NCodR: A multi-class support vector machine classification to distinguish non-coding RNAs in Viridiplantae.

Non-coding RNAs (ncRNAs) are major players in the regulation of gene expression. This study analyses seven classes of ncRNAs in plants using sequence and secondary structure-based RNA folding measures. We observe distinct regions in the distribution of AU content along with overlapping regions for different ncRNA classes. Additionally, we find similar averages for minimum folding energy index across various ncRNAs classes except for pre-miRNAs and lncRNAs. Various RNA folding measures show similar trends among the different ncRNA classes except for pre-miRNAs and lncRNAs. We observe different k-mer repeat signatures of length three among various ncRNA classes. However, in pre-miRs and lncRNAs, a diffuse pattern of k-mers is observed. Using these attributes, we train eight different classifiers to discriminate various ncRNA classes in plants. Support vector machines employing radial basis function show the highest accuracy (average F1 of ~96%) in discriminating ncRNAs, and the classifier is implemented as a web server, NCodR.

Genome-wide prediction of cauliflower miRNAs and lncRNAs and their roles in post-transcriptional gene regulation.

MAIN CONCLUSION: We have predicted miRNAs, their targets and lncRNAs from the genome of Brassica oleracea along with their functional annotation. Selected miRNAs and their targets are experimentally validated. Roles of these non-coding RNAs in post-transcriptional gene regulation are also deciphered. Cauliflower (Brassica oleracea var. Botrytis) is an important vegetable crop for its dietary and medicinal values with rich source of vitamins, dietary fibers, flavonoids and antioxidants. MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs), which regulate gene expression by inhibiting translation or by degrading messenger RNAs (mRNAs). On the other hand, long non-coding RNAs (lncRNAs) are responsible for the up regulation and the down regulation of transcription. Although the genome of cauliflower is reported, yet the roles of these ncRNAs in post-transcriptional gene regulation (PTGR) remain elusive. In this study, we have computationally predicted 355 miRNAs, of which 280 miRNAs are novel compared to miRBase 22.1. All the predicted miRNAs belong to 121 different families. We have also identified 934 targets of 125 miRNAs along with their functional annotation. These targets are further classified into biological processes, molecular functions and cellular components. Moreover, we have predicted 634 lncRNAs, of which 61 are targeted by 30 novel miRNAs. Randomly chosen 10 miRNAs and 10 lncRNAs are experimentally validated. Five miRNA targets including squamosa promoter-binding-like protein 9, homeobox-leucine zipper protein HDG12-like, NAC domain-containing protein 100, CUP-SHAPED COTYLEDON 1 and kinesin-like protein NACK2 of four miRNAs including bol-miR156a, bol-miR162a, bol-miR164d and bol-miR2673 are also experimentally validated. We have built network models of interactions between miRNAs and their target mRNAs, as well as between miRNAs and lncRNAs. Our findings enhance the knowledge of non-coding genome of cauliflower and their roles in PTGR, and might play important roles in improving agronomic traits of this economically important crop.

Differential responses of Phaseolus vulgaris cultivars following mungbean yellow mosaic India virus infection.

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.

Molecular Cloning of an Amino Acid Permease Gene and Structural Characterization of the Protein in Common Bean (Phaseolus vulgaris L.).

Plants synthesize amino acids by collateral metabolic pathways using primary elements carbon and oxygen from air, hydrogen from water in soil and nitrogen from soil. Following synthesis, amino acids are immediately used for metabolism, transient storage or transported to the phloem. Different families of transporters have been identified for import of amino acids into plant cells. The first identified amino acid transporter, amino acid permease 1 (AAP1) in Arabidopsis belongs to a family of eight members and transports acidic, neutral, and basic amino acids. Legumes fix atmospheric nitrogen through a symbiotic relationship with root nodules bacteria. Following fixation, nitrogen is reduced to amino acids and is exported via different amino acid transporters. However, information is lacking about the structure of these important classes of amino acid transporter proteins in plant. We have amplified AAP from Phaseolus vulgaris, an economically important leguminous plant grown all over the world, and sequenced. The sequence has been characterized in silico and a three-dimensional structure of AAP has been predicted and validated. The information obtained not only enhances the knowledge about the structure of an amino acid permease gene in P. vulgaris, but will also help in designing protein-ligand studies using this protein as well.

Tiny Yet Indispensable Plant MicroRNAs Are Worth to Explore as Key Components for Combating Genotoxic Stresses.

