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KEY MESSAGE: The investigation of MYMIV-infected mung bean leaf apoplast revealed viral genome presence, increased EVs secretion, and altered stress-related metabolite composition, providing comprehensive insights into plant-virus interactions. The apoplast, an extracellular space around plant cells, plays a vital role in plant-microbe interactions, influencing signaling, defense, and nutrient transport. While the involvement of apoplast and extracellular vesicles (EVs) in RNA virus infection is documented, the role of the apoplast in plant DNA viruses remains unclear. This study explores the apoplast's role in mungbean yellow mosaic India virus (MYMIV) infection. Our findings demonstrate the presence of MYMIV genomic components in apoplastic fluid, suggesting potential begomovirus cell-to-cell movement via the apoplast. Moreover, MYMIV infection induces increased EVs secretion into the apoplast. NMR-based metabolomics reveals altered metabolic profiles in both apoplast and symplast in response to MYMIV infection, highlighting key metabolites associated with stress and defense mechanisms. The data show an elevation of α- and ß-glucose in both apoplast and symplast, suggesting a shift in glucose utilization. Interestingly, this increase in glucose does not contribute to the synthesis of phenolic compounds, potentially influencing the susceptibility of mung bean to MYMIV. Fructose levels increase in the symplast, while apoplastic sucrose levels rise significantly. Symplastic aspartate levels increase, while proline exhibits elevated concentration in the apoplast and reduced concentration in the cytosol, suggesting a role in triggering a hypersensitive response. These findings underscore the critical role of the apoplast in begomovirus infection, providing insights for targeted viral disease management strategies.

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Yellow mosaic disease (YMD) remains a major constraint in mungbean (Vigna radiata (L.)) production; while short-duration genotypes offer multiple crop cycles per year and help in escaping terminal heat stress, especially during summer cultivation. A comprehensive genotyping by sequencing (GBS)-based genome-wide association studies (GWAS) analysis was conducted using 132 diverse mungbean genotypes for traits like flowering time, YMD resistance, soil plant analysis development (SPAD) value, trichome density, and leaf area. The frequency distribution revealed a wide range of values for all the traits. GBS studies identified 31,953 high-quality single nucleotide polymorphism (SNPs) across all 11 mungbean chromosomes and were used for GWAS. Structure analysis revealed the presence of two genetically distinct populations based on ΔK. The linkage disequilibrium (LD) varied throughout the chromosomes and at r2 = 0.2, the mean LD decay was estimated as 39.59 kb. Two statistical models, mixed linear model (MLM) and Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) identified 44 shared SNPs linked with various candidate genes. Notable candidate genes identified include FPA for flowering time (VRADI10G01470; chr. 10), TIR-NBS-LRR for mungbean yellow mosaic India virus (MYMIV) resistance (VRADI09G06940; chr. 9), E3 ubiquitin-protein ligase RIE1 for SPAD value (VRADI07G28100; chr. 11), WRKY family transcription factor for leaf area (VRADI03G06560; chr. 3), and LOB domain-containing protein 21 for trichomes (VRADI06G04290; chr. 6). In-silico validation of candidate genes was done through digital gene expression analysis using Arabidopsis orthologous (compared with Vigna radiata genome). The findings provided valuable insight for marker-assisted breeding aiming for the development of YMD-resistant and early-maturing mungbean varieties.

