Deciphering the Role of Mucosal Immune Responses and the Cervicovaginal Microbiome in Resistance to HIV Infection in HIV-Exposed Seronegative (HESN) Women.

The female genital tract (FGT) is an important site of human immunodeficiency virus (HIV) infection. Discerning the nature of HIV-specific local immune responses is crucial for identifying correlates of protection in HIV-exposed seronegative (HESN) individuals. The present study involved a comprehensive analysis of soluble immune mediators, secretory immunoglobulins (sIg), natural killer (NK) cells, CXCR5+ CD8+ T cells, T follicular helper (Tfh) cells, and T regulatory cells (Tregs) in the vaginal mucosa as well as the nature and composition of the cervicovaginal microbiome in HESN women. We found significantly elevated antiviral cytokines, soluble immunoglobulins, and increased frequencies of activated NK cells, CXCR5+ CD8+ T cells, and Tfh cells in HESN females compared to HIV-unexposed healthy (UH) women. Analysis of the genital microbiome of HESN women revealed a greater bacterial diversity and increased abundance of Gardnerella spp. in the mucosa. The findings suggest that the female genital tract of HESN females represents a microenvironment equipped with innate immune factors, antiviral mediators, and critical T cell subsets that protect against HIV infection. IMPORTANCE The vast majority of human immunodeficiency virus (HIV) infections across the world occur via the sexual route. The genital tract mucosa is thus the primary site of HIV replication, and discerning the nature of HIV-specific immune responses in this compartment is crucial. The role of the innate immune system at the mucosal level in exposed seronegative individuals and other HIV controllers remains largely unexplored. This understanding can provide valuable insights to improve vaccine design. We investigated mucosal T follicular helper (Tfh) cells, CXCR5+ CD8+ T cells, natural killer (NK) cells subsets, soluble immune markers, and microbiome diversity in HIV-exposed seronegative (HESN) women. We found a significantly higher level of mucosal CXCR5+ CD8+ T cells, CD4+ Tfh cells, activated NK cell subsets, and antiviral immune cell mediators in HESN women. We also found a higher abundance of Gardnerella spp., microbiome dysbiosis, and decreased levels of inflammatory markers to be associated with reduced susceptibility to HIV infection. Our findings indicate that increased distribution of mucosal NK cells, CXCR5+ CD8+ T cells, Tfh cells, and soluble markers in HIV controllers with a highly diverse cervicovaginal microbiome could contribute effectively to protection against HIV infection. Overall, our findings imply that future vaccine design should emphasize inducing these highly functional cell types at the mucosal sites.

Brief Report: Vaginal Viral Shedding With Undetectable Plasma HIV Viral Load in Pregnant Women Receiving 2 Different Antiretroviral Regimens: A Randomized Clinical Trial.

BACKGROUND: Pregnant women using antiretrovirals (ARVs) may have persistent vaginal viral shedding, which could be associated with sexual and perinatal HIV transmission. However, there are scant data on vaginal viral load (VVL) in pregnant women with undetectable plasma viral load (PVL). METHODS: This study was a post hoc analysis of an open-label randomized trial to evaluate the virologic response of 2 ART regimens. The participants were ART-naive women living with HIV initiating ART regimens between 20 and 36 weeks of pregnancy recruited at 19 clinical sites in 6 countries. Participants were randomized to receive 400 mg of raltegravir 2 times a day or 600 mg of efavirenz 4 times a day in addition to 150 mg of lamivudine and 300 mg of zidovudine 2 times a day. VVL and PVL tests were performed at every study visit. The primary outcome measures were HIV-1 PVL and VVL at maternal study week 4 and rates of perinatal HIV transmission. RESULTS: A total of 408 were enrolled, of whom 323 had VVL samples 4 weeks after enrollment and were included in this analysis. Among women with undetectable/nonquantifiable PVL during ART, the overall rate of quantifiable VVL at week 4 was 2.54% (7/275). Of the 275 with nonquantifiable PVL, 99.1% (115/116) and 96.2% (153/159) had nonquantifiable VVL in the efavirenz and raltegravir arms, respectively. None of the 7 women with quantifiable VVL at the week 4 study visit transmitted HIV to their infants. CONCLUSIONS: Detectable VVL in pregnant women with undetectable/nonquantifiable PVL while receiving ART was rare and not associated with perinatal HIV transmission.

First complete genome sequence of an isolate of cowpea severe mosaic virus from South America.

