Integrated Transcriptome and Metabolome Dissecting Interaction between Vitis vinifera L. and Grapevine Fabavirus.

Grapevine fabavirus (GFabV) is a novel member of the Fabavirus genus associated with chlorotic mottling and deformation symptoms in grapevines. To gain insights into the interaction between GFabV and grapevines, V. vinifera cv. 'Summer Black' infected with GFabV was investigated under field conditions through physiological, agronomic, and multi-omics approaches. GFabV induced significant symptoms on 'Summer Black', and caused a moderate decrease in physiological efficiency. In GFabV-infected plants, alterations in carbohydrate- and photosynthesis-related genes might trigger some defense responses. In addition, secondary metabolism involved in plant defense was progressively induced by GFabV. Jasmonic acid and ethylene signaling were down-regulated in GFabV-infected leaves and berries along with the expression of proteins related to LRR and protein kinases, suggesting that GFabV can block the defense in healthy leaves and berries. Furthermore, this study provided biomarkers for early monitoring of GFabV infection in grapevines, and contributed to a better understanding of the complex grapevine-virus interaction.

Genome sequence and characterization of a novel fabavirus infecting Cirsium setidens (gondre) in South Korea.

The complete nucleotide sequence of a novel gondre (Cirsium setidens)-infecting virus, provisionally named "cirsium virus A" (CiVA), was determined by high-throughput and Sanger sequencing, revealing a genome organization typical of fabaviruses. RNA1 and RNA2 are 5,828 and 3,478 nucleotides long, excluding the 3'-terminal poly(A) tails, each containing a single open reading frame. The highest sequence identity values for the CiVA coat protein and proteinase-polymerase, compared with known fabavirus sequences, were 59.09% and 69.68%, respectively, falling below the current thresholds for Fabavirus species demarcation. Our findings support classifying CiVA as a novel putative member of the genus Fabavirus, subfamily Comovirinae, family Secoviridae.

Genetic plasticity in RNA2 is associated with pathogenic diversification of broad bean wilt virus 2.

Broad bean wilt virus 2 (BBWV2) is an evolutionarily successful RNA virus with an extensive host range and worldwide distribution that causes severe damage to crops. While numerous BBWV2 isolates from various plant species have been identified and their genome sequences determined, little information is available on the virulence and symptomatic characteristics corresponding to the genomic sequences. In this study, we provide integrated information on the molecular and pathogenic characteristics of three genetically distant BBWV2 isolates: BBWV2-PC, -LS2, and P3 obtained from Gentiana scabra, Leonurus sibiricus, and Pisum sativum, respectively. Phylogenetic and diversity analyses of the BBWV2 population included 42 isolates from various host species and revealed that RNA2 has higher genetic plasticity than RNA1 and may have evolved under host-imposed constraints. In addition, we generated an infectious cDNA clone of BBWV2-PC RNA2 (pBBWV2-PC-R2). Pseudo-recombination analysis of pBBWV2-PC-R2 further demonstrated that RNA2 determines the pathogenic characteristics of the PC isolate.

Phylodynamics and Codon Usage Pattern Analysis of Broad Bean Wilt Virus 2.

Broad bean wilt virus 2 (BBWV-2), which belongs to the genus Fabavirus of the family Secoviridae, is an important pathogen that causes damage to broad bean, pepper, yam, spinach and other economically important ornamental and horticultural crops worldwide. Previously, only limited reports have shown the genetic variation of BBWV2. Meanwhile, the detailed evolutionary changes, synonymous codon usage bias and host adaptation of this virus are largely unclear. Here, we performed comprehensive analyses of the phylodynamics, reassortment, composition bias and codon usage pattern of BBWV2 using forty-two complete genome sequences of BBWV-2 isolates together with two other full-length RNA1 sequences and six full-length RNA2 sequences. Both recombination and reassortment had a significant influence on the genomic evolution of BBWV2. Through phylogenetic analysis we detected three and four lineages based on the ORF1 and ORF2 nonrecombinant sequences, respectively. The evolutionary rates of the two BBWV2 ORF coding sequences were 8.895 × 10-4 and 4.560 × 10-4 subs/site/year, respectively. We found a relatively conserved and stable genomic composition with a lower codon usage choice in the two BBWV2 protein coding sequences. ENC-plot and neutrality plot analyses showed that natural selection is the key factor shaping the codon usage pattern of BBWV2. Strong correlations between BBWV2 and broad bean and pepper were observed from similarity index (SiD), codon adaptation index (CAI) and relative codon deoptimization index (RCDI) analyses. Our study is the first to evaluate the phylodynamics, codon usage patterns and adaptive evolution of a fabavirus, and our results may be useful for the understanding of the origin of this virus.

