Alternative splicing shapes the transcriptome complexity in blackgram [Vigna mungo (L.) Hepper].

Vigna mungo, a highly consumed crop in the pan-Asian countries, is vulnerable to several biotic and abiotic stresses. Understanding the post-transcriptional gene regulatory cascades, especially alternative splicing (AS), may underpin large-scale genetic improvements to develop stress-resilient varieties. Herein, a transcriptome based approach was undertaken to decipher the genome-wide AS landscape and splicing dynamics in order to establish the intricacies of their functional interactions in various tissues and stresses. RNA sequencing followed by high-throughput computational analyses identified 54,526 AS events involving 15,506 AS genes that generated 57,405 transcripts isoforms. Enrichment analysis revealed their involvement in diverse regulatory functions and demonstrated that transcription factors are splicing-intensive, splice variants of which are expressed differentially across tissues and environmental cues. Increased expression of a splicing regulator NHP2L1/SNU13 was found to co-occur with lower intron retention events. The host transcriptome is significantly impacted by differential isoform expression of 1172 and 765 AS genes that resulted in 1227 (46.8% up and 53.2% downregulated) and 831 (47.5% up and 52.5% downregulated) transcript isoforms under viral pathogenesis and Fe2+ stressed condition, respectively. However, genes experiencing AS operate differently from the differentially expressed genes, suggesting AS is a unique and independent mode of regulatory mechanism. Therefore, it can be inferred that AS mediates a crucial regulatory role across tissues and stressful situations and the results would provide an invaluable resource for future endeavours in V. mungo genomics.

Deciphering the role of alternative splicing as modulators of defense response in the MYMIV-Vigna mungo pathosystem.

Alternative splicing (AS) is a crucial regulatory mechanism that impacts transcriptome and proteome complexity under stressful situations. Although its role in abiotic stresses is somewhat understood, our understanding of the mechanistic regulation of pre-messenger RNA splicing in plant-pathogen interaction is meager. To comprehend this unexplored immune reprogramming mechanism, transcriptome profiles of Mungbean Yellow Mosaic India Virus (MYMIV)-resistant and susceptible Vigna mungo genotypes were analyzed for AS genes that may underlie the resistance mechanism. Results revealed a repertoire of AS-isoforms accumulated during pathogenic infestation, with intron retention being the most common AS mechanism. Identification of 688 differential alternatively spliced (DAS) genes in the resistant host elucidates its robust antiviral response, whereas 322 DAS genes were identified in the susceptible host. Enrichment analyses confirmed DAS transcripts pertaining to stress, signaling, and immune system pathways have undergone maximal perturbations. Additionally, a strong regulation of the splicing factors has been observed both at the transcriptional and post-transcriptional levels. qPCR validation of candidate DAS transcripts with induced expression upon MYMIV infection demonstrated a competent immune response in the resistant background. The AS-impacted genes resulted either in partial/complete loss of functional domains or altered sensitivity to micro-RNA-mediated gene silencing. A complex regulatory module, miR7517-ATAF2, has been identified in an aberrantly spliced ATAF2 isoform that exposes an intronic miR7517 binding site, thereby suppressing the negative regulator to enhance the defense reaction. The present study establishes AS as a noncanonical immune reprogramming mechanism that operates in parallel, thereby offering an alternative strategy for developing yellow mosaic-resistant V. mungo cultivars.

Eisosome protein Pil1 regulates mitochondrial morphology, mitophagy, and cell death in Saccharomyces cerevisiae.