Plants being sessile are always exposed to various stresses including biotic and abiotic stresses. Some of these stresses are genotoxic to cells causing DNA damage by forming lesions which include altered bases, cross-links, and breaking of DNA strands, which in turn hamper the genomic integrity. In order to survive through all these adverse conditions, plants have evolved different DNA repair mechanisms. As seen from the mammalian system and different human diseases, various microRNAs (miRNAs) can target the 3'-untranslated region of mRNAs that code for the proteins involved in DNA repair pathways. Since miRNAs play an important role in plant cells by regulating various metabolic pathways, it can also be possible that miRNAs play an important role in DNA repair pathways too. However, till date, only a handful of plant miRNAs have been identified to play important role in combating genotoxic stresses in plants. Limitation of information regarding involvement of miRNAs in DNA repair as well as in ROS scavenging prompted us to gather information about plant miRNAs specific for these tasks. This mini-review aims to present pertinent literature dealing with different genotoxic stresses that cause genome instability as well as plant specific responses to survive the damage. This is intertwined with the involvement of miRNAs in genotoxic stress in plants, challenges of applying miRNAs as a tool to combat DNA damage along with ways to overcome these challenges, and finally, the future prospective of these understudied aspects.

Identification and characterization of differentially expressed Phaseolus vulgaris miRNAs and their targets during mungbean yellow mosaic India virus infection reveals new insight into Phaseolus-MYMIV interaction.

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.

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.

Genome-wide identification of miRNAs and lncRNAs in Cajanus cajan.

BACKGROUND: Non-coding RNAs (ncRNAs) are important players in the post transcriptional regulation of gene expression (PTGR). On one hand, microRNAs (miRNAs) are an abundant class of small ncRNAs (~22nt long) that negatively regulate gene expression at the levels of messenger RNAs stability and translation inhibition, on the other hand, long ncRNAs (lncRNAs) are a large and diverse class of transcribed non-protein coding RNA molecules (> 200nt) that play both up-regulatory as well as down-regulatory roles at the transcriptional level. Cajanus cajan, a leguminosae pulse crop grown in tropical and subtropical areas of the world, is a source of high value protein to vegetarians or very poor populations globally. Hence, genome-wide identification of miRNAs and lncRNAs in C. cajan is extremely important to understand their role in PTGR with a possible implication to generate improve variety of crops. RESULTS: We have identified 616 mature miRNAs in C. cajan belonging to 118 families, of which 578 are novel and not reported in MirBase21. A total of 1373 target sequences were identified for 180 miRNAs. Of these, 298 targets were characterized at the protein level. Besides, we have also predicted 3919 lncRNAs. Additionally, we have identified 87 of the predicted lncRNAs to be targeted by 66 miRNAs. CONCLUSIONS: miRNA and lncRNAs in plants are known to control a variety of traits including yield, quality and stress tolerance. Owing to its agricultural importance and medicinal value, the identified miRNA, lncRNA and their targets in C. cajan may be useful for genome editing to improve better quality crop. A thorough understanding of ncRNA-based cellular regulatory networks will aid in the improvement of C. cajan agricultural traits.

Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting.

Non-coding RNAs (ncRNAs) have emerged as versatile master regulator of biological functions in recent years. MicroRNAs (miRNAs) are small endogenous ncRNAs of 18-24 nucleotides in length that originates from long self-complementary precursors. Besides their direct involvement in developmental processes, plant miRNAs play key roles in gene regulatory networks and varied biological processes. Alternatively, long ncRNAs (lncRNAs) are a large and diverse class of transcribed ncRNAs whose length exceed that of 200 nucleotides. Plant lncRNAs are transcribed by different RNA polymerases, showing diverse structural features. Plant lncRNAs also are important regulators of gene expression in diverse biological processes. There has been a breakthrough in the technology of genome editing, the CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) technology, in the last decade. CRISPR loci are transcribed into ncRNA and eventually form a functional complex with Cas9 and further guide the complex to cleave complementary invading DNA. The CRISPR-Cas technology has been successfully applied in model plants such as Arabidopsis and tobacco and important crops like wheat, maize, and rice. However, all these studies are focused on protein coding genes. Information about targeting non-coding genes is scarce. Hitherto, the CRISPR-Cas technology has been exclusively used in vertebrate systems to engineer miRNA/lncRNAs, but it is still relatively unexplored in plants. While briefing miRNAs, lncRNAs and applications of the CRISPR-Cas technology in human and animals, this review essentially elaborates several strategies to overcome the challenges of applying the CRISPR-Cas technology in editing ncRNAs in plants and the future perspective of this field.

Computational prediction of miRNAs and their targets in Phaseolus vulgaris using simple sequence repeat signatures.