RNA2-encoded VP37 protein of Broad bean wilt virus 1 is a determinant of pathogenicity, host susceptibility, and a suppressor of post-transcriptional gene silencing.

Broad bean wilt virus 1 (BBWV-1, genus Fabavirus, family Secoviridae) is a bipartite, single-stranded positive-sense RNA virus infecting many horticultural and ornamental crops worldwide. RNA1 encodes proteins involved in viral replication whereas RNA2 encodes two coat proteins (the large and small coat proteins) and two putative movement proteins (MPs) of different sizes with overlapping C-terminal regions. In this work, we determined the role played by the small putative BBWV-1 MP (VP37) on virus pathogenicity, host specificity, and suppression of post-transcriptional gene silencing (PTGS). We engineered a BBWV-1 35S-driven full-length cDNA infectious clone corresponding to BBWV-1 RNA1 and RNA2 (pBBWV1-Wt) and generated a mutant knocking out VP37 (pBBWV1-G492C). Agroinfiltration assays showed that pBBWV1-Wt, as the original BBWV-1 isolate, infected broad bean, tomato, pepper, and Nicotiana benthamiana, whereas pBBWV1-G492C did not infect pepper and tomato systemically. Also, pBBWV1-G492C induced milder symptoms in broad bean and N. benthamiana than pBBWV1-Wt. Differential retrotranscription and amplification of the (+) and (-) strands showed that pBBWV1-G492C replicated in the agroinfiltrated leaves of pepper but not in tomato. All this suggests that VP37 is a determinant of pathogenicity and host specificity. Transient expression of VP37 through a potato virus X (PVX) vector enhanced PVX symptoms and induced systemic necrosis associated with programmed cell death in N. benthamiana plants. Finally, VP37 was identified as a viral suppressor of RNA silencing by transient expression in N. benthamiana 16c plants and movement complementation of a viral construct based on turnip crinkle virus (pTCV-GFP).

Genetic variability of grapevine fabavirus variants and development of a broad-spectrum assay for their detection.

Complete RNA1 and RNA2 sequences of two and nearly complete genome sequences of six new variants of grapevine fabavirus found in Japan were compared to those of previously reported variants. Negative selection pressure was suggested, and no recombination events were detected in either RNA1 or RNA2. The first 18 nucleotides in both RNAs were predicted to form a stem-loop structure. The variants could be genetically divided into four groups based on RNA1 and two based on RNA2. A broad-spectrum reverse transcription polymerase chain reaction assay using a primer pair designed based on an RNA2 consensus sequence was able to detect all of the known variants.

Mikania Micrantha Wilt Virus Alters Insect Vector's Host Preference to Enhance Its Own Spread.

As an invasive weed, Mikaniamicrantha Kunth has caused serious damage to natural forest ecosystems in South China in recent years. Mikania micrantha wilt virus (MMWV), an isolate of the Gentian mosaic virus (GeMV), is transmitted by Myzuspersicae (Sulzer) in a non-persistent manner and can effectively inhibit the growth of M. micrantha. To explore the MMWV-M. micrantha-M. persicae interaction and its impact on the invasion of M. micrantha, volatile compounds (VOCs) emitted from healthy, mock-inoculated, and MMWV-infected plants were collected, and effects on host preference of the apterous and alate aphids were assessed with Y-shaped olfactometers. Gas chromatography-mass spectrometry (GC-MS) analysis indicated that MMWV infection changed the VOC profiles, rendering plants more attractive to aphids. Clip-cages were used to document the population growth rate of M.persicae fed on healthy, mock-inoculated, or MMWV-infected plants. Compared to those reared on healthy plants, the population growth of M. persicae drastically decreased on the MMWV-infected plants. Plant host choice tests based on visual and contact cues were also conducted using alate M.persicae. Interestingly, the initial attractiveness of MMWV-infected plants diminished, and more alate M. persicae moved to healthy plants. Taken together, MMWV appeared to be able to manipulate its plant host to first attract insect vectors to infected plants but then repel viruliferous vectors to promote its own dispersal. Its potential application for invasive weed management is discussed.

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing.

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species.

Movement protein of broad bean wilt virus 2 serves as a determinant of symptom severity in pepper.