Mitochondrial morphology and dynamics maintain mitochondrial integrity by regulating its size, shape, distribution, and connectivity, thereby modulating various cellular processes. Several studies have established a functional link between mitochondrial dynamics, mitophagy, and cell death, but further investigation is needed to identify specific proteins involved in mitochondrial dynamics. Any alteration in the integrity of mitochondria has severe ramifications that include disorders like cancer and neurodegeneration. In this study, we used budding yeast as a model organism and found that Pil1, the major component of the eisosome complex, also localizes to the periphery of mitochondria. Interestingly, the absence of Pil1 causes the branched tubular morphology of mitochondria to be abnormally fused or aggregated, whereas its overexpression leads to mitochondrial fragmentation. Most importantly, pil1Δ cells are defective in mitophagy and bulk autophagy, resulting in elevated levels of reactive oxygen species and protein aggregates. In addition, we show that pil1Δ cells are more prone to cell death. Yeast two-hybrid analysis and co-immunoprecipitations show the interaction of Pil1 with two major proteins in mitochondrial fission, Fis1 and Dnm1. Additionally, our data suggest that the role of Pil1 in maintaining mitochondrial shape is dependent on Fis1 and Dnm1, but it functions independently in mitophagy and cell death pathways. Together, our data suggest that Pil1, an eisosome protein, is a novel regulator of mitochondrial morphology, mitophagy, and cell death.

Exposure to Low UV-B Dose Induces DNA Double-Strand Breaks Mediated Onset of Endoreduplication in Vigna radiata (L.) R. Wilczek Seedlings.

Multiple lines of evidence indicate that solar UV-B light acts as an important environmental signal in plants, regulating various cellular and metabolic activities, gene expression, growth and development. Here, we show that low levels of UV-B (4.0 kJ m-2) significantly influence plant response during early seedling development in the tropical legume crop Vigna radiata (L.) R. Wilczek. Exposure to low doses of UV-B showed relatively less growth inhibition yet remarkably enhanced lateral root formation in seedlings. Both low and high (8.0 kJ m-2) doses of UV-B treatment induced DNA double-strand breaks and activated the SOG1-related ATM-ATR-mediated DNA damage response pathway. These effects led to G2-M-phase arrest with a compromised expression of the key cell cycle regulators, including CDKB1;1, CDKB2;1 and CYCB1;1, respectively. However, along with these effects, imbibitional exposure of seeds to a low UV-B dose resulted in enhanced accumulation of FZR1/CCS52A, E2Fa and WEE1 kinase and prominent induction of endoreduplication in 7-day-old seedlings. Low dose of UV-B mediated phenotypical responses, while the onset of endoreduplication appeared to be regulated at least in part via UV-B induced reactive oxygen species accumulation. Transcriptome analyses further revealed a network of co-regulated genes associated with DNA repair, cell cycle regulation and oxidative stress response pathways that are activated upon exposure to low doses of UV-B.

Complex molecular mechanisms underlying MYMIV-resistance in Vigna mungo revealed by comparative transcriptome profiling.

Mungbean Yellow Mosaic India Virus (MYMIV)-infection creates major hindrance in V. mungo cultivation and poses significant threat to other grain legume production. Symptoms associated include severe patho-physiological alterations characterized by chlorotic foliar lesion accompanied by reduced growth. However, dissection of the host's defense machinery remains a tough challenge due to limited of host's genomic resources. A comparative RNA-Seq transcriptomes of resistant (VM84) and susceptible (T9) plants was carried out to identify genes potentially involved in V. mungo resistance against MYMIV. Distinct gene expression landscapes were observed in VM84 and T9 with 2158 and 1679 differentially expressed genes (DEGs), respectively. Transcriptomic responses in VM84 reflect a prompt and intense immune reaction demonstrating an efficient pathogen surveillance leading to activation of basal and induced immune responses. Functional analysis of the altered DEGs identified multiple regulatory pathways to be activated or repressed over time. Up-regulation of DEGs including NB-LRR, WRKY33, ankyrin, argonaute and NAC transcription factor revealed an insight on their potential roles in MYMIV-resistance; and qPCR validation shows a propensity of their accumulation in VM84. Analyses of the current RNA-Seq dataset contribute immensely to decipher molecular responses that underlie MYMIV-resistance and will aid in the improvement strategy of V. mungo and other legumes through comparative functional genomics.

