Groundnut Bud Necrosis Virus Research Articles

Complete Genome Sequence of Mulberry Vein Banding Associated Virus, a New Tospovirus Infecting Mulberry.

Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5’-/3’-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.

Tomato spotted wilt virus in Sri Lanka : emerging problems of tospoviruses

Tomato spotted wilt virus (TSWV), the type species of the genus Tospovirus has long been reported infecting economically important horticultural crops such as tomato, groundnut, pepper, potato and soybean, all over the world. In Sri Lanka, characteristic tospovirus symptoms such as bud necrosis, axillary shoot proliferation and ring spots were observed on leaves of groundnut in Angunakolapalassa, and concentric rings on leaves and fruits of tomato, in Ambalantota and Gannoruwa. Sap extracted from both tomato and groundnut plants was serologically positive for TSWV infection in a double antibody sandwich enzyme-linked immunosorbentassay (DASELISA), but not for Groundnut bud necrosis virus (GBNV). Nucleocapsid (N) genes of both isolates were amplified in Reverse Transcription Polymerase Chain Reactions (RT-PCR) using primers specific for TSWV and GBNV. The sequences obtained showed 98% amino acid sequence identity to the N gene of the Brazilian isolate of TSWV-BR-01. This study confirms the presence of TSWV infecting groundnut and tomato in Sri Lanka.

GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm interacts with NP via its N-terminal unfolded region and the NSm–NP complex could in turn interact with the ER membrane via the C-terminal coiled coil domain of NSm to form vesicles that are targeted to PD and there by assist the cell to cell movement of the viral genome complex.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

Multiple and mixed infections with yellow mosaic, leaf crinkle and bud necrosis disease complex in mungbean: A threat to cultivation of mungbean in India

The occurrence, incidence, yield losses and diagnostics of yellow mosaic disease (YMD) caused Mungbean yellow mosaic India virus (MYMIV), bud necrosis disease (BND) caused by Groundnut bud necrosis virus (GBNV) and Urdbean leaf crinkle disease complex (ULCD) were studied in mungbean (Vigna radiata) in Delhi condition. Eighteen mungbean cultivars were grown for successive four years from 2006 to 2009. Overall disease incidence of 11.6–18.5% for YMD; 14.4–20.5% for ULCD and 9.4–14.5% for BND were estimated. The mixed infection was studied in kharif season and overall it was 10.5% for YMD+ULCD, 8.3% for YMD+BND, 6.8% for ULCD+BND and 4.3% for YMD+ULCD+BND. None of the mungbean cultivars exhibited resistance to any of the three diseases. Yield loss estimated in susceptible mungbean cv. PS 16 was up to 61.1, 91.0 and 76.8% over control for YMD, ULCD and BND, respectively. Mixed infection with YMD and BND caused 92.8% yield losses. Biological and molecular detection using enzyme linked Immuno-sorbent assay (ELISA), polymerase chain reaction (PCR) and nucleotide analysis determined that MYMIV is the causal agent of YMD and GBNV of BND. Although etiology of ULCD was not confirmed, however, seed and sap transmission study showed that it is caused by an infectious agent.

Tospo viral infection instigates necrosis and premature senescence by micro RNA controlled programmed cell death in Vigna unguiculata

Tospoviruses are the only plant infecting viruses belonging to the family Bunyavirideae. The most characteristic symptoms of tospoviral infection in different hosts are necrosis and premature senescence. To understand the mechanistic model of symptom development and the role of programmed cell death (PCD) during infection, we compared the levels of biochemical intermediates and stress responsive micro RNAs (miRNAs) with their target transcripts in Vigna unguiculata during Groundnut bud necrosis virus (GBNV) infection at the infection and systemic site. Appearance of chlorotic spots was the first visual symptom of GBNV infection. These spots turned to necrotic as the infection progressed. Premature senescence, starting from appearance of yellow mosaic followed by complete yellowing was observed in the systemic leaves. Necrosis (cell death) in the form of hypersensitive response (HR) was observed at the infection site, as judged by the accumulation of reactive oxygen species (ROS). In addition, we also found increased activities of antioxidant enzymes, such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) that are associated with detoxification of ROS. At this stage, down-regulation of miR398a regulating COX-5b, (Cytochrome C oxidase subunit 5b), responsible for oxidative stress resistance was also observed. The other three studied stress responsive miRNAs; miR319a.2, miR399f, miR482 also showed down-regulation with respective induction in the expression of transcripts they regulate; metacaspase-8, PHO2 (PHOSPATE 2; ubiquitin conjugating enzyme) and an NBS-LRR gene, respectively. Production of plant stress signals and differential expression of stress indicators during the HR response at infection (inoculation) sites and PCD at systemic sites is discussed.