BACKGROUND: MicroRNAs (miRNAs) are endogenous, noncoding, short RNAs directly involved in regulating gene expression at the post-transcriptional level. In spite of immense importance, limited information of P. vulgaris miRNAs and their expression patterns prompted us to identify new miRNAs in P. vulgaris by computational methods. Besides conventional approaches, we have used the simple sequence repeat (SSR) signatures as one of the prediction parameter. Moreover, for all other parameters including normalized Shannon entropy, normalized base pairing index and normalized base-pair distance, instead of taking a fixed cut-off value, we have used 99% probability range derived from the available data. RESULTS: We have identified 208 mature miRNAs in P. vulgaris belonging to 118 families, of which 201 are novel. 97 of the predicted miRNAs in P. vulgaris were validated with the sequencing data obtained from the small RNA sequencing of P. vulgaris. Randomly selected predicted miRNAs were also validated using qRT-PCR. A total of 1305 target sequences were identified for 130 predicted miRNAs. Using 80% sequence identity cut-off, proteins coded by 563 targets were identified. The computational method developed in this study was also validated by predicting 229 miRNAs of A. thaliana and 462 miRNAs of G. max, of which 213 for A. thaliana and 397 for G. max are existing in miRBase 20. CONCLUSIONS: There is no universal SSR that is conserved among all precursors of Viridiplantae, but conserved SSR exists within a miRNA family and is used as a signature in our prediction method. Prediction of known miRNAs of A. thaliana and G. max validates the accuracy of our method. Our findings will contribute to the present knowledge of miRNAs and their targets in P. vulgaris. This computational method can be applied to any species of Viridiplantae for the successful prediction of miRNAs and their targets.

Molecular modeling of protein-protein interaction to decipher the structural mechanism of nonhost resistance in rice.

Nonhost resistance (NHR) is the most common and durable form of plant resistance to disease-causing organisms. A successful example of NHR is the cloning of a maize R gene Rxo1 in rice and validating its function in conferring bacterial streak resistance in transgenic rice lines. In order to understand the structural mechanism of NHR in rice, we built the model of the protein-protein interaction between the encoded Rxo1 (RXO1) and AvrRXO1 (avirulence protein of rice pathogen, Xanthomonas oryzae pv. oryzicola). Interestingly, although a RXO1 homolog in rice (RHR) is present, it does not interact with AvrRXO1 in nature. We have confirmed that the specificity of RXO1-AvrRXO1 interaction originates from the structured leucine rich repeat (LRR) domain of RXO1, facilitating the recognition process, while the absence of such ordered LRR region makes RHR unfavorable to recognize AvrRXO1. We postulate that the RXO1-AvrRXO1 complex formation is a three step process where electrostatic interactions, shape complementarity and short-range interactions play an important role. The presence of the structural and physicochemical properties essential for the protein-protein recognition process empowers RXO1 to mediate NHR, which the host protein RHR lacks and consequently loses its specificity to bind with AvrRXO1. To the best of our knowledge, this is the first report on the understanding of NHR in rice from the structural perspective of protein-protein interaction.

The phylogeny and biogeographic history of ashes (fraxinus, oleaceae) highlight the roles of migration and vicariance in the diversification of temperate trees.

The cosmopolitan genus Fraxinus, which comprises about 40 species of temperate trees and shrubs occupying various habitats in the Northern Hemisphere, represents a useful model to study speciation in long-lived angiosperms. We used nuclear external transcribed spacers (nETS), phantastica gene sequences, and two chloroplast loci (trnH-psbA and rpl32-trnL) in combination with previously published and newly obtained nITS sequences to produce a time-calibrated multi-locus phylogeny of the genus. We then inferred the biogeographic history and evolution of floral morphology. An early dispersal event could be inferred from North America to Asia during the Oligocene, leading to the diversification of the section Melioides sensus lato. Another intercontinental dispersal originating from the Eurasian section of Fraxinus could be dated from the Miocene and resulted in the speciation of F. nigra in North America. In addition, vicariance was inferred to account for the distribution of the other Old World species (sections Sciadanthus, Fraxinus and Ornus). Geographic speciation likely involving dispersal and vicariance could also be inferred from the phylogenetic grouping of geographically close taxa. Molecular dating suggested that the initial divergence of the taxonomical sections occurred during the middle and late Eocene and Oligocene periods, whereas diversification within sections occurred mostly during the late Oligocene and Miocene, which is consistent with the climate warming and accompanying large distributional changes observed during these periods. These various results underline the importance of dispersal and vicariance in promoting geographic speciation and diversification in Fraxinus. Similarities in life history, reproductive and demographic attributes as well as geographical distribution patterns suggest that many other temperate trees should exhibit similar speciation patterns. On the other hand, the observed parallel evolution and reversions in floral morphology would imply a major influence of environmental pressure. The phylogeny obtained and its biogeographical implications should facilitate future studies on the evolution of complex adaptive characters, such as habitat preference, and their possible roles in promoting divergent evolution in trees.