Screening and Identification of putative long non coding RNAs from transcriptome data of a high yielding blackgram (Vigna mungo), Cv. T9.

Blackgram (Vigna mungo) is one of primary legumes cultivated throughout India, Cv.T9 being one of its common high yielding cultivar. This article reports RNA sequencing data and a pipeline for prediction of novel long non-coding RNAs from the sequenced data. The raw data generated during sequencing are available at Sequence Read Archive (SRA) of NCBI with accession number- SRX1558530.

Identification, characterization and gene expression analyses of important flowering genes related to photoperiodic pathway in bamboo.

BACKGROUND: Bamboo is an important member of the family Poaceae and has many inflorescence and flowering features rarely observed in other plant groups. It retains an unusual form of perennialism by having a long vegetative phase that can extend up to 120 years, followed by flowering and death of the plants. In contrast to a large number of studies conducted on the annual, reference plants Arabidopsis thaliana and rice, molecular studies to characterize flowering pathways in perennial bamboo are lacking. Since photoperiod plays a crucial role in flower induction in most plants, important genes involved in this pathway have been studied in the field grown Bambusa tulda, which flowers after 40-50 years. RESULTS: We identified several genes from B. tulda, including four related to the circadian clock [LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), ZEITLUPE (ZTL) and GIGANTEA (GI)], two circadian clock response integrators [CONSTANS A (COA), CONSTANS B (COB)] and four floral pathway integrators [FLOWERING LOCUS T1, 2, 3, 4 (FT1, 2, 3, 4)]. These genes were amplified from either gDNA and/or cDNA using degenerate as well as gene specific primers based on homologous sequences obtained from related monocot species. The sequence identity and phylogenetic comparisons revealed their close relationships to homologs identified in the temperate bamboo Phyllostachys edulis. While the four BtFT homologs were highly similar to each other, BtCOA possessed a full-length B-box domain that was truncated in BtCOB. Analysis of the spatial expression of these genes in selected flowering and non-flowering tissue stages indicated their possible involvement in flowering. The diurnal expression patterns of the clock genes were comparable to their homologs in rice, except for BtZTL. Among multiple BtCO and BtFT homologs, the diurnal pattern of only BtCOA and BtFT3, 4 were synchronized in the flower inductive tissue, but not in the non-flowering tissues. CONCLUSION: This study elucidates the photoperiodic regulation of bamboo homologs of important flowering genes. The finding also identifies copy number expansion and gene expression divergence of CO and FT in bamboo. Further studies are required to understand their functional role in bamboo flowering.

Molecular modeling and simulation of three important components of Plant Pathogen Interaction cascade in Vigna mungo.

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.

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.

Molecular phylogeny of 21 tropical bamboo species reconstructed by integrating non-coding internal transcribed spacer (ITS1 and 2) sequences and their consensus secondary structure.

The unavailability of the reproductive structure and unpredictability of vegetative characters for the identification and phylogenetic study of bamboo prompted the application of molecular techniques for greater resolution and consensus. We first employed internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) sequences to construct the phylogenetic tree of 21 tropical bamboo species. While the sequence alone could grossly reconstruct the traditional phylogeny amongst the 21-tropical species studied, some anomalies were encountered that prompted a further refinement of the phylogenetic analyses. Therefore, we integrated the secondary structure of the ITS sequences to derive individual sequence-structure matrix to gain more resolution on the phylogenetic reconstruction. The results showed that ITS sequence-structure is the reliable alternative to the conventional phenotypic method for the identification of bamboo species. The best-fit topology obtained by the sequence-structure based phylogeny over the sole sequence based one underscores closer clustering of all the studied Bambusa species (Sub-tribe Bambusinae), while Melocanna baccifera, which belongs to Sub-Tribe Melocanneae, disjointedly clustered as an out-group within the consensus phylogenetic tree. In this study, we demonstrated the dependability of the combined (ITS sequence+structure-based) approach over the only sequence-based analysis for phylogenetic relationship assessment of bamboo.

