Ranjeet R. Kumar

Diagnostic assays for two closely related tospovirus species, Watermelon bud necrosis virus and Groundnut bud necrosis virus and identification of new natural hosts

Watermelon bud necrosis virus (WBNV) and Groundnut bud necrosis virus (GBNV) are two closely related tospovirus species infecting several crops in India. In the present study, specific diagnostic assays for these two most predominant tospoviruses were developed by comparing host reactions and genome sequence information. Bioassay was developed by sap transmission of both the viruses to a set of host plants, cowpea, groundnut and watermelon. WBNV induced only local symptoms including chlorotic spots on cowpea cv. Pusa Komal, typical chlorotic ring spots on cv. C-152, bud necrosis on watermelon and no symptoms on groundnut. Whereas, GBNV induced both local and systemic symptoms including chlorotic/necrotic spots and veinal necrosis on both the cowpea cultivars, systemic yellowing and bud necrosis on groundnut and no symptoms on watermelon. Single and duplex RT-PCR was developed for diagnosis of WBNV and GBNV by designing primers based on differential sequence from Gn/Gc genes located on M RNA and from nucleocapsid protein (NP) gene located on S RNA genome. The duplex RT-PCR using NP gene specific primers was successfully utilised for the diagnosis of natural infection of GBNV in dahlia and muskmelon and mixed infection of GBNV and WBNV in chrysanthemum. The amplified products of duplex RT-PCR were sequenced and phylogenetic analysis confirmed the authenticity of the RT-PCR assay as well as confirmed infection of WBNV and GBNV in the new natural hosts. The bioassay and RT-PCR developed in this study will be useful in detecting single and mixed infection of GBNV and WBNV strains in different crops.

Mechanism of action of hydrogen peroxide in wheat thermotolerance - interaction between antioxidant isoenzymes, proline and cell membrane

Terminal heat stress causes an array of physiological, biochemical and morphological changes in plants, which affect plant growth and development. It has very severe effect on the pollen viability and seed setting in wheat. In the present investigation, an altered expression of H 2 O 2 (0.9 μg/g in C-306 and 0.75 μg/g in HD2329) was observed with the highest accumulation at seed hardening stage and against heat shock (HS) of 42°C for 2 h. With the increase in H 2 O 2 accumulation, an increase in the number of isoenzymes of superoxide dismutase and catalase were observed with high activities under differential heat shock. A decrease in the proline accumulation was observed under differential heat shock. Exogenous application of H 2 O 2 (10 mmole/L) leads to increase in the accumulation of intracellular H 2 O 2 and further an increase in the number of isoenzymes of superoxide dismutase (SOD) and catalase (CAT) was observed. The tolerant cultivar was more responsive to exogenous application of H 2 O 2 compared to susceptible cultivar. The percentage decrease in cell membrane stability under differential heat shock was low in H2O2 treated plants compared to non-treated. The results from this study suggest a potential role for H 2 O 2 in regulating the activity of antioxidant enzymes and accumulation of proline inside cells and in turn influence the cell membrane stability under heat stress. All the defense associated genes were observed to be very responsive to intracellular H 2 O 2 , which gives inference that H 2 O 2 has regulatory role to play in controlling the expression and activities of these proteins under abiotic stresses. Key words : Antioxidant enzymes, wheat, heat stress, H 2 O 2 , proline, catalase, superoxide dismutase, cell membrane stability, reactive oxygen species.