In this study, the complete nucleotide sequence of a Brazilian isolate of cowpea severe mosaic virus (CPSMV) is presented for the first time. To date, the CPSMV-DG isolate, from the USA, is the only one with the complete known genome. High-throughput sequencing (Illumina HiSeq) and Sanger sequencing of the total RNA extract from a cowpea plant collected in Teresina city, Brazil, revealed the genome sequence of the CPSMV-Ter1 isolate. RNA-1 and RNA-2 are, respectively, 5921 and 3465 nucleotides (nt) long without the poly(A) tail, and show 77.91% and 76.08% nt sequence identity with CPSMV-DG, considered the type isolate of the species. The open reading frames (ORFs) were determined and the cleavage sites of the polyproteins were predicted. Although the two isolates show a similar genomic organization, there was a low percentage of sequence identity between Ter1 and DG. Furthermore, pairwise comparisons of a partial RNA-1 fragment between CPSMV-Ter1 and 11 CPSMV isolates from Brazil indicated 94.6 to 94.8% nt and 98.9% to 99.4% aa sequence identities.

Comparative RNA-Seq analysis unfolds a complex regulatory network imparting yellow mosaic disease resistance in mungbean [Vigna radiata (L.) R. Wilczek].

Yellow Mosaic Disease (YMD) in mungbean [Vigna radiata (L.) R. Wilczek] is one of the most damaging diseases in Asia. In the northern part of India, the YMD is caused by Mungbean Yellow Mosaic India Virus (MYMIV), while in southern India this is caused by Mungbean Yellow Mosaic Virus (MYMV). The molecular mechanism of YMD resistance in mungbean remains largely unknown. In this study, RNA-seq analysis was conducted between a resistant (PMR-1) and a susceptible (Pusa Vishal) mungbean genotype under infected and control conditions to understand the regulatory network operating between mungbean-YMV. Overall, 76.8 million raw reads could be generated in different treatment combinations, while mapping rate per library to the reference genome varied from 86.78% to 93.35%. The resistance to MYMIV showed a very complicated gene network, which begins with the production of general PAMPs (pathogen-associated molecular patterns), then activation of various signaling cascades like kinases, jasmonic acid (JA) and brassinosteroid (BR), and finally the expression of specific genes (like PR-proteins, virus resistance and R-gene proteins) leading to resistance response. The function of WRKY, NAC and MYB transcription factors in imparting the resistance against MYMIV could be established. The string analysis also revealed the role of proteins involved in kinase, viral movement and phytoene synthase activity in imparting YMD resistance. A set of novel stress-related EST-SSRs are also identified from the RNA-Seq data which may be used to find the linked genes/QTLs with the YMD resistance. Also, 11 defence-related transcripts could be validated through quantitative real-time PCR analysis. The identified gene networks have led to an insight about the defence mechanism operating against MYMIV infection in mungbean which will be of immense use to manage the YMD resistance in mungbean.

Rapid and visual detection of milk vetch dwarf virus using recombinase polymerase amplification combined with lateral flow strips.

BACKGROUND: Milk vetch dwarf virus (MDV) is an important ssDNA virus which causes yellowing, stunting and leaf rolling symptoms on legumes. In China, the virus causes great economic losses and has recently been found to infect tobacco. The expansion of its host range and its ability to spread rapidly has given rise to the urgent need for a sensitive, specific and rapid diagnostic assay that can assist in effective disease control. METHODS: Assays based on the polymerase chain reaction combined with lateral flow strip detection (PCR-LFS) and recombinase polymerase amplification combined with LFS (RPA-LFS) were developed targeting the coat protein (CP) gene of MDV. RESULTS: The PCR and RPA assays could detect respectively 103 copies or 101 copies of MDV by agarose gel electrophoresis. The PCR-LFS and RPA-LFS assays developed could both detect as few as 101 copies per reaction at 37 °C. Both methods could detect MDV in crude leaf extracts. CONCLUSIONS: The RPA-LFS assay developed is a rapid, sensitive and specific method for detecting MDV, which is convenient and has great potential for use in the field.

Identification, characterization, and expression analysis of cowpea (Vigna unguiculata [L.] Walp.) miRNAs in response to cowpea severe mosaic virus (CPSMV) challenge.