High throughput sequencing reveals modulation of microRNAs in Vigna mungo upon Mungbean Yellow Mosaic India Virus inoculation highlighting stress regulation.

MicroRNAs (miRNAs) are 20-24 nucleotides long non-coding RNAs known to play important regulatory roles during biotic and abiotic stresses by controlling gene expression. Blackgram (Vigna mungo), an economically important grain legume is highly susceptible to pathogenic begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) and resulting in high yield loss. In this study two different leaf-small-RNA libraries were prepared from the pooled RNA at three different time points of resistant V. mungo inbred line VM84 inoculated either with viruliferous or non-viruliferous whiteflies carrying MYMIV and performed high-throughput Illumina sequencing. Sequencing followed by bioinformatics analysis of the small RNA reads indicated that the expression patterns of most of the known and novel miRNAs were altered in resistant line over mock-inoculated sample during the plant virus incompatible interaction. Highly altered miRNAs belong to the families of miR156, miR159, miR160, miR166, miR398, miR1511, miR1514, miR2118 and novel vmu-miRn7, vmu-miRn8, vmu-miRn13 and vmu-miRn14. These results were validated using qPCR, and most of the miRNAs showed similar pattern of expression like that of Illumina reads. The expression patterns of some selected known and novel miRNAs were also compared between the infected MYMIV-resistant and -susceptible genotypes and most of these were modulated after MYMIV-inoculation. Target transcripts like NB-LRR, NAC, MYB, Zinc finger, CCAAT-box transcription factor, fructose 2-6 bisphosphate, HDZIP protein that confers immune response were predicted as targets amongst identified miRNAs using psRNATarget server. Some selected target transcripts including NB-LRR, ARF, SOD, SPB, Basic blue copper protein were validated and their differential expression were demonstrated between MYMIV-resistant and -susceptible V. mungo by qPCR data analyses. In the present study we have identified miRNAs that implicate in the regulation of MYMIV-induced stress response in V. mungo; and generated genomic resources for a non-model legume with the aid of bioinformatics tools supplemented by experimental validation.

Bamboo Flowering from the Perspective of Comparative Genomics and Transcriptomics.

Bamboos are an important member of the subfamily Bambusoideae, family Poaceae. The plant group exhibits wide variation with respect to the timing (1-120 years) and nature (sporadic vs. gregarious) of flowering among species. Usually flowering in woody bamboos is synchronous across culms growing over a large area, known as gregarious flowering. In many monocarpic bamboos this is followed by mass death and seed setting. While in sporadic flowering an isolated wild clump may flower, set little or no seed and remain alive. Such wide variation in flowering time and extent means that the plant group serves as repositories for genes and expression patterns that are unique to bamboo. Due to the dearth of available genomic and transcriptomic resources, limited studies have been undertaken to identify the potential molecular players in bamboo flowering. The public release of the first bamboo genome sequence Phyllostachys heterocycla, availability of related genomes Brachypodium distachyon and Oryza sativa provide us the opportunity to study this long-standing biological problem in a comparative and functional genomics framework. We identified bamboo genes homologous to those of Oryza and Brachypodium that are involved in established pathways such as vernalization, photoperiod, autonomous, and hormonal regulation of flowering. Additionally, we investigated triggers like stress (drought), physiological maturity and micro RNAs that may play crucial roles in flowering. We also analyzed available transcriptome datasets of different bamboo species to identify genes and their involvement in bamboo flowering. Finally, we summarize potential research hurdles that need to be addressed in future research.

Analyses of MYMIV-induced transcriptome in Vigna mungo as revealed by next generation sequencing.

Mungbean Yellow Mosaic Virus (MYMIV) is the viral pathogen that causes yellow mosaic disease to a number of legumes including Vigna mungo. VM84 is a recombinant inbred line resistant to MYMIV, developed in our laboratory through introgression of resistance trait from V. mungo line VM-1. Here we present the quality control passed transcriptome data of mock inoculated (control) and MYMIV-infected VM84, those have already been submitted in Sequence Read Archive (SRX1032950, SRX1082731) of NCBI. QC reports of FASTQ files generated by 'SeqQC V2.2' bioinformatics tool.