Groundnut bud necrosis virus encoded NSm associates with membranes via its C-terminal domain.

Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200–250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.

First Report of Capsicum chlorosis virus Infecting Waxflower (Hoya calycina Schlecter) in the United States.

In February 2013, an ornamental waxflower (Hoya calycina Schlecter) with leaves displaying concentric chlorotic and necrotic rings surrounding sunken, necrotic lesions typical of tospovirus infection was observed at a community garden in Honolulu, HI. Symptomatic leaf tissue tested negative for Tomato spotted wilt virus (TSWV), a common tospovirus in Hawaii, using a TSWV ImmunoStrips (AgDia, Elkhart, IN) assay following the manufacturer's instructions. Double-stranded RNAs were isolated from a symptomatic leaf and reverse transcribed using random primers (2). The cDNA was then used as template in a universal tospovirus PCR assay using primers gL3637 and gL4435c, which amplify sequences of the L segment encoding the RNA-dependent RNA polymerase of tospoviruses (1). An ~800-bp product was amplified and cloned using pGEM-T Easy (Promega, Madison, WI). Three clones were selected and found to be identical by dye-terminator sequencing performed at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. Following primer sequence trimming, the 773-bp sequence (GenBank Accession No. KF030938) was found to be 97, 88, and 87% identical to Capsicum chlorosis virus (CaCV; a tentative species in the family Bunyaviridae, genus Tospovirus) strains Ch-Har (GU199334), TwTom1 (HM021140), and AIT (DQ256124), respectively. To confirm the presence of CaCV, the cDNA was also used as template in a universal tospovirus PCR assay with primers 3'T12 and TsMCR2 which amplify a region of the S segment of tospoviruses (3). The amplification product from this assay was cloned and sequenced as described above and found to be 93 to 98% identical to CaCV nucleotide sequences present in GenBank. Attempts to detect the CaCV strain in waxflower using a watermelon silver mottle virus and groundnut bud necrosis virus triple antibody sandwich ELISA (AgDia) were unsuccessful. No other plants in the community garden had typical tospovirus-like symptoms; however, samples from tomato (Solanum lycopersicum L.; two samples), chili pepper (Capsicum spp.; four samples), eggplant (Solanum melongena L.; one sample), and passionfruit (Passiflora edulis Sims; one sample) with virus-like symptoms were collected from the garden and had RNA isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). No tospoviruses were detected in any of these samples with the RT-PCR assay using primers gL3637 and gL4435. The waxflower plant infected with CaCV was immediately removed by community garden members and destroyed, preventing any additional serological or biological assays to be performed. CaCV is transmitted by several species of thrips, including Thrips palmi, which is present in Hawaii. Waxflower is not native to Hawaii and it is unclear whether CaCV entered Hawaii in this plant or whether it was infected by viruliferous thrips. A survey for CaCV in known hosts is essential to determine the geographic distribution of CaCV in Hawaii, as this virus poses a considerable threat to tomato, chili pepper, and phalaenopsis orchid production in Hawaii and the United States.

First report of Groundnut bud necrosis virus infecting wild species of Vigna, based on NP gene sequence characteristics

Symptoms such as chlorotic and necrotic spots on leaves, necrosis of the stems and petioles, broadly resembling those induced by Groundnut bud necrosis virus (GBNV) in cultivated species of Vigna were observed in twelve accessions of wild species/sub-species of Vigna ( V. umbellata, V. glabrescens, V. hainiana, V. mungo var. mungo, V. radiata var. radiata and V. radiata var. sublobata ) grown at the Indian Institute of Pulses Research, Kanpur, India during August-November 2011. Disease incidence ranged between 7.13- 40.3%. The identity of the virus was confirmed on the basis of symptoms on diagnostic host (cowpea cv. Pusa Komal) and nucleocapsid protein ( NP ) gene sequence characteristics. Analysis of NP gene of intra-field isolates of GBNV revealed 1-4% diversity in nucleotide and 0-4% in amino acids. However, diversity among intra-field isolates and isolates from cultivated Vigna ranged 2-7% at nucleotide level and 0-6 at amino acids level. This paper reports for the first time GBNV infection in these wild species/sub-species of Vigna and analyze the intra-field diversity in the NP gene of 12 GBNV isolates.

Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection

Cocktail of polyclonal antibodies (PAb) were produced that will help in multiple virus detection and overcome the limitation of individual virus purification, protein expression and purification as well as immunization in multiple rabbits. A dual fusion construct was developed using conserved coat protein (CP) sequences of Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) in an expression vector, pET-28a(+). The fusion protein (∼40kDa) was expressed in Escherichia coli and purified. Likewise, a triple fusion construct was developed by fusing conserved CP sequences of CMV and PRSV with conserved nucleocapsid protein (N) sequence of Groundnut bud necrosis virus (GBNV) and expressed as a fusion protein (∼50kDa) in pET-28a(+). PAb made separately to each of these three viruses recognized the double and triple fusion proteins in Western blot indicating retention of desired epitopes for binding with target antibodies. The fusion proteins (∼40kDa and ∼50kDa) were used to produce cocktail of PAb by immunizing rabbits, which simultaneously detected natural infection of CMV and PRSV or CMV, PRSV and GBNV in Cucurbitaceous, Solanaceous and other hosts in DAC-ELISA. This is the first report on production of a cocktail of PAb to recombinant fusion protein of two or three distinct viruses.

First Report of a Tospovirus in Mulberry.

Mulberry (Morus alba L.) is an economically important crop grown widely throughout Asia. Various virus-like symptoms including mosaics, vein banding, and chlorotic ringspots have been observed and reported on mulberry trees in China and Japan for decades. However, the etiology of mulberry viral diseases is generally understudied, although two mulberry-infecting viruses, Mulberry latent virus (genus Carlavirus) (2) and Mulberry ringspot virus (genus Nepovirus) (3), have been partially characterized. In a recent (2010 to 2011) field survey in Guangxi Province, China, supported by the local government, the incidence of virus-like diseases of mulberry ranged between 40 and 80%. To identify the viruses infecting mulberry, deep sequencing of small RNAs (4) was conducted using an Illumina Genome Analyzer. Small RNAs were isolated from five samples of mulberry leaves showing various virus-like symptoms and sequenced. Among the contigs assembled, a 445-bp contig (GenBank Accession No. JX268597) was found to share 76.6% nucleotide identity and 83.0% amino acid identity to Groundnut bud necrosis virus (genus Tospovirus, family Bunyaviridae; Accession Nos. U42555 and AAC55521). To obtain a longer cDNA fragment of this virus, a reverse transcription (RT)-PCR was done with primers MV-N-F (5'-AAGCCATCAATGTGCCTCCGGA-3') and MV-N-R (5'-AACACCATGTCTACCGTCCGTC-3') that align to the S-RNA sequence encompassing the nucleocapsid (N) gene and a portion of the intergenic region (IGR) of the Tospovirus. PCR products of about 1,000 bp were successfully amplified from the total RNA of the three mulberry samples (sl-1, xcsy-1, and xcsy-4) showing vein banding symptoms, but not from asymptomatic mulberry (jk-1). These PCR products were cloned and sequenced. The lengths of the amplicons were 1,027 bp (isolate sl-1, JX173786), 987 bp (isolate xcsy-1, JX173787), and 979 bp (isolate xcsy-4, JX173788) and the partial IGRs of the sl-1, xcsy-1, and xcsy-4 isolates were 187 bp, 147 bp, and 139 bp, respectively. The coding regions for the N protein were 831 bp and the deduced proteins of 277 amino acid residues were 100% identical for all three isolates. Since the N protein of this virus shared up to only 74.4% identity to other tospoviruses (74.4% to Capsicum chlorosis virus, ABB83818; and 71.5% to Watermelon bud necrosis virus, ABY79095), it may represent a new member of the Tospovirus genus, temporarily named Mulberry vein banding virus (MuVBV), according to the species demarcation criteria for the Bunyaviridae (1). To the best of our knowledge, this is the first report of a Tospovirus infecting M. alba. In an RT-PCR screening of 48 randomly selected mulberry samples suspected to be virus-infected, 32 were MuVBV-positive. Giving the high incidence and the high yield loss associated with Tospovirus and the presence of thrips, suspected vectors for the virus, MuVBV may represent a substantial threat to the silkworm industry in China.

Molecular characterisation of bud necrosis virus isolates infecting tomato (Lycopersicon esculentum Mill.) by coat protein gene analysis in Andhra Pradesh

The complete nucleotide sequence was determined for coat protein (CP) gene of Groundnut bud necrosis virus (GBNV) infecting tomato in Andhra Pradesh (AP). The sequenced region in GBNV-To isolate contained a single open reading frame of 831 bases that could potentially code for a CP of 276 amino acids. The CP gene sequence of GBNV-To has 93–99% sequence homology with that of the gene sequences of other GBNV isolates prevalent on different hosts in India. In the present study, it is determined that the GBNV-To isolate of tomato prevalent in AP is regarded as the same strain of GBNV already existing in India.

