. Presence of the three viruses were verified by RT-PCR detection using designed specific primers for PBNSPaV (Forward: 5'-CAACAAAACTCCCACAGCGG-3 [positions 4014-4033, NC_009992.1] / Reverse: 5'-GCCAAAAGAAGTGCTGGTGG-3' [positions 4659-4640, NC_009992.1]), MuVA (Forward: 5'-AAGAGAATTACTTCAATGCCCTC-3' [positions 171-194, NC_040568.1] / Reverse: 5'-GATATCCAAGATACGATTAGCCAG-3' [positions 533-510, NC_040568.1]), and PLPaV (Forward: 5'-GCTATATCTCAACAACTGCAAGAA-3 [positions 5798-5821, KY867750.1] / Reverse: 5'- GAGTGATACATAGTCCACAGAGAT-3'[ positions 6045-6022, KY867750.1]). The amplified 626, 350 and 251 bp fragments of PBNSPaV, MuVA and PLPaV had 91.47%, 98.07% and 81.89% sequence identity to their respective reference sequences. This is the first report of PBNSPaV and MuVA infecting mei in China, and more importantly, the first report of a new host for PLPaV. In addition, 30 collected leaf samples from Nanjing and Wuhan were analyzed by RT-PCR and 15, 6, and 5 samples tested positive to PLPaV, PBNSPaV, and MuVA, respectively. Although it is difficult to link a particular virus with the observed symptoms due to mixed infections, the symptoms were significantly associated with viral infection because almost all symptomatic leaf samples were virus(es)-positive. Further studies would be required to determine the distribution and impact of these viruses on mei trees and other stone fruits species and to understand the possibility that mei trees may play a role in PLPaV epidemiology.

Lipid flippases promote antiviral silencing and the biogenesis of viral and host siRNAs in Arabidopsis.

Dicer-mediated processing of virus-specific dsRNA into short interfering RNAs (siRNAs) in plants and animals initiates a specific antiviral defense by RNA interference (RNAi). In this study, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in Arabidopsis thaliana Using whole-genome sequencing and a computational pipeline, we identified aminophospholipid transporting ATPase 2 (ALA2) and the related ALA1 in the type IV subfamily of P-type ATPases as key components of antiviral RNAi. ALA1 and ALA2 are flippases, which are transmembrane lipid transporter proteins that transport phospholipids across cellular membranes. We found that the ala1/ala2 single- and double-mutant plants exhibited enhanced disease susceptibility to cucumber mosaic virus when the virus-encoded function to suppress RNAi was disrupted. Notably, the antiviral activity of both ALA1 and ALA2 was abolished by a single amino acid substitution known to inactivate the flippase activity. Genetic analysis revealed that ALA1 and ALA2 acted to enhance the amplification of the viral siRNAs by RNA-dependent RNA polymerase (RdRP) 1 (RDR1) and RDR6 and of the endogenous virus-activated siRNAs by RDR1. RNA virus replication by plant viral RdRPs occurs inside vesicle-like membrane invaginations induced by the recruitment of the viral RdRP and host factors to subcellular membrane microdomains enriched with specific phospholipids. Our results suggest that the phospholipid transporter activity of ALA1/ALA2 may be necessary for the formation of similar invaginations for the synthesis of dsRNA precursors of highly abundant viral and host siRNAs by the cellular RdRPs.

Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease.

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

Knockout of SlDCL2b attenuates the resistance of tomato to potato spindle tuber viroid infection.

RNA interference, or RNA silencing, is an important defence mechanism against viroid infection in plants. Plants encode multiple DICER-LIKE (DCL) proteins that are key components of the RNA silencing pathway. However, the roles of different DCLs in defence responses against viroid infection remain unclear. Here, we determined the function of tomato DCL2b (SlDCL2b) in defence responses against potato spindle tuber viroid (PSTVd) infection using SlDCL2b loss-of-function tomato mutant plants. Compared with wild-type plants, mutant plants were more susceptible to PSTVd infection, developing more severe symptoms earlier and accumulating higher levels of PSTVd RNAs. Moreover, we verified the feedback mechanism for the regulation of SlDCL2b expression by miR6026. Functional blocking of tomato miR6026, by expressing its target mimics, can enhance resistance to PSTVd infection in tomato plants. These findings deepen the current understanding of RNAi-based resistance against viroid infection and provide a potentially new strategy for viroid control.