Transcript dynamics at early stages of molecular interactions of MYMIV with resistant and susceptible genotypes of the leguminous host, Vigna mungo.

Initial phases of the MYMIV-Vigna mungo interaction is crucial in determining the infection phenotype upon challenging with the virus. During incompatible interaction, the plant deploys multiple stratagems that include extensive transcriptional alterations defying the virulence factors of the pathogen. Such molecular events are not frequently addressed by genomic tools. In order to obtain a critical insight to unravel how V. mungo respond to Mungbean yellow mosaic India virus (MYMIV), we have employed the PCR based suppression subtractive hybridization technique to identify genes that exhibit altered expressions. Dynamics of 345 candidate genes are illustrated that differentially expressed either in compatible or incompatible reactions and their possible biological and cellular functions are predicted. The MYMIV-induced physiological aspects of the resistant host include reactive oxygen species generation, induction of Ca2+ mediated signaling, enhanced expression of transcripts involved in phenylpropanoid and ubiquitin-proteasomal pathways; all these together confer resistance against the invader. Elicitation of genes implicated in salicylic acid (SA) pathway suggests that immune response is under the regulation of SA signaling. A significant fraction of modulated transcripts are of unknown function indicating participation of novel candidate genes in restricting this viral pathogen. Susceptibility on the other hand, as exhibited by V. mungo Cv. T9 is perhaps due to the poor execution of these transcript modulation exhibiting remarkable repression of photosynthesis related genes resulting in chlorosis of leaves followed by penalty in crop yield. Thus, the present findings revealed an insight on the molecular warfare during host-virus interaction suggesting plausible signaling mechanisms and key biochemical pathways overriding MYMIV invasion in resistant genotype of V. mungo. In addition to inflate the existing knowledge base, the genomic resources identified in this orphan crop would be useful for integrating MYMIV-tolerance trait in susceptible cultivars of V. mungo.

Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing.

MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformatic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resources for better understanding of miRNA mediated post-transcriptional gene regulation.

Functional characterization of a serine-threonine protein kinase from Bambusa balcooa that implicates in cellulose overproduction and superior quality fiber formation.

BACKGROUND: Molecular markers allow rapid identification of biologically important germplasm/s having desired character. Previously we have reported a genotype specific molecular marker, Balco1128 [GenBank ID EU258678] of Bambusa balcooa containing an ORF (375 bp) having high similarity with receptor like cytoplasmic kinase of Arabidopsis and Oryza. Balco1128 was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. Under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development. RESULTS: The full length cDNA (3342 bp) of BbKst, a serine-threonine protein kinase was isolated from B. balcooa comprising of six LRR domains at the N-terminal end and a kinase domain at the C-terminal end. Bacteria-expressed BbKst-kinase domain (3339 bp long) showed Mg(2+) dependent kinase activity at pH 7.0, 28°C. Bioinformatics study followed by phospho-amino analysis further confirmed that BbKst-kinase belongs to the serine/threonine protein kinase family. Transcript analysis of the BbKst gene following RNA slot blot hybridization and qPCR revealed higher expression of BbKst during initiation and elongation stages of fiber development. Tissue specific expression studies showed much higher expression of BbKst transcript in stems and internodes of B. balcooa than in leaves and rhizomes. Southern analysis revealed single copy insertion of BbKst in most of the Agrobacterium mediated transgenic tobacco plants. Real-time PCR detected 150-200 fold enhanced expression of BbKst in different T1 tobacco lines than that of the vector transformed plants. Heterologous expression of BbKst under control of 35S promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. Number of xylary fibers was higher in transgenic T0 and T1 plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower Runkel ratio of the fibers. CONCLUSIONS: This finding clearly demonstrated that BbKst gene (GenBank ID JQ432560) encodes a serine/threonine protein kinase. BbKst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant.