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Mungbean yellow mosaic India virus (MYMIV) is one of the most serious commonly occurring yellow mosaic virus (YMV's) group in majority of the pulses especially black gram and green gram in southern India compared to previously reported mungbean yellow mosaic virus. In January 2020 Desmodium laxiflorum and Abelmoscus moschatus showing mosaic symptoms and vein yellowing were collected from Guntur and Prakasam districts respectively in Andhra Pradesh. PCR analysis using MYMIV and betasatellite specific primers gave desired expected amplification from the infected samples of A. moschatus (YMV-ABEL) whereas only MYMIV specific amplification was obtained in D. laxiflorum (YMV-DES). However, no PCR amplification was obtained in respective healthy leaf samples of both plants. Sequence analysis showed that the CP sequence of YMV-ABEL and YMV-DES showed a similarity of 99.19% with MYMIV (KP677496) and 99.75% with MYMIV (JN181003) respectively. The full-length betasatellite (1356 bp) showed highest identity of 90% with bhendi yellow vein mosaic betasatellite (BYVMB) (GU111977). Phylogenetic analysis clustered the test isolates with south Indian isolates of MYMIV whereas the betasatellite sequence clustered with various isolates of BYVMB, tomato leaf curl New Delhi virus betasatellite and okra leaf curl betasatellite reported from India and Pakistan. To the best of our knowledge, this is the first report of a MYMIV in D. laxiflorum and A. moschatus and MYMIV betasatellite complex in A. moschatus.

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In plants, RNA interference (RNAi) is triggered by double-stranded RNA (dsRNA). Accordingly, various RNA silencing technologies involving hpRNA, artificial microRNA (miRNA), and virus-induced gene silencing (VIGS) are used for controlling the expression of genes. Such manipulations help understanding gene functions and crop improvement biotechnology. A typical hpRNA construct is comprised of an intron splicable perfect inverted repeat of the target gene sequences under the control of a strong promoter. Geminiviruses, especially Mungbean Yellow Mosaic India Virus (MYMIV) cause devastating diseases in legume plants including cowpea, incurring severe crop loss. RNAi, involving hpRNA construct as transgene, is used to control these diseases at the early stages of geminivirus infection in the host, preventing symptom development and viral DNA accumulation. In this chapter, we describe a detailed protocol for the identification of geminivirus isolates from the filed grown cowpea plants, characterization of virus isolates under the laboratory conditions, design and construct RNAi vectors for effective suppression of viral target genes, and consequent development of transgenic cowpea using Agrobacterium-mediated transformation protocol. These transgenics are subsequently evaluated for resistance to MYMIV.

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PURPOSE: Mungbean yellow mosaic India virus (MYMIV) is a serious constraint in the mungbean which is a potential source of easily digestible high-quality proteins, fibers, minerals, and vitamins in Asian countries. Developing resistant cultivars is the most cost-effective, eco-friendly, and sustainable approach to protect mungbean from MYMIV damage. Mutation breeding provides a quick and cost-effective way of developing resistance as lack of genetic variability is the biggest bottleneck for other traditional breeding tools. MATERIALS AND METHODS: Outstanding but MYMIV-sensitive varieties of mungbean, viz., MH 2-15 and MH 318 were mutagenized through various individual and combined doses of gamma-rays and Ethyl methanesulfonate (EMS) and evaluated in M2 and M3 generations for the appearance of resistance reactions. This was subsequently validated through marker-assisted genotyping using previously reported Yellow Mosaic Disease (YMD) linked markers. RESULTS: The phenotyping in M3 generation yielded 64 MYMIV resistant mutants whereas, marker-assisted genotyping identified the 22 mutants with true resistance. Markers YR4, CYR1, and CEDG180 were found associated with MYMIV resistance whereas, DMB-SSR158 did not show any amplification. Among identified resistant mutants, ten lines exhibited at par and two revealed a little higher seed yield over controls. CONCLUSIONS: The mutagenesis created significant variability in MYMIV resistance as well as seed yield per plant. YR4, CYR1, and CEDG180 are found to be linked with the MYMIV loci in the mungbean and could be utilized for MYMIV resistance breeding. Mutant M-37 from MH 2-15 and M-104 from MH 318 exhibited more seed yield along with MYMIV resistance which upon further validation can be released as a variety. The induced mutagenesis integrated with powerful emerging molecular and next-generation sequencing (NGS) tools would be highly helpful in breeding mungbean for durable resistance against threatening MYMIV.