Selection and Validation of Reference Genes for Gene Expression Studies Using Quantitative Real-Time PCR in Prunus Necrotic Ringspot Virus-Infected Cucumis sativus.

Several members of the genus Ilarvirus infect fruit trees and are distributed worldwide. Prunus necrotic ringspot virus (PNRSV) is one of the most prevalent viruses, causing significant losses. Cucumissativus can be infected by several ilarviruses, leading to obvious symptoms, including PNRSV, which suggests that cucumbers could be good hosts for the study of the pathogenesis of ilarviruses. Real-time quantitative PCR is an optimal choice for studying gene expression because of its simplicity and its fast and high sensitivity, while its accuracy is highly dependent on the stability of the reference genes. In this study, we assessed the stability of eleven reference genes with geNorm, NormFinder, ΔCt method, BestKeeper, and the ranking software, RefFinder. The results indicated that the combined use of EF1α and F-BOX was the most accurate normalization method. In addition, the host genes AGO1, AGO4, and RDR6 were selected to test the reliability of the reference genes. This study provides useful information for gene expression analysis during PNRSV infection and will facilitate gene expression studies associated with ilarvirus infection.

Detection and Simultaneous Differentiation of Three Co-infected Viruses in Zanthoxylum armatum.

Green Sichuan pepper (Zanthoxylum armatum) is an important economic fruit crop, which is widely planted in the southwest region of China. Recently, a serious disease, namely flower yellowing disease (FYD), broke out, and the virus of green Sichuan pepper nepovirus (GSPNeV) was identified to be highly correlated with the viral symptoms. Meanwhile, green Sichuan pepper idaeovirus (GSPIV) and green Sichuan pepper enamovirus (GSPEV) were also common viruses infecting green pepper. In our research, specific primers were designed according to the reported sequences of the three viruses, and a multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for the simultaneous detection of GSPNeV, GSPIV, and GSPEV was established. The annealing temperature, extension time, and cycle number affecting the multiplex RT-PCR reaction were adjusted and optimized. Sensitivity analysis showed that the system could detect the three viruses simultaneously from the complementary deoxyribonucleic acid (cDNA) samples diluted by 10-3. The results of the ten samples detected by the multiplex RT-PCR system were consistent with the results of a single PCR, indicating that the method can be successfully used for rapid detection of field samples.

Identification of Silencing Suppressor Protein Encoded by Strawberry Mottle Virus.

Strawberry mottle virus (SMoV) is associated with strawberry decline disease, causing losses to fruit yield and quality. In this study, using a screening system that enables detection of both local and systemic plant host (RNA silencing) defense responses, we found that Pro2Glu and P28, encoded by SMoV RNA2 genome, functioned to suppress local and systemic RNA silencing triggered by single- but not double-stranded GFP RNA. Subcellular localization assay revealed that both Pro2Glu and P28 were localized to nucleus and cytoplasm. The deletion of 11 amino acid residues at the C-terminus destabilized Pro2Glu protein, and the disruption of two conserved GW motifs deprived Pro2Glu of ability to suppress RNA silencing. Additionally, SMoV Pro2Glu and P28 enhanced the accumulation of potato virus X (PVX) in Nicotiana benthamiana 22 days post-infiltration, and P28 exacerbated significantly the symptoms of PVX. Collectively, these data indicate that the genome of SMoV RNA2 encodes two suppressors of RNA silencing. This is the first identification of a stramovirus suppressor of RNA silencing.