Defining reference genes for qPCR normalization to study biotic and abiotic stress responses in Vigna mungo.

KEY MESSAGE: Expression of ACT, EF1A; H2A, EF1A, ACT and 18S, TUB showed stability under MYMIV, salinity and drought stress, respectively; these are recommended as reference genes for qPCR normalization in Vigna mungo. Accurate gene expression profiling through qPCR depends on selection of appropriate reference gene(s) for normalization. Due to lack of unanimous internal standard, suitable constitutively expressed reference genes are selected that exhibit stable expression under diverse experimental conditions. In this communication, a comparative evaluation of stability among seven V. mungo genes encoding actin (ACT), histone H2A (H2A), elongation factor 1-alpha (EF1A), 18S rRNA (18S), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin (CYP) and tubulin (TUB) under biotic (MYMIV) and abiotic (drought and salinity) stress conditions has been attempted. Specificity and amplification efficiency for each primer pair were verified; however, cumulative assessment of their accumulated transcripts revealed no uniformity. Therefore, individual stability and suitability of these seven candidates have been assessed in silico, by two widely used algorithms, geNorm and Normfinder. Based on the computed results, high stability was obtained for ACT and EF1A during MYMIV stress, while H2A, EFIA and ACT were found to be most suitable in salinity stress experiments and TUB and 18S during drought treatments. Combinations of ACT/TUB or ACT/EFIA were recommended for their use in the pooled analysis, while expression of 18S and CYP showed greater variations and therefore considered unsuitable as reference genes. Additionally, precise quantification of the target gene VmPRX under these stresses was shown to be a function of reference genes' stability, which tends to get affected when normalized with the least stable genes. Hence, use of these normalizers will facilitate accurate and reliable analyses of gene expression in V. mungo.

Proteomics approach combined with biochemical attributes to elucidate compatible and incompatible plant-virus interactions between Vigna mungo and Mungbean Yellow Mosaic India Virus.

BACKGROUND: Vigna mungo, a tropical leguminous plant, highly susceptible to yellow mosaic disease caused by Mungbean Yellow Mosaic India Virus (MYMIV) resulting in high yield penalty. The molecular events occurring during compatible and incompatible interactions between V. mungo and MYMIV pathosystem are yet to be explored. In this study biochemical analyses in conjunction with proteomics of MYMIV-susceptible and -resistant V. mungo genotypes were executed to get an insight in the molecular events during compatible and incompatible plant-virus interactions. RESULTS: Biochemical analysis revealed an increase in phenolics, hydrogen peroxide and carbohydrate contents in both compatible and incompatible interactions; but the magnitudes were higher during incompatible interaction. In the resistant genotype the activities of superoxide dismutase and ascorbate peroxidase increased significantly, while catalase activity decreased. Comparative proteome analyses using two-dimensional gel electrophoresis coupled with mass spectrometry identified 109 differentially abundant proteins at 3, 7 and 14 days post MYMIV-inoculation. Proteins of several functional categories were differentially changed in abundance during both compatible and incompatible interactions. Among these, photosynthesis related proteins were mostly affected in the susceptible genotype resulting in reduced photosynthesis rate under MYMIV-stress. Differential intensities of chlorophyll fluorescence and chlorophyll contents are in congruence with proteomics data. It was revealed that Photosystem II electron transports are the primary targets of MYMIV during pathogenesis. Quantitative real time PCR analyses of selected genes corroborates with respective protein abundance during incompatible interaction. The network of various cellular pathways that are involved in inducing defense response contains several conglomerated cores of nodal proteins, of which ascorbate peroxidase, rubisco activase and serine/glycine hydroxymethyl transferase are the three major hubs with high connectivity. These nodal proteins play the crucial role of key regulators in bringing about a coordinated defense response in highly orchestrated manner. CONCLUSIONS: Biochemical and proteomic analyses revealed early accumulation of the defense/stress related proteins involved in ROS metabolism during incompatible interaction. The robustness in induction of defense/stress and signal transduction related proteins is the key factor in inducing resistance. The mechanism of MYMIV-resistance in V. mungo involves redirection of carbohydrate flux towards pentose phosphate pathway. Some of these identified, differentially regulated proteins are also conferring abiotic stress responses illustrating harmony amongst different stress responses. To the best of our knowledge, this is the lone study deciphering differential regulations of V. mungo leaf proteome upon MYMIV infection elucidating the mode of resistance response at the biochemical level.