Broad bean wilt virus 2 (BBWV2, genus Fabavirus, family Secoviridae) has a wide host range and infects many economically important crops. Various isolates of BBWV2 have been identified from diverse host plants, and their molecular and biological characteristics have been investigated. In our previous study, we demonstrated that BBWV2 RNA2 contains a symptom determinant(s) capable of enhancing symptom severity by utilizing infectious full-length cDNA clones of two distinct strains of BBWV2, pBBWV2-PAP1 (a severe strain) and pBBWV2-RP1 (a mild strain). In the present study, to identify the symptom determinant(s) of BBWV2, we exploited disease responses of pBBWV2-PAP1- and pBBWV2-RP1-derived chimeric viruses and amino acid substitution mutant viruses in Nicotiana benthamiana and pepper (Capsicum annuum Quarri) and demonstrated that the movement protein (MP) encoded in BBWV RNA2 is the determinant of disease symptom severity in both plants. A single amino acid substitution in the MP was sufficient for changing symptom severity of BBWV2. Our finding provides a role for the MP as a symptom determinant in BBWV2 and increases the understanding of the basis of molecular interactions between host plants and BBWV2.

Deep sequencing reveals the first fabavirus infecting peach.

A disease causing smaller and cracked fruit affects peach [Prunus persica (L.) Batsch], resulting in significant decreases in yield and quality. In this study, peach tree leaves showing typical symptoms were subjected to deep sequencing of small RNAs for a complete survey of presumed causal viral pathogens. The results revealed two known viroids (Hop stunt viroid and Peach latent mosaic viroid), two known viruses (Apple chlorotic leaf spot trichovirus and Plum bark necrosis stem pitting-associated virus) and a novel virus provisionally named Peach leaf pitting-associated virus (PLPaV). Phylogenetic analysis based on RNA-dependent RNA polymerase placed PLPaV into a separate cluster under the genus Fabavirus in the family Secoviridae. The genome consists of two positive-sense single-stranded RNAs, i.e., RNA1 [6,357 nt, with a 48-nt poly(A) tail] and RNA2 [3,862 nt, with a 25-nt poly(A) containing two cytosines]. Biological tests of GF305 peach indicator seedlings indicated a leaf-pitting symptom rather than the smaller and cracked fruit symptoms related to virus and viroid infection. To our knowledge, this is the first report of a fabavirus infecting peach. PLPaV presents several new molecular and biological features that are absent in other fabaviruses, contributing to an overall better understanding of fabaviruses.

High throughput sequencing reveals a novel fabavirus infecting sweet cherry.

The genus Fabavirus currently consists of five species represented by viruses that infect a wide range of hosts but none reported from temperate climate fruit trees. A virus with genomic features resembling fabaviruses (tentatively named Prunus virus F, PrVF) was revealed by high throughput sequencing of extracts from a sweet cherry tree (Prunus avium). PrVF was subsequently shown to be graft transmissible and further identified in three other non-symptomatic Prunus spp. from different geographical locations. Two genetic variants of RNA1 and RNA2 coexisted in the same samples. RNA1 consisted of 6,165 and 6,163 nucleotides, and RNA2 consisted of 3,622 and 3,468 nucleotides.

Molecular and biological characterization of highly infectious transcripts from full-length cDNA clones of broad bean wilt virus 1.

Broad bean wilt virus 1 (BBWV-1), genus Fabavirus, has a genome composed of two single-stranded positive-sense RNAs of ∼5.8 (RNA1) and 3.4kb (RNA2). Full-length cDNA clones of both genomic RNAs (pBenR1 and pBenR2) from BBWV-1 isolate Ben were constructed under the control of the T7 promoter. In vitro derived capped transcripts were infectious in Nicotiana benthamiana, Chenopodium quinoa and Vicia faba plants. The biological activity of viral transcripts was not affected by extra bases at the 5'-terminus introduced during in vitro transcription. Virions derived from the infectious cDNA clones displayed similar viral infectivity and accumulation, as well as symptom induction as the wild-type BBWV-1 isolate.

Mutual association of Broad bean wilt virus 2 VP37-derived tubules and plasmodesmata obtained from cytological observation.