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Phaseolus vulgaris, commonly known as French bean is a vital leguminous crop worldwide and India stood 1st rank in dry bean and 4th rank in green bean production worldwide (FAOSTAT 2017). However, this production is severely affected by Mungbean yellow mosaic India virus (MYMIV) infection. Hence it is very important to identify MYMIV tolerant P. vulgaris cultivars. MYMIV infection results in the production of reactive oxygen species and plant cells have evolved complex defense mechanisms at different levels to overcome the damage. Our study for the first time focused on the changes at the morphological and biochemical level, as well as on the relative quantification of MYMIV genes in nine cultivars of P. vulgaris after MYMIV infection. Highest growth and the highest accumulation of four antioxidants of cv. 'Anupam' after MYMIV infection, established that cv. 'Anupam' was less affected by MYMIV infection amongst all nine cultivars. Relative quantification studies also correlated well with these results. Additionally, there is a consistent level of photosynthetic pigments content in mock- and MYMIV-treated seedlings of cv. 'Anupam' over early infection period. Combining all the results we conclude that cv. 'Anupam' is a MYMIV tolerant cultivar.

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The present study aimed to detect the marker-trait association of a selected diverse panel of 127 mungbean genotypes against mungbean yellow mosaic India virus (MYMIV). Virus-specific primers pairs viz., AC-abut/AV-abut and BC-abut/BV-abut confirmed the involvement of MYMIV in yellow mosaic disease development and the same was validated through restriction digestion analysis. 256 genome-wide microsatellite markers were screened on a test panel in which 93 polymorphic markers were used in association studies. Population structure analysis led to formation of six distinct subpopulations. 1097 alleles were detected among 127 test genotypes whereas number of alleles ranged 2-22 and PIC values ranged 0.27-0.92%, indicating ample amount of variation at genome level. 15 microsatellite markers were detected as associated with MYMIV resistance, among them three microsatellites explained 11-14% phenotypic variation. The specific regions close to CEDG293, DMB-SSR008 and DMB-SSR059 associated with MYMIV resistance were detected, located on linkage group 2, 4 and 9 and may prove useful in marker-assisted mungbean improvement programme for enhancing MYMIV resistance.

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Mungbean yellow mosaic India virus (MYMIV) belonging to the family Geminiviridae and the genus Begomovirus is a severe pathogen of tropical legumes including soybean. The absence of genetically mapped loci conferring resistance together with the genetic diversity of begomoviruses infecting soybean warrants the utilization of RNA interference (RNAi) technology to develop virus resistance. However, viral suppressors of RNAi (VSRs) reduce the effectiveness of RNA silencing. Here, we report the effectiveness of Agrobacterium-mediated transient expression of shRNA, targeting a conserved region of AC2 ORF (a VSR) of MYMIV, in conferring virus resistance in soybean. Transient expression of shRNA showed progressive reduction of the viral titre estimated by the MYMIV-derived AC2 gene copy numbers from the initial inoculum by approximately 80-fold 20 days post-application. In addition, the newly emerging leaves exhibited symptom recovery. Thus, this study proves that AC2 of MYMIV is a potent target gene for obtaining RNAi-mediated virus resistance in soybean. Agro-infiltration-based delivery of shRNA was an efficient means of gene silencing and could pave way for the development of transgenic virus-resistant soybean genotype.

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Mungbean yellow mosaic India virus (MYMIV) infecting soybean and other legumes causes yellow mosaic disease (YMD). Evaluation of soybean genotypes for YMD resistance involves field screening at disease hot spots or in a protected environment using infectious clones or viruliferous whiteflies as sources of virus inocula. Development of efficient virus inoculation and quantification protocols to screen soybean genetic stocks against YMD is imperative for breeding resistant varieties. Binary plasmids harbouring complete, tandem dimeric genomic components DNA A and DNA B of MYMIV-soybean isolate were engineered. The infectivity of the clones was demonstrated in soybean genotypes JS335 and UPSM534 that display contrasting YMD resistance. As a follow-up, soybean germplasm lines, breeding lines, and representative cultivars that were initially screened at an YMD hot-spot were then subjected to Agrobacterium-based infection with MYMIV. Quantitative real time polymerase chain reaction (qRT-PCR) based copy number analysis of MYMIV genomic components allowed soybean genotypes to be classified into three discrete categories; resistant, moderately resistant and susceptible to the viral infection. Thus, a soybean germplasm disease screening system based on agro-infection and qRT-PCR based quantification of MYMIV was developed to facilitate breeding YMD resistant soybean. The implications of this study for obtaining YMD resistant soybean cultivars are discussed.