Characterization of an Isolate of Citrus Concave Gum-Associated Virus from Apples in China and Development of an RT-RPA Assay for the Rapid Detection of the Virus.

Apple (Malus domestica) fruits exhibiting bright stripe symptoms were identified in Weihai City, Shandong Province, China. To investigate the virome in the apple samples, the method of high throughput sequencing (HTS) was used to identify the viruses. It was found that the sequence of citrus concave gum-associated virus (CCGaV) was involved in the apple transcriptome dataset. The full-length genome of the CCGaV-Weihai isolate contained two segments, the RNA1 was 6674 nt in size containing a conserved RNA-dependent RNA polymerase (RdRp), and the RNA2 was ambisense, 2706 nt in length, encoding a movement protein (MP) and a coat protein (CP). Sequence alignment and phylogenetic analyses indicated that CCGaV-Weihai was more closely related to CCGaV-H2799 isolated from the apple host in the United States and distantly related to CCGaV-CGW2 from Citrus sinensis in Italy, indicating a possibly geographical and host differentiation of CCGaV isolates. This was the first identification and characterization of CCGaV infecting apples in China. Additionally, a rapid and sensitive reverse transcription recombinase polymerase amplification (RT-RPA) assay technique was established for CCGaV detection in apple plants. The RT-RPA of CCGaV was not affected by other common viruses in apple plants and is about 10-fold more sensitive than the conventional reverse transcription polymerase chain reaction method, which can be used in large-scale testing.

Spatial Virome Analysis of Zanthoxylum armatum Trees Affected With the Flower Yellowing Disease.

Zanthoxylum armatum is an important woody crop with multiple applications in pharmaceutics, cosmetics, and food industries. With continuous increases in the plantation area, integrated pest management is required for scale production when diseases caused by biotic factors such as pests and pathogens have become new problems, one of which is the infectious flower yellowing disease (FYD). Here, isolates of a new illarvirus (3) and a new nepovirus-associated subviral satellite RNA (12) were identified in Z. armatum, in addition to 38 new isolates of four previously reported RNA viruses. Sequence variation can be observed in viral/subviral quasispecies and among predominant isolates from the same or different samples and geographic origins. Intriguingly, RNA sequencing of different diseased trees invariably showed an extraordinary pattern of particularly high reads accumulation of the green Sichuan pepper-nepovirus (GSPNeV) and the satellite RNA in symptomatic tissues. In addition, we also examined small RNAs of the satellite RNA, which show similar patterns to those of coinfecting viruses. This study provides further evidence to support association of the FYD with viral/subviral infections and deepens our understanding of the diversity and molecular characteristics of the viruses and satellite, as well as their interactions with the host.

Nitric Oxide as a Downstream Signaling Molecule in Brassinosteroid-Mediated Virus Susceptibility to Maize Chlorotic Mottle Virus in Maize.

Maize chlorotic mottle virus (MCMV) infection causes growth abnormalities in maize. Transcriptome sequencing was conducted to compare the global gene expression of MCMV-inoculated plants with that of mock-inoculated plants. Data analyses showed that brassinosteroid (BR)-associated genes were upregulated after MCMV infection. Exogenous 2,4-epibrassinolide (BL) or brassinazole (BRZ) applications indicated that BR pathway was involved in the susceptibility to MCMV infection. In addition, treatment of BL on maize induced the accumulation of nitric oxide (NO), and the changes of NO content played positive roles in the disease incidence of MCMV. Moreover, MCMV infection was delayed when the BL-treated plants were applied with NO scavenger, which suggested that BR induced the susceptibility of maize to MCMV infection in a NO-dependent manner. Further investigation showed the maize plants with knock-down of DWARF4 (ZmDWF4, a key gene of BR synthesis) and nitrate reductase (ZmNR, a key gene of NO synthesis) by virus-induced gene silencing displayed higher resistance to MCMV than control plants. Taken together, our results suggest that BR pathway promotes the susceptibility of maize to MCMV in a NO-dependent manner.