The movement protein VP37 of broad bean wilt virus 2 (BBWV 2) forms tubules in the plasmodesmata (PD) for the transport of virions between cells. This paper reports a mutual association between the BBWV 2 VP37-tubule complex and PD at the cytological level as determined by transmission electron microscopy. The generation of VP37-tubules within different PD leads to a different occurrence frequency as well as different morphology lines of virus-like particles. In addition, the frequency of VP37-tubules was different between PD found at different cellular interfaces, as well as between single-lined PD and branched PD. VP37-tubule generation also induced structural alterations of PD as well as modifications to the cell wall (CW) in the vicinity of the PD. A structural comparison using three-dimensional (3D) electron tomography (ET), determined that desmotubule structures found in the center of normal PD were absent in PD containing VP37-tubules. Using gold labeling, modification of the CW by callose deposition and cellulose reduction was observable on PD containing VP37-tubule. These cytological observations provide evidence of a mutual association of MP-derived tubules and PD in a natural host, improving our fundamental understanding of interactions between viral MP and PD that result in intercellular movement of virus particles.

A determinant of disease symptom severity is located in RNA2 of broad bean wilt virus 2.

Broad bean wilt virus 2 (BBWV2), which belongs to the genus Fabavirus, is a destructive pathogen of many economically important horticultural and ornamental crops. In this study, we constructed infectious full-length cDNA clones of two distinct isolates of BBWV2 under control of the cauliflower mosaic virus 35S promoter. BBWV2-PAP1 isolated from paprika (Capsicum annuum var. gulosum) induces severe disease symptoms in various pepper varieties, whereas BBWV2-RP1 isolated from red pepper (Capsicum annuum L.) causes mild symptoms. Agrobacterium-mediated inoculation of the infectious cDNA clones of BBWV2-PAP1 and RP1 resulted in the same symptoms as the original virus isolates. The infectious cDNA clones of BBWV2-PAP1 and RP1 were used to examine the symptoms induced by pseudorecombinants between the two isolates to localize in which of the two genomic RNAs are the symptom severity determinants in BBWV2. The pseudorecombinant of RP1-RNA1 and PAP1-RNA2 induced severe symptoms, similar to those caused by the parental isolate PAP1, whereas the pseudorecombinant of PAP1-RNA1 and RP1-RNA2 induced mild symptoms, similar to those caused by the parental isolate RP1. Our results suggest that BBWV2 RNA2 contains a symptom determinant(s) capable of enhancing symptom severity.

[Molecular identification and sequence analysis of broad bean wilt virus 2 isolates from atractylodes macrocephala Koidz].

To identity the pathogen that causes the mosaic and yellowing symptoms on Atractylodes macrocephala Koidz in Jiangxian, Shanxi province, biological inoculation, sequence-independent amplification (SIA),RT-PCR and other identification methods were used. The results showed that the chlorotic and necrosis symptoms occurred in the indicator plant Chenopodium quinoa after it was infected with the pathogen,and the same symptoms appeared after the reinoculation of healthy Atractylodes macrocephala Koidz; this reflected that the disease was likely to be caused by a virus. The results of SIA and sequencing showed that Broad bean wilt virus 2 (BBWV2) was present in severely mosaic Atractylodes macrocephala Koidz leaves. To further characterize the BBWV2 isolate from Atractylodes macrocephala (BBWV2-Am), the polyprotein partial gene encoded by BBWV2-Am RNA2 was cloned and sequenced. Sequence alignments showed that the nucleotide sequence identity of BBWV2-Am SCP and LCP genes ranged from 79.3% to 87.2% and from 80.1% to 89.2% compared to other BBWV2 strains,respectively; the deduced amino acid sequence similarities of the two gene products ranged from 91.2% to 95.7% and from 89.44 to 95.5%, respectively,compared to those of other BBWV2 strains. Phylogenetic comparisons showed that BBWV2-Am was most likely to be related to BBWV2-Rg,but formed an independent branch. This is the first report of BBWV2 in Atractylodes macrocephala Koidz.

Molecular identification and sequence analysis of broad bean wilt virus 2 isolates from atractylodes macrocephala Koidz / 病毒学报