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Phaseolus vulgaris is an economically important legume in tropical and subtropical regions of Asia, Africa, Latin-America and parts of USA and Europe. However, its production gets severely affected by mungbean yellow mosaic India virus (MYMIV). We aim to identify and characterize differentially expressed miRNAs during MYMIV-infection in P. vulgaris. A total of 422 miRNAs are identified of which 292 are expressed in both MYMIV-treated and mock-treated samples, 109 are expressed only in MYMIV-treated and 21 are expressed only in mock-treated samples. Selected up- and down-regulated miRNAs are validated by RT-qPCR. 3367 target ORFs are identified for 270 miRNAs. Selected targets are validated by 5' RLM-RACE. Differentially expressed miRNAs regulate transcription factors and are involved in improving stress tolerance to MYMIV. These findings will provide an insight into the role of miRNAs during MYMIV infection in P. vulgaris in particular and during any biotic stress conditions in Leguminosae family in general.

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A new isolate (Mg-mungbean-1) of yellow mosaic virus (YMV) was identified and characterized from mungbean growing in mid-hill condition of Meghalaya, India. Full genome of components (DNA A and DNA B; NCBI accessions number KU95030 and KU95031, respectively) of the virus were amplified through rolling circle amplification and sequenced. Both, DNA A and DNA B shared a common region (CR) with 90.4% similarity. The DNA A of Mg-mungbean-1 showed maximum (97.59%) nucleotide identity with mungbean yellow mosaic India virus (MYMIV) isolate (HF922628) reported from West Bengal, India and DNA B showed ~ 96% nucleotide identity with mungbean yellow mosaic virus (MYMV) isolates having variant DNA B. Phylogenetic tree of DNA A also identified Mg-mungbean-1 as a MYMIV. Based on DNA B the current isolate grouped with the variant Indian MYMV isolates. One recombination event in the CR of DNA B of Mg-mungbean-1 was detected, where MYMV:India:clonePB1 and MYMIV:India:cloneMBB-B31 have been identified as major and minor parents, respectively. Overall, the current study indicated occurrence of an isolate of MYMIV with a recombinant DNA B component on mungbean from mid-hills of Meghalaya, India. To the best of our knowledge this is the first molecular characterization of YMV from northeast India.

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Plant pathogen interaction plays a great role in plant immunity. The regulation of various components of plant pathogen interactions is quite complicated and is very important in establishing relationship among components of this system. Yellow Mosaic Disease is common among legumes such as Vigna mungo. Mungbean Yellow Mosaic India Virus (MYMIV) and whitefly (Bemisia tabaci) is a vector causing the disease. Therefore, it is of interest to document the molecule models of three different components of Plant Pathogen interaction cascade- MAP kinase1, MAP kinase 2 and WRKY33 from V. mungo resistant to MYMIV. Both the MAP kinases were sequenced for this study while WRKY 33 was extracted and modeled from transcripts generated from two different transcriptome libraries, one set MYMIV- challenged, the other fed with aviruliferous whitefly. Post simulation studies revealed that MAPKs contained less percentage of disordered residues and were structurally more stable and than WRKY33.