First report of Groundnut bud necrosis virus infecting onion (Allium cepa)

The natural occurrence of Groundnut bud necrosis virus (GBNV) on onion (Allium cepa L.) was detected by enzyme linked immunosorbent assay using an antiserum raised against GBNV and reverse transcription polymerase chain reaction using coat protein gene specific primers. Sequence analysis showed 93–100 % and 95–100 % identity at nucleotide and amino acid levels respectively with other reported GBNV isolates.

Biological characterization and variability of the nucleocapsid protein gene of Groundnut bud necrosis virus isolates infecting pea from India

A disease of pea characterized by browning in veins, leaves and stems, mostly in growing tips, and brown circular spots on pods, was recorded in four districts of Uttar Pradesh, India. The causal agent of this disease was detected by reverse transcription-polymerase chain reaction (RT-PCR) using primers pair HRP 26/HRP 28 and identified as Groundnut bud necrosis virus (GBNV) on the basis of nucleocapsid protein (NP) gene sequence. Virus isolates from Bareilly (BRY), Kanpur (KNP), Udham Singh Nagar (USN) and Shahjahanpur (SJP) were designated as GBNV-[Pea_BRY], GBNV-[Pea_KNP], GBNV-[Pea_USN] and GBNV-[Pea_SJP] and their NP genes sequenced. The sequence data of each isolate were deposited at NCBI database (JF281101-JF281104). The complete nucleotide sequence of the NP genes of all the GBNV isolates had a single open reading frame of 831 nucleotides and 276 amino acids. The isolates had among them 2% variability at amino acid level and 2‒3 variability at nucleotide level, but had variability with other GBNV isolates of fabaceous hosts in the range of 0‒6% at amino acid level and 1‒8% at nucleotide level. Though this variation in nucleotide sequences of GBNV isolates from fabaceous hosts is within the limits of species demarcation for tospoviruses, formation of a separate cluster within the GBNV isolates indicates the possibility of distinct variants in GBNV.

Scientific Opinion on the pest categorisation of the tospoviruses

The European Commission requested EFSA's Panel on Plant Health to perform the pest categorisation for the 24 viruses of the Tospovirus genus for the EU territory. The following tospoviruses were analysed: Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), Polygonum ringspot virus (PolRSV), Groundnut ringspot virus (GRSV), Tomato chlorotic spot virus (TCSV), Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Melon severe mosaic virus (MSMV), Tomato yellow (fruit) ring virus (TYRV), Tomato zonate spot virus (TZSV), Groundnut yellow spot virus (GYSV), Groundnut chlorotic fan-spot virus (GCFSV), Groundnut bud necrosis virus (GBNV), Zucchini lethal chlorosis virus (ZLCV), Capsicum chlorosis virus (CaCV), Watermelon bud necrosis virus (WBNV), Watermelon silver mottle virus (WSMoV), Tomato necrotic ringspot virus (TNRV), Calla lily chlorotic spot virus (CCSV), Melon yellow spot virus (MYSV), Soybean vein necrosis associated virus (SVNaV), Bean necrotic mosaic virus (BeNMV) and Pepper necrotic spot virus (PNSV). In reaching its conclusions, the Panel considered four parameters to be of critical importance in the risk assessment area: (i) the presence of a tospovirus, (ii) the existence of host plants, (iii) the existence of thrips vector species and (iv) the potential for damage to crops grown in Europe. Based on its analysis, the Panel concluded that the 24 viruses analysed could be allocated to four different risk groups. Seven viruses (GRSV, TCSV, ANSV, CSNV, MSMV, TYRV, TZSV) for which both thrips species vectors and natural or experimental hosts crops are present in the EU territory were considered by the Panel to represent the highest risk to the EU territory. In contrast, three viruses (INSV, IYSV and PolRSV) already present in the risk assessment area were not considered by the Panel to pose a risk justifying the development of full risk assessments.