Detection and Characterization of Cucumis melo Cryptic Virus, Cucumis melo Amalgavirus 1, and Melon Necrotic Spot Virus in Cucumis melo.

Three RNA viruses-Cucumis melo cryptic virus (CmCV), Cucumis melo amalgavirus 1 (CmAV1), and melon necrotic spot virus (MNSV)-were identified from a melon (Cucumis melo) transcriptome dataset. CmCV has two dsRNA genome segments; dsRNA-1 is 1592 bp in size, containing a conserved RNA-dependent RNA polymerase (RdRp), and dsRNA-2 is 1715 bp in size, and encodes a coat protein (CP). The sequence alignment and phylogenetic analyses of the CmCV RdRp and CP indicated CmCV clusters with approved or putative deltapartitiviruses in well-supported monophyletic clade. The RdRp of CmCV shared an amino acid sequence identity of 60.7% with the closest RdRp of beet cryptic virus 3, and is <57% identical to other partitiviruses. CmAV1 is a nonsegmented dsRNA virus with a genome of 3424 bp, including two partially overlapping open reading frames (ORFs) encoding a putative CP and RdRp. The sequence alignment and phylogenetic analyses of CmAV1 RdRp revealed that it belongs to the genus Amalgavirus in the family Amalgaviridae. The RdRp of CmAV1 shares 57.7% of its amino acid sequence identity with the most closely related RdRp of Phalaenopsis equestris amalgavirus 1, and is <47% identical to the other reported amalgaviruses. These analyses suggest that CmCV and CmAV1 are novel species in the genera Amalgavirus and Deltapartitivirus, respectively. These findings enrich our understanding of new plant dsRNA virus species.

Characterization of maize translational responses to sugarcane mosaic virus infection.

Sugarcane mosaic virus (SCMV) frequently causes dramatic losses in maize production as the main pathogen of maize dwarf mosaic disease. It is important to understand the translational responses in maize to SCMV infection since viruses have to recruit host translation apparatus to express their proteins. However, due to technical limitations, research on virus translation lags far behind that on transcription. Here, we studied the relationship between systemic symptom expression and virus accumulation and found that both SCMV RNA and proteins accumulated rapidly during the systemic infection process in which varying degrees of chlorosis to mosaic symptoms developed on non-inoculated leaves. In addition, we applied ribosome profiling, which couples polysomal mRNA isolation with high-throughput sequencing, on the symptomatic leaves infected with SCMV to unravel the translational responses of maize to viral infection on a genome-wide scale. The results showed that only the genomic positive-stranded RNA of SCMV was involved in translation, and SCMV only occupied a small amount of translational resources of host plant at the early stage of infection. Further analyses on a global gene expression and gene ontology (GO) enrichment revealed that photosynthesis and metabolism were dramatically repressed at both transcriptional and translational levels. Altogether, our results laid a foundation for dissecting the molecular mechanism of plant translational responses to viral infection.

Functional Scanning of Apple Geminivirus Proteins as Symptom Determinants and Suppressors of Posttranscriptional Gene Silencing.

Apple geminivirus (AGV) is a recently identified geminivirus which is isolated from the apple tree in China. We carried out functional scanning of apple geminivirus proteins as symptom determinants and suppressors of posttranscriptional gene silencing (PTGS). Our results indicated that AGV V2 is an important virulence factor localized to the nucleus and cytoplasm that suppresses PTGS and induces severe symptoms of crinkling and necrosis. AGV C1 is also a virulence determinant which elicits systemic necrosis when expressed from a PVX-based vector. The AGV C4 is targeted to cytoplasm, plasma membrane, nucleus, and chloroplasts. The inoculation of PVX-C4 on N. benthamiana induced severe upward leaf curling, which implied that AGV C4 also functions as a symptom determinant, and mutation analyses suggested that the acylated residues on Gly2 and Cys8 play important roles in its subcellular localization and symptom development.