Molecular marker-assisted genotyping of mungbean yellow mosaic India virus resistant germplasms of mungbean and urdbean.

Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus begomovirus causes the yellow mosaic disease in a number of economically important edible grain legumes including mungbean (Vigna radiata), urdbean (Vigna mungo) and soybean (Glycine max). The disease is severe, critical, open spread and inflicts heavy yield losses annually. The objective of this study is to develop molecular markers linked to MYMIV-resistance to facilitate genotyping of urdbean and mungbean germplasms for MYMIV-reaction. Resistance-linked molecular markers were successfully developed from consensus motifs of other resistance (R) gene or R gene homologue sequences. Applying linked marker-assisted genotyping, plant breeders can carry out repeated genotyping throughout the growing season in absence of any disease incidence. Two MYMIV-resistance marker loci, YR4 and CYR1, were identified and of these two CYR1 is completely linked with MYMIV-resistant germplasms and co-segregating with MYMIV-resistant F2, F3 progenies of urdbean. The present study demonstrated that these two markers could be efficiently employed together in a multiplex-PCR-reaction for genotyping both V. mungo and V. radiata germplasms from field grown plants and also directly from the seed stock. This method of genotyping would save time and labour during the introgression of MYMIV-resistance through molecular breeding, as methods of phenotyping against begomoviruses are tedious, labour and time intensive.

New motifs within the NB-ARC domain of R proteins: probable mechanisms of integration of geminiviral signatures within the host species of Fabaceae family and implications in conferring disease resistance.

The Gemini viruses are a group of plant infectious agents, of which mungbean yellow mosaic India virus (MYMIV) belongs to the bipartite subgroup of Gemini virus and causes serious yield penalty in the leguminous group of plants. In this investigation we have isolated two resistant gene homologues (RGHs; AY301990, AY301991) from two MYMIV-resistant lines of Vigna mungo and V. radiata that have high homology with a MYMIV-resistant linked marker, VMYR1 (AY 297425). These three resistance factors also have similarity with 221 reported R gene/RGH sequences in the NB-ARC domain of the family Fabaceae. NB-ARC domain is an ancient, highly conserved domain of a class of plant disease resistance genes/proteins. Out of 221 in silico translated protein sequences, multialignment of 188 sequences without large insertion or truncation, unlike that of the rest 33, illustrated presence of both TIR and non-TIR subfamilies of NB-ARC domain. A critical analysis of these sequences revealed eight new conserved motifs, in addition to the reported conserved motifs within the NB-ARC domains, which are hitherto not reported. Further analysis of these eight motifs with the aid of PRINTS and PROSITE databases revealed signatures of geminiviral coat protein (GVCP) within the favoured allele, R gene or RGHs. GVCP signatures are absent within the NB-ARC domain of three species of Medicago, which are non-host to Gemini virus. These observations tempted us to predict probable mechanism of integration of GVCP within the plant R gene/RGHs and their implications in conferring geminiviral disease resistance to the host plants. Our conjecture is that these signatures were integrated during plant pathogen interaction and are being maintained within this conserved domain through active selection of the favoured allele. We comprehensively addressed the biological significance of GVCP signatures, which probably provides additional defense against Gemini viruses through degradation of homologous transcript of the virus.

Theoretical model of the three-dimensional structure of a disease resistance gene homolog encoding resistance protein in Vigna mungo.

Plant disease resistance (R) genes, the key players of innate immunity system in plants encode 'R' proteins. 'R' protein recognizes product of avirulance gene from the pathogen and activate downstream signaling responses leading to disease resistance. No three dimensional (3D) structural information of any 'R' proteins is available as yet. We have reported a 'R' gene homolog, the 'VMYR1', encoding 'R' protein in Vigna mungo. Here, we describe the homology modeling of the 'VMYR1' protein. The model was created by using the 3D structure of an ATP-binding cassette transporter protein from Vibrio cholerae as a template. The strategy for homology modeling was based on the high structural conservation in the superfamily of P-loop containing nucleoside triphosphate hydrolase in which target and template proteins belong. This is the first report of theoretical model structure of any 'R' proteins.