First Report of Groundnut bud necrosis virus in Tomato in Bangladesh

An unusual disease of tomato characterized by leaf mottling and necrotic streaks on veins, shortened internodes, necrosis of terminal buds, and concentric rings on fruits was observed during 2010 to 2011 surveys in tomato growing regions of Godagari Upzila, Rajshahi district, Bangladesh. Disease incidence in popularly grown F1 hybrid cultivars, which include Sobal, Abhiruchi, Salamat, Bangobir, and BARI hybrid tomato-5 and -6 in about 40 commercial fields, ranged from 40 to 90%. Extracts from the field samples (n = 10) reacted with polyclonal antiserum to Groundnut bud necrosis virus (GBNV) in direct antigen coated ELISA, suggesting the association of a tospovirus antigenically related to serogroup IV topsovirus (1). To identify whether the tospovirus was a distinct virus species, ELISA-positive samples were subjected to total RNA extraction with an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA) followed by reverse transcription (RT)-PCR with tospovirus-specific primers (5'-ATGGTTGAAAAGAGCAAGAATGATGC-3') and degenerate primer (5'-CTTCTTATGAAGTGTACTCACCATAAGTCATCC-3') derived from the conserved sequences of GBNV, Watermelon bud necrosis virus (WBNV), and Capsicum chlorosis virus (CaCV) (2). The RT-PCR product was cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced at Department of Biochemistry, University of Delhi, South Campus, Delhi, India (GenBank Accession No. JQ692083). The sequences of cloned fragments were assembled. Analysis of the 477-bp region of the nucleocapsid protein (N) gene revealed that the tomato tospovirus shared maximum identity both at the nucleotide (96%) and amino acid (97%) levels with the corresponding region of GBNV. In contrast, only 78 to 81% and 85 to 87% identity at nucleotide and amino acid levels, respectively, was observed with the corresponding region of the N genes of CaCV, WBNV, and Watermelon silver mottle virus. These results suggested the association of GBNV with the diseased tomato samples. To our knowledge, this is the first report of GBNV infecting tomato in Bangladesh and regular surveys are necessary to ascertain the prevalence and incidence of GBNV in other crops.

Sequence Diversity in the NSm Gene of Groundnut Bud Necrosis Virus Isolates Originating from Different Hosts and Locations in India

AbstractThe movement protein (NSm) gene of Groundnut bud necrosis virus (GBNV) isolates from pea, mungbean, cowpea, French bean, tomato and potato collected from different locations of India were compared to study their diversity. The NSm gene sequences of all the GBNV isolates were highly conserved and had only 0–3% diversity in amino acids and 0–10% in nucleotides. Comparison of amino acid sequence of NSm gene of 25 GBNV isolates revealed the presence of many conserved regions. Both ‘D‐motif’ and ‘G‐residue’, the conserved regions of ‘30K superfamily’ of virus movement protein, were present in all the isolates. Clustering of the GBNV isolates does not appear to be based on their place of origin and host plant species.

Mixed Groundnut bud necrosis virus and Okra yellow vein mosaic virus Infection of Okra in India

To our knowledge, this is the first report of Groundnut bud necrosis virus (GBNV, genus Tospovirus) infection in okra and also its mixed infection with Okra yellow vein mosaic virus (OYVMV, genus Geminivirus) in India. It appears that the GBNV is expanding its host range from Fabaceae and Solanaceae families to crops belonging to Malvaceae family. The potential impact of mixed infection of GBNV and OYVMV on economical yield in okra warrants continuous monitoring of the diseases in field. Accepted for publication 3 October 2012. Published 23 October 2012.

Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-011-1110-0) contains supplementary material, which is available to authorized users.

Interference in plant defense and development by non-structural protein NSs of Groundnut bud necrosis virus

Groundnut bud necrosis virus (GBNV) infects a large number of leguminous and solanaceous plants. To elucidate the biological function of the non-structural protein encoded by the S RNA of GBNV (NSs), we studied its role in RNA silencing suppression and in viral pathogenesis. Our results demonstrated that GBNV NSs functions as a suppressor of RNA silencing using the agroinfiltration patch assay. An in silico analysis suggested the presence of pro-apoptotic protein Reaper-like sequences in the GBNV NSs, which were known to be present in animal infecting bunyaviruses. Utilizing NSs mutants, we demonstrated that a Leu-rich domain was required for RNA silencing suppression activity, but not the non-overlapping Trp/GH3 motif of the Reaper-like sequence. To investigate the role of NSs in symptom development we generated transgenic tomato expressing the GBNV NSs and showed that the expression of NSs in tomato mimics symptoms induced by infection with GBNV, such as leaf senescence and necrosis. As leaf senescence is controlled by miR319 regulation of the transcription factor TCP1, we assessed the accumulation of both RNAs in transgenic NSs-expressing and GBNV-infected tomato plants. In both types of plants the levels of miR319 decreased, while the levels of TCP1 transcripts increased. We propose that GBNV-NSs affects miRNA biogenesis through its RNA silencing suppressor activity and interferes with TCP1-regulated leaf developmental pathways.