Identification of Putative RuBisCo Activase (TaRca1)—The Catalytic Chaperone Regulating Carbon Assimilatory Pathway in Wheat (Triticum aestivum) under the Heat Stress

RuBisCo activase (Rca) is a catalytic chaperone involved in modulating the activity of RuBisCo (key enzyme of photosynthetic pathway). Here, we identified eight novel transcripts from wheat through data mining predicted to be Rca and cloned a transcript of 1.4 kb from cv. HD2985, named as TaRca1 (GenBank acc. no. KC776912). Single copy number of TaRca1 was observed in wheat genome. Expression analysis in diverse wheat genotypes (HD2985, Halna, PBW621, and HD2329) showed very high relative expression of TaRca1 in Halna under control and HS-treated, as compared to other cultivars at different stages of growth. TaRca1 protein was predicted to be chloroplast-localized with numerous potential phosphorylation sites. Northern blot analysis showed maximum accumulation of TaRca1 transcript in thermotolerant cv. during mealy-ripe stage, as compared to thermosusceptible. Decrease in the photosynthetic parameters was observed in all the cultivars, except PBW621 in response to HS. We observed significant increase in the Rca activity in all the cultivars under HS at different stages of growth. HS causes decrease in the RuBisCo activity; maximum reduction was observed during pollination stage in thermosusceptible cvs. as validated through immunoblotting. We observed uniform carbon distribution in different tissues of thermotolerant cvs., as compared to thermosusceptible. Similarly, tolerance level of leaf was observed maximum in Halna having high Rca activity under HS. A positive correlation was observed between the transcript and activity of TaRca1 in HS-treated Halna. Similarly, TaRca1 enzyme showed positive correlation with the activity of RuBisCo. There is, however, need to manipulate the thermal stability of TaRca1 enzyme through protein engineering for sustaining the photosynthetic rate under HS—a novel approach toward development of “climate-smart” crop.

SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.

Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sangers sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat—a novel step toward the development of “climate-smart” wheat.

Molecular Mapping of Loci Affecting the Contents of Three Major Fatty Acids in Indian Mustard (Brassica juncea L)

The fatty acid constituents of mustard oil are palmitic, stearic, oleic, linoleic, linolenic and erucic acids. With the objective of mapping loci influencing the content of these fatty acids, a population of F6 generation recombinant inbred lines (RILs) derived from an inter-varietal cross of mustard was analyzed. Transgressive variation was evident for all the six fatty acids analysed irrespective of the levels of differences between the parents. The frequency distribution was normal for the linolenic acid, linoleic acid and stearic acid contents, while deviation from normality was observed for the other three fatty acids. The content of erucic acid was negatively correlated with the contents of all other fatty acids, which were positively correlated. Based on single marker analysis and interval mapping, two loci each for linoleic, linolenic and erucic acids were mapped to marker intervals on three linkage groups. Position of log of odds ratio (LOD) peaks suggested presence of common, linked and independently segregating loci for the fatty acid contents. The percentage of phenotypic variance explained by individual quantitative trait loci (QTLs) ranged from 10.5 to 19.5%, whereas the cumulative action of loci detected for different traits accounted for 16.3 to 27.6% of the variance. The additive effect for an individual locus ranged from 1.09 to 4.33. Presence of the favourable alleles at both the contributing loci in most of the RILs with a high linolenic acid content and of the unfavourable alleles in the lines with a low linolenic acid content indicated the possibility of pyramiding useful genes from phenotypically similar parental lines.

Understanding the Diversity of Aspergillus by Next-Generation Sequencing

Abstract Aspergillus sp. has great importance in the present scenario in human and plants by producing immunosuppressive secondary metabolites, aflatoxins, carcinogenic, and teratogenic. The diversity of Aspergillus in the present scenario remains poorly understood. The assessment of true biodiversity of Aspergillus and other fungi in nature is essential for the understanding of their biology. There are several methods available for the detection of biodiversity, such as morphological character, biochemical molecules, and molecular molecules. Recent technological advancements have allowed significant progress in documenting fungal diversity. The use of DNA-based techniques, mainly DNA sequencing, for detecting fungi in the environment revolutionized our view of how and where fungi live. The most widely used genomic region for delimiting fungal species is the internal transcribed spacer (ITS), a short DNA segment with enough intraspecific variation that is suitable for diagnosing different species. Because of the high speed, low cost, and significant technical advantages, next-generation sequencing techniques (NGSTs) have been extensively used in fungal diversity in the past decade. Next-generation sequencing (NGS) has enormous potential to further our understanding of microbial biodiversity. In addition, NGSTs eliminate the need for cloning DNA, thus reducing the bias often associated with this step. There are some initial improvements in the Aspergillus diversity assessments by NGSTs, which speedup the sequencing and also save time.