KEY MESSAGE: Cowpea miRNAs and Argonaute genes showed differential expression patterns in response to CPSMV challenge Several biotic stresses affect cowpea production and yield. CPSMV stands out for causing severe negative impacts on cowpea. Plants have two main induced immune systems. In the basal system (PTI, PAMP-triggered immunity), plants recognize and respond to conserved molecular patterns associated with pathogens (PAMPs). The second type (ETI, Effector-triggered immunity) is induced after plant recognition of specific factors from pathogens. RNA silencing is another important defense mechanism in plants. Our research group has been using biochemical and proteomic approaches to learn which proteins and pathways are involved and could explain why some cowpea genotypes are resistant whereas others are susceptible to CPSMV. This current study was conducted to determine the role of cowpea miRNA in the interaction between a resistant cowpea genotype (BRS-Marataoã) and CPSMV. Previously identified and deposited plant microRNA sequences were used to find out all possible microRNAs in the cowpea genome. This search detected 617 mature microRNAs, which were distributed in 89 microRNA families. Next, 4 out of these 617 miRNAs and their possible target genes that encode the proteins Kat-p80, DEAD-Box, GST, and SPB9, all involved in the defense response of cowpea to CPSMV, had their expression compared between cowpea leaves uninoculated and inoculated with CPSMV. Additionally, the differential expression of genes that encode the Argonaute (AGO) proteins 1, 2, 4, 6, and 10 is reported. In summary, the studied miRNAs and AGO 2 and AGO4 associated genes showed differential expression patterns in response to CPSMV challenge, which indicate their role in cowpea defense.

Seed transmission of a distinct soybean yellow mottle mosaic virus strain identified from India in natural and experimental hosts.

Soybean yellow mottle mosaic virus (SYMMV) is a newly identified member of the genus Gammacarmovirus from grain legumes in India. As the modes of transmission of this virus have not been described, we assessed the possibility of SYMMV to be transmitted through seed collected from field infected mungbean plants and mechanically sap inoculated French bean plants using serological and molecular techniques followed by progeny assays. Direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) and reverse transcription polymerase chain reaction (RT-PCR) results are inconsistent with field infected mungbean seed tissues to ensure seed transmissibility irrespective of seed number used. Seed from mechanical sap inoculated French bean showed higher absorbance values in DAC-ELISA and amplification corresponding to replicase, movement and coat protein regions of SYMMV genome. The relative accumulation of SYMMV was higher in pod walls, immature seed and stamens and stigma of mechanical sap inoculated French bean. Progeny assays with infected seed revealed the seed transmissibility of SYMMV at the rate of 63.33% in mungbeanand 73.33% in French bean. Mechanical sap inoculation of mungbean progeny seedlings on French bean cv. Pusa Parvati produced characteristic symptoms of SYMMV. The results obtained from this study demonstrate that SYMMV is seed borne in nature and can be transmitted to next generation seedlings. This is the first report of seed transmission of SYMMV in mungbean and French bean.

Plant Virus Nanoparticles for Vaccine Applications.

In the rapidly evolving field of nanotechnology, plant virus nanoparticles (pVNPs) are emerging as powerful tools in diverse applications ranging from biomedicine to materials science. The proteinaceous structure of plant viruses allows the capsid structure to be modified by genetic engineering and/or chemical conjugation with nanoscale precision. This means that pVNPs can be engineered to display peptides and proteins on their external surface, including immunodominant peptides derived from pathogens allowing pVNPs to be used for active immunization. In this context, pVNPs are safer than VNPs derived from mammalian viruses because there is no risk of infection or reversion to pathogenicity. Furthermore, pVNPs can be produced rapidly and inexpensively in natural host plants or heterologous production platforms. In this review, we discuss the use of pVNPs for the delivery of peptide antigens to the host immune in pre-clinical studies with the final aim of promoting systemic immunity against the corresponding pathogens. Furthermore, we described the versatility of plant viruses, with innate immunostimulatory properties, in providing a huge natural resource of carriers that can be used to develop the next generation of sustainable vaccines.

H2O2Accumulation, Host Cell Death and Differential Levels of Proteins Related to Photosynthesis, Redox Homeostasis, and Required for Viral Replication Explain the Resistance of EMS-mutagenized Cowpea to Cowpea Severe Mosaic Virus.