Isolation, characterization, and structure analysis of a non-TIR-NBS-LRR encoding candidate gene from MYMIV-resistant Vigna mungo.

Yellow mosaic disease of Vigna mungo caused by Mungbean yellow mosaic India virus (MYMIV) is still a major threat in the crop production. A candidate disease resistance (R) gene, CYR1 that co-segregates with MYMIV-resistant populations of V. mungo has been isolated. CYR1 coded in silico translated protein sequence comprised of 1,176 amino acids with coiled coil structure at the N-terminus, central nucleotide binding site (NBS) and C-terminal leucine-rich repeats (LRR) that belongs to non-TIR-NBS-LRR subfamily of plant R genes. CYR1 transcript was unambiguously expressed during incompatible plant virus interactions. A putative promoter-like sequence present upstream of this candidate gene perhaps regulates its expression. Enhanced transcript level upon MYMIV infection suggests involvement of this candidate gene in conferring resistance against the virus. In silico constructed 3D models of NBS and LRR regions of this candidate protein and MYMIV-coat protein (CP) revealed that CYR1-LRR forms an active pocket and successively interacts with MYMIV-CP during docking, like that of receptor-ligand interaction; indicating a critical role of CYR1 as signalling molecule to protect V. mungo plants from MYMIV. This suggests involvement of CYR1 in recognizing MYMIV-effector molecule thus contributing to incompatible interaction. This study is the first stride to understand molecular mechanism of MYMIV resistance.

Identification of genes involved in bamboo fiber development.

Recently bamboo has gained reputation as a major resource of non-wood fiber. The present study was undertaken to generate information about fiber development process in bamboo (Bambusa balcooa) using PCR-based suppressive subtractive hybridization (PCR-SSH) technique, as molecular mechanism of its fiber development is yet to be explored. SSH was performed between cDNA isolated from leaf (as driver) and internodes (as tester) of B. balcooa which indicated up-regulation of 521 ESTs. Among these 41 were contigs and 65 ESTs were singletons. On the basis of BLASTX search 307 ESTs with known functions were classified into several functional categories including transport, metabolism, information, perception and response to stimuli while others were either non-significant (120) or hypothetical proteins (94). A total of 51 out of 307 functional ESTs were found fiber specific and their global distribution among different plant species like maize, rice, cotton and Arabidopsis ESTs were determined. Net distributions and differential expression patterns of 13 important B. balcooa fiber specific cDNAs among different internodes during bamboo development were studied using RNA slot-blot, semi-quantitative RT-PCR and real time PCR. In-situ localization of mRNA transcript for few selected bamboo fiber ESTs namely, V1Bb147 (protein kinase-like protein) and V1Bb88 (myb domain-containing protein) were detected using Confocal Laser Scanning Microscope. Transcript levels of these genes exhibited an orchestral turn-over during bamboo development, suggesting their close association with fiber development, an event associated with several metabolic and physiological changes. The results clearly suggest that these genes are involved in several concerted mechanisms involving Ca(+) signaling pathway, cell wall synthesis, hormone regulation, system maintaining cell turgor pressure and cytoskeleton synthesis pathway accountable for bamboo fiber development signifying fiber development as a complex but ordered metabolic process involving differential expression of large scale fiber associated genes. This is the first report on systematic analysis of genes involved in bamboo fiber development.