Isolation and Characterization of Plastidial Localized, Seed Expressive Lysophosphatidyl Acyl Transferase (LPAAT) cDNA from Brassica juncea L

In order to characterize different forms of Lysophosphatidyl acyl transferase (LPAAT) gene and to study its cellular location, PCR amplification of cDNA isolated from B. juncea was carried out using LPAAT gene specific primers. The amplicon obtained was of 1009 by and showed homology with BAT2 clone of B. napus. A multiple sequence analysis of LPAAT derived from different sources revealed that there were ten strongly conserved domains among different isoforms of LPAAT products with two most prominent conserved domain PEGTRS and NH. Southern blot analysis showed that there were two different isoforms of LPAAT gene in B. juncea. Northern blot analysis showed that maximum expression of the transcript was in case of seeds and low level in buds. However, the transcript was not detectable in leaves.

Differentiation of the American and Oriental Maydeae Accessions Using the Maize Simple Sequence Repeat (SSR) Markers

The present study evaluated the utility of maize microsatellite markers in genotype differentiation and analysis of genetic diversity among the Oriental Maydeae accessions (Coix aquatica and Chionachne koenigii) vis-à-vis the American Maydeae (maize, teosintes and maize-teosinte derivatives). Analysis of 27 microsatellite loci distributed throughout the maize genome led to the detection of 89 SSR alleles at average of 3.30 per locus. Polymorphism information content (PIC) values and observed genetic distances showed the existence of large variability among the various accessions. The maize SSR markers, particularly nc013, phi059, bnlg1523 and umc1014, were highly effective in differentiating the Oriental Maydeae accessions from the American Maydeae. Cluster analysis and Principal Component Analysis based on the SSR data clearly indicated significant genetic divergence between the Oriental Maydeae and the American Maydeae accessions, as well as between Coix aquatica and Chionachne koenigii. Molecular analyses would be valuable in gaining a better understanding of genera belonging to the Oriental Maydeae, the lesser known wild relatives of maize.

Characterization of novel heat-responsive transcription factor (TaHSFA6e) gene involved in regulation of heat shock proteins (HSPs) — A key member of heat stress-tolerance network of wheat

Heat stress has an adverse effect on the quality and quantity of agriculturally important crops, especially wheat. The tolerance mechanism has not been explored much in wheat and very few genes/ TFs responsive to heat stress is available on public domain. Here, we identified, cloned and characterized a putative TaHSFA6e TF gene of 1.3 kb from wheat cv. HD2985. We observed an ORF of 368 aa with Hsf DNA binding signature domain in the amino acid sequence. Single copy number of TaHSFA6e was observed integrated in the genome of wheat. Expression analysis of TaHSFA6e under differential HS showed maximum transcripts in wheat cv. Halna (thermotolerant) in response to 38 °C for 2 h during pollination and grain-filling stages, as compared to PBW343, HD2329 and HD2985. Putative target genes of TaHSFA6e (HSP17, HSP70 and HSP90) showed upregulation in response to differential HS (30 & 38 °C, 2 h) during pollination and grain-filling stages. Small HSP17 was observed most triggered in Halna under HS. We observed increase in the catalase, guaiacol peroxidase, total antioxidant capacity (TAC), and decrease in the lipid peroxidation in thermotolerant cvs. (Halna, HD2985), as compared to thermosusceptible (PBW343, HD2329) under differential HS. Multiple stresses (heat - 38 °C, 2 h, and drought - 100 mL of 20% polyethylene Glycol 