Infection with Cowpea severe mosaic virus (CPSMV) represents one of the main limitations for cowpea (Vigna unguiculata L. Walp.) productivity due to the severity of the disease symptoms, frequency of incidence, and difficulties in dissemination control. This study aimed to identify the proteins and metabolic pathways associated with the susceptibility and resistance of cowpea plants to CPSMV. Therefore, we treated the seeds of a naturally susceptible cowpea genotype (CE-31) with the mutagenic agent ethyl methane sulfonate (EMS) and compared the secondary leaf proteomic profile of the mutagenized resistant plants inoculated with CPSMV (MCPI plant group) to those of the naturally susceptible cowpea genotype CE-31 inoculated (CPI) and noninoculated (CPU) with CPSMV. MCPI responded to CPSMV by accumulating proteins involved in the oxidative burst, increasing H2O2 generation, promoting leaf cell death (LCD), increasing the synthesis of defense proteins, and decreasing host factors important for the establishment of CPSMV infection. In contrast, CPI accumulated several host factors that favor CPSMV infection and did not accumulate H2O2 or present LCD, which allowed CPSMV replication and systemic dissemination. Based on these results, we propose that the differential abundance of defense proteins and proteins involved in the oxidative burst, LCD, and the decrease in cowpea protein factors required for CPSMV replication are associated with the resistance trait acquired by the MCPI plant group.

Elucidating the functional aspects of different domains of bean common mosaic virus coat protein.

The coat protein (CP) is the only structural protein present in the polyprotein of bean common mosaic virus. The well known characteristics of the CP are self-oligomerization and nucleic acid binding activity. The studies of the coat protein mutants revealed that the oligomeric property of CP solely depends on the amino-terminal residues and the nucleic acid binding domain present at the 194-202 residue position. The 3'UTR RNA of the virus showed high binding affinity with the RNA binding domain as compared to the 5'UTR RNA. Further, the intrinsic fluorescence study of the CP also suggested that the N- and C-terminal of CP contains a highly disordered region. The present study also illustrates that the coat protein contains a conserved RNA binding pocket among the potyviruses, but displays divergent oligomerization propensities due to the difference in residue at the N- and C-terminal.

Detection of QTLs associated with mungbean yellow mosaic virus (MYMV) resistance using the interspecific cross of Vigna radiata × Vigna umbellata.

Mungbean (Vigna radiata) and ricebean (V. umbellata) were utilized to obtain an inter-specific recombinant inbred line (RIL) population with the objective of detecting quantitative trait loci (QTL) associated with mungbean yellow mosaic virus (MYMV) resistance. To precisely map QTLs, accurate genetic linkage maps are essential. In the present study, genotyping-by-sequencing (GBS) platform was utilized to develop the genetic linkage map. The map contained 538 single nucleotide polymorphism (SNP) markers, consisted of 11 linkage groups and spanned for 1291.7 cM with an average marker distance of 2.40 cM. The individual linkage group ranged from 90.2 to 149.1 cM in length, and the SNP markers were evenly distributed in the genetic linkage map, with 30-79 SNP markers per chromosome. The QTL analysis using the genetic map and 2 years (2015 and 2016) of phenotyping data identified five QTLs with phenotypic variation explained (PVE) from 10.11 to 20.04%. Of these, a QTL on chromosome 4, designated as qMYMV4-1, was major and stably detected in the same marker interval in both years. This QTL region harbours possible candidate genes for controlling MYMV resistance. The linkage map and QTL/gene (s) for MYMV resistance identified in this study should be useful for QTL fine mapping and cloning for further studies.

Providence virus: An animal virus that replicates in plants or a plant virus that infects and replicates in animal cells?

INTRODUCTION: Emerging viral diseases, most of which are zoonotic, pose a significant threat to global health. There is a critical need to identify potential new viral pathogens and the challenge is to identify the reservoirs from which these viruses might emerge. Deep sequencing of invertebrate transcriptomes has revealed a plethora of viruses, many of which represent novel lineages representing both plant and animal viruses and little is known about the potential threat that these viruses pose. METHODS: Providence virus, an insect virus, was used to establish a productive infection in Vigna unguiculata (cowpea) plants. Providence virus particles purified from these cowpea plants were used to infect two mammalian cell lines. FINDINGS: Here, we present evidence that Providence virus, a non-enveloped insect RNA virus, isolated from a lepidopteran midgut cell line can establish a productive infection in plants as well as in animal cells. The observation that Providence virus can readily infect both plants and mammalian cell culture lines demonstrates the ability of an insect RNA virus to establish productive infections across two kingdoms, in plants and invertebrate and vertebrate animal cell lines. CONCLUSIONS: The study highlights the potential of phytophagous insects as reservoirs for viral re-assortment and that plants should be considered as reservoirs for emerging viruses that may be potentially pathogenic to humans.

Nanopore sequencing of a novel bipartite New World begomovirus infecting cowpea.