To identity the pathogen that causes the mosaic and yellowing symptoms on Atractylodes macrocephala Koidz in Jiangxian, Shanxi province, biological inoculation, sequence-independent amplification (SIA),RT-PCR and other identification methods were used. The results showed that the chlorotic and necrosis symptoms occurred in the indicator plant Chenopodium quinoa after it was infected with the pathogen,and the same symptoms appeared after the reinoculation of healthy Atractylodes macrocephala Koidz; this reflected that the disease was likely to be caused by a virus. The results of SIA and sequencing showed that Broad bean wilt virus 2 (BBWV2) was present in severely mosaic Atractylodes macrocephala Koidz leaves. To further characterize the BBWV2 isolate from Atractylodes macrocephala (BBWV2-Am), the polyprotein partial gene encoded by BBWV2-Am RNA2 was cloned and sequenced. Sequence alignments showed that the nucleotide sequence identity of BBWV2-Am SCP and LCP genes ranged from 79.3% to 87.2% and from 80.1% to 89.2% compared to other BBWV2 strains,respectively; the deduced amino acid sequence similarities of the two gene products ranged from 91.2% to 95.7% and from 89.44 to 95.5%, respectively,compared to those of other BBWV2 strains. Phylogenetic comparisons showed that BBWV2-Am was most likely to be related to BBWV2-Rg,but formed an independent branch. This is the first report of BBWV2 in Atractylodes macrocephala Koidz.

Broad bean wilt virus 2 encoded VP53, VP37 and large capsid protein orchestrate suppression of RNA silencing in plant.

Viruses encode RNA silencing suppressors to counteract host RNA silencing-mediated defense responses. In this study, we demonstrate that VP53, VP37 and LCP encoded by RNA2 of broad bean wilt virus 2 (BBWV-2), a member of the genus Fabavirus, are strong suppressors of RNA silencing triggered by single-stranded sense RNA. They, however, had no effect on suppression of RNA silencing induced by double-stranded RNA. We provide evidence that these three suppressors can significantly limit the accumulation of small interfering RNAs (siRNAs) in tissues where the GFP gene has been silenced, and prevent the long distance spread of the induced silencing signal. Gel mobility shift assays showed that all three suppressors could bind ssRNA in a size-specific manner. Interestingly, VP37 and LCP, but not VP53, could reverse the silencing of a GFP gene in leaf tissue. Furthermore, these three proteins are capable of enhancing pathogenicity of potato virus X. Collectively, our findings indicate that viruses employ a more sophisticated strategy to overcome the host defense response mediated through suppression of RNA silencing during virus infection. As far as we are aware, this is the first report of RNA silencing suppressors encoded by a virus in the genus Fabavirus.

Detection and identification of Fabavirus species by one-step RT-PCR and multiplex RT-PCR.

The genus Fabavirus of the family Secoviridae comprises a group of poorly characterized viruses. To date, only five species have been described: Broad bean wilt virus 1 (BBWV-1), Broad bean wilt virus 2 (BBWV-2), Lamium mild mosaic virus (LMMV), Gentian mosaic virus (GeMV) and Cucurbit mild mosaic virus (CuMMV). The development is described of two RT-PCR procedures for the detection and identification of Fabavirus species: a one-step RT-PCR using a single pair of conserved primers for the detection of all fabaviruses, and a one-step multiplex RT-PCR using species-specific primers for the simultaneous detection and identification of the above-mentioned species of the genus Fabavirus. These methods were applied successfully to field samples and the results were compared with those obtained by molecular hybridization and ELISA. The combination of the two techniques enables rapid, sensitive and reliable identification of the five known fabavirus species, as well as the possibility of discovering new species of this genus.

Genetic variability and evolution of broad bean wilt virus 1: role of recombination, selection and gene flow.

Analysis of four genomic regions from 37 geographically diverse isolates of broad bean wilt virus 1 (BBWV-1) showed high genetic diversity in comparison to most plant viruses. Comparison of synonymous and nonsynonymous substitutions of the small coat protein gene (SCP) revealed negative selection for most amino acid positions. Phylogenetic analysis of SCP showed that some BBWV-1 isolates from distant geographical areas were genetically close, suggesting long-distance migration. Analysis of genetic differentiation revealed high gene flow between Spanish and Near Eastern subpopulations, which were separated from North-Central and South-Eastern European subpopulations. Finally, putative recombinant and reassortant genomes were also identified.

The complete genome sequence of Lamium mild mosaic virus, a member of the genus Fabavirus.

Lamium mild mosaic virus (LMMV) is the only one of the five members of the genus Fabavirus for which there are no nucleotide sequence data. In this study, the complete genome sequence of LMMV was determined and compared with the available complete genome sequences of other members of the genus Fabavirus. The genome was the largest of the genus but maintained the typical organization, with RNA 1 of 6080 nucleotides (nt), RNA 2 of 4065 nt, and an unusually long 3' untranslated region in RNA 2 of 603 nt. Phylogenetic analysis of the amino acid sequences of the protease-polymerase (Pro-Pol) region and the two coat proteins confirmed that LMMV belongs to a distinct species within the genus Fabavirus.