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Vigna mungo (Urdbean) is cultivated in the tropical and sub-tropical continental region of Asia. It is not only important source of dietary protein and nutritional elements, but also of immense value to human health due to medicinal properties. Yellow mosaic disease caused by Mungbean Yellow Mosaic India Virus is known to incur huge loss to crop, adversely affecting crop yield. Contrasting genotypes are ideal source for knowledge discovery of plant defence mechanism and associated candidate genes for varietal improvement. Whole genome sequence of this crop is yet to be completed. Moreover, genomic resources are also not freely accessible, thus available transcriptome data can be of immense use. V. mungo Transcriptome database, accessible at http://webtom.cabgrid.res.in/vmtdb/ has been developed using available data of two contrasting varieties viz., cv. VM84 (resistant) and cv. T9 (susceptible). De novo assembly was carried out using Trinity and CAP3. Out of total 240,945 unigenes, 165,894 (68.8%) showed similarity with known genes against NR database, and remaining 31.2% were found to be novel. We found 22,101 differentially expressed genes in all datasets, 44,335 putative genic SSR markers, 4105 SNPs and Indels, 64,964 transcriptional factor, 546 mature miRNA target prediction in 703 differentially expressed unigenes and 137 pathways. MAPK, salicylic acid-binding protein 2-like, pathogenesis-related protein and NBS-LRR domain were found which may play an important role in defence against pathogens. This is the first web genomic resource of V. mungo for future genome annotation as well as ready to use markers for future variety improvement program.

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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.

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Mungbean Yellow Mosaic India Virus (MYMIV) is one of the most prevalent pathogen that limits soybean production in India. In this study RILs derived from JS335, dominant but MYMIV susceptible variety and PI171443, donor of MYMIV resistance gene in most of the MYMIV resistant varieties released in India and F2 population derived from SL525, a resistant variety released for northern India and NRC101, a susceptible genotype were used to study the inheritance of MYMIV resistance and map the gene responsible for MYMIV resistance. F1s were found to be completely susceptible. F2:3 and RILs population segregated to fit a ratio of 1:2:1 and 1:1 indicating that a single recessive gene controlled resistance to MYMIV. BSA was performed using 144 polymorphic SSR markers. MYMIV resistance gene was mapped on chr 6 (LG C2) within a 3.5-cM genome region between two SSR markers GMAC7L and Satt322 whose size was estimated to be 77.115 kb (position of 12,259,594-12,336,709 bp). This is the first report on linkage mapping of MYMIV resistance gene in soybean. This will be helpful in breeding soybean varieties for resistance against MYMIV responsible for wide spread damage to soybean crop in India using Marker Assisted Selection.

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Yellow mosaic disease (YMD) is one of the major diseases affecting mungbean (Vigna radiata (L.) Wilczek). In this study, we report the mapping of the quantitative trait locus (QTL) for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean. An F8 recombinant inbred line (RIL) mapping population was generated in Thailand from a cross between NM10-12-1 (MYMIV resistance) and KPS2 (MYMIV susceptible). One hundred and twenty-two RILs and their parents were evaluated for MYMIV resistance in infested fields in India and Pakistan. A genetic linkage map was developed for the RIL population using simple sequence repeat (SSR) markers. Composite interval mapping identified five QTLs for MYMIV resistance: three QTLs for India (qYMIV1, qYMIV2 and qYMIV3) and two QTLs for Pakistan (qYMIV4 and qYMIV5). qYMIV1, qYMIV2, qYMIV3, qYMIV4 and qYMIV5 explained 9.33%, 10.61%, 12.55%, 21.93% and 6.24% of variation in disease responses, respectively. qYMIV1 and qYMIV4 appeared to be the same locus and were common to a major QTL for MYMIV resistance in India identified previously using a different resistant mungbean.

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Yellow mosaic disease of cultivated legumes in South-East Asia, is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Efforts to engineer resistance against the genus Begomovirus are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a complementary-sense gene (ACI) encoding Replication initiation Protein (Rep) to develop resistance against soybean isolate of Mungbean yellow mosaic India virus-[India:New Delhi:Soybean 2:1999], a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that the legume host plants co-agroinoculated with infectious constructs of soybean isolate of Mungbean yellow mosaic India virus [India:New Delhi:Soybean 2:1999] along with this antisense Rep gene construct show resistance to the virus.