A new bipartite begomovirus (family Geminiviridae) was detected on cowpea (Vigna unguiculata) plants exhibiting bright golden mosaic symptoms on leaves under field conditions in Brazil. Complete consensus sequences of DNA-A and DNA-B components of an isolate of the virus (PE-088) were obtained by nanopore sequencing and confirmed by Sanger sequencing. The genome components presented the typical genomic organization of New World (NW) begomoviruses. Pairwise sequence comparisons revealed low levels of identity with other begomovirus species previously reported infecting cowpea around the world. Phylogenetic analysis using complete sequences of DNA-A components revealed that the closest relatives of PE-088 (85-87% nucleotide sequence identities) were three legume-infecting begomoviruses from Brazil: bean golden mosaic virus, macroptilium common mosaic virus and macroptilium yellow vein virus. According to the current classification criteria, PE-088 represents a new species in the genus Begomovirus, tentatively named as cowpea bright yellow mosaic virus (CoBYMV).

A resistant cowpea (Vigna unguiculata [L.] Walp.) genotype became susceptible to cowpea severe mosaic virus (CPSMV) after exposure to salt stress.

In nature, plants are simultaneously challenged by biotic and abiotic stresses. However, little is known about the effects of these combined stresses for most crops. This work aimed to evaluate the responsed of the virus-resistant cowpea genotype BRS-Marataoã to the exposure of salt stress combined with CPSMV infection. Cowpea plants were exposed to 200 mM NaCl either simultaneously (SV plant group) or 24 h prior to the CPSMV infection [S(24 h)V plant group]. Physiological, biochemical, and proteomic analyses at 2 and 6 days post salt stress (DPS) revealed that cowpea significantly reprogrammed its cellular metabolism. Indeed, plant size, photosynthetic parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) and chlorophyll and carotenoid contents were reduced in S(24 h)V compared to SV. Moreover, accumulation of viral particles at 6 DPS in S(24 h)V was observed indicating that the salt stress imposed prior to virus infection favors viral particle proliferation. Proteomic analysis showed differential contents of 403 and 330 proteins at 2 DPS and 6 DPS, respectively, out of 733 differentially abundant proteins between the two plant groups. The altered leaf proteins are involved in energy and metabolism, photosynthesis, stress response, and oxidative burst. BIOLOGICAL SIGNIFICANCE: This is an original study in which a virus-resistant cowpea genotype (BRS-Marataoã) was (i) exposed simultaneously to 200 mM NaCl and inoculation with CPSMV (SV plant group) or (ii) exposed to 200 mM NaCl stress 24 h prior to inoculation with CPSMV [S(24 h)V plant group]. The purpose was to shed light on how this CPSMV resistant cowpea responded to the combined stresses. Numerous key proteins and associated pathways were altered in the cowpea plants challenged with both stresses, but unexpectedly, the salt stress imposed 24 h prior to CPSMV inoculation allowed viral proliferation, turning the cowpea genotype from resistant to susceptible.

Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte.

The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified different pathways leading to assembled virions from their components, experimental evidence is severely lacking. Here, we use time-resolved small-angle X-ray scattering to uncover the nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging their full RNA genome. We reveal the formation of amorphous complexes via an en masse pathway and their relaxation into virions via a synchronous pathway. The binding energy of capsid subunits on the genome is moderate (~7kBT0, with kB the Boltzmann constant and T0 = 298 K, the room temperature), while the energy barrier separating the complexes and the virions is high (~ 20kBT0). A synthetic polyelectrolyte can lower this barrier so that filled capsids are formed in conditions where virions cannot build up. We propose a representation of the dynamics on a free energy landscape.

Exogenous application of methyl jasmonate induces defense response and develops tolerance against mungbean yellow mosaic India virus in Vigna mungo.

Vigna mungo (L.)Hepper is an economically important leguminous crop in south-east Asia. However, its production is severely affected by Mungbean yellow mosaic India virus (MYMIV). It is well established that methyl jasmonate (MeJA) is effective in inducing resistance against pathogens in several plants. To assess the role of MeJA in developing MYMIV tolerance in V. mungo, we analysed time-dependent biochemical and molecular responses of MYMIV susceptible V. mungo after exogenous application of different MeJA concentrations, followed by MYMIV infection. Our analysis revealed that exogenous application of different concentrations of MeJA resulted in decreased levels of malondialdehyde with higher membrane stability index values in MYMIV susceptible V. mungo, suggesting the protective role of MeJA through restoring the membrane stability. Moreover, the level of expression of different antioxidative enzymes revealed that exogenous MeJA is also very effective in ROS homeostasis maintenance. Enhanced expressions of the defence marker genes lipoxygenase and phenylalanine ammonia-lyase and the reduced expression of the MYMIV coat-protein encoding gene in all MeJA treated plants post MYMIV infection revealed that exogenous application of MeJA is effective for MYMIV tolerance in V. mungo. Our findings provide new insights into the physiological and molecular mechanisms of MYMIV tolerance in Vigna induced by MeJA.

RNAi-derived transgenic resistance to Mungbean yellow mosaic India virus in cowpea.

Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea.

Molecular and biochemical characterization of a Vigna mungo MAP kinase associated with Mungbean Yellow Mosaic India Virus infection and deciphering its role in restricting the virus multiplication.

Yellow Mosaic Disease caused by the begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) severely affects many economically important legumes. Recent investigations in Vigna mungo - MYMIV incompatible interaction identified a MAPK homolog in the defense signaling pathway. An important branch of immunity involves phosphorylation by evolutionary conserved Mitogen-activated protein kinases (MAPK) that transduce signals of pathogen invasion to downstream molecules leading to diverse immune responses. However, most of the knowledge of MAPKs is derived from model crops, and functions of these versatile kinases are little explored in legumes. Here we report characterization of a MAP kinase (VmMAPK1), which was induced upon MYMIV-inoculation in resistant V. mungo. Phylogenetic analysis revealed that VmMAPK1 is closely related to other plant-stress-responsive MAPKs. Both mRNA and protein of VmMAPK1 were accumulated upon MYMIV infection. The VmMAPK1 protein localized in the nucleus as well as cytoplasm and possessed phosphorylation activity in vitro. A detailed biochemical characterization of purified recombinant VmMAPK1 demonstrated an intramolecular mechanism of autophosphorylation and self-catalyzed phosphate incorporation on both threonine and tyrosine residues. The Vmax and Km values of recombinant VmMAPK1 for ATP were 6.292nmol/mg/min and 0.7978µM, respectively. Furthermore, the ability of VmMAPK1 to restrict MYMIV multiplication was validated by its ectopic expression in transgenic tobacco. Importantly, overexpression of VmMAPK1 resulted in the considerable upregulation of defense-responsive marker PR genes. Thus, the present data suggests the critical role of VmMAPK1 in suppressing MYMIV multiplication presumably through SA-mediated signaling pathway and inducing PR genes establishing the significant implications in understanding MAP kinase gene function during Vigna-MYMIV interaction; and hence paves the way for introgression of resistance in leguminous crops susceptible to MYMIV.

Complete genomic characterization of milk vetch dwarf virus isolates from cowpea and broad bean in Anhui province, China.

Cowpea and broad bean plants showing severe stunting and leaf rolling symptoms were observed in Hefei city, Anhui province, China, in 2014. Symptomatic plants from both species were shown to be infected with milk vetch dwarf virus (MDV) by PCR. The complete genomes of MDV isolates from cowpea and broad bean were sequenced. Each of them had eight genomic DNAs that differed between the two isolates by 10.7% in their overall nucleotide sequences. In addition, the MDV genomes from cowpea and broad bean were associated with two and three alphasatellite DNAs, respectively. This is the first report of MDV on cowpea in China and the first complete genome sequences of Chinese MDV isolates.

Complete genome sequences of cowpea polerovirus 1 and cowpea polerovirus 2 infecting cowpea plants in Burkina Faso.

The full-length genome sequences of two novel poleroviruses found infecting cowpea plants, cowpea polerovirus 1 (CPPV1) and cowpea polerovirus 2 (CPPV2), were determined using overlapping RT-PCR and RACE-PCR. Whereas the 5845-nt CPPV1 genome was most similar to chickpea chlorotic stunt virus (73% identity), the 5945-nt CPPV2 genome was most similar to phasey bean mild yellow virus (86% identity). The CPPV1 and CPPV2 genomes both have a typical polerovirus genome organization. Phylogenetic analysis of the inferred P1-P2 and P3 amino acid sequences confirmed that CPPV1 and CPPV2 are indeed poleroviruses. Four apparently unique recombination events were detected within a dataset of 12 full polerovirus genome sequences, including two events in the CPPV2 genome. Based on the current species demarcation criteria for the family Luteoviridae, we tentatively propose that CPPV1 and CPPV2 should be considered members of novel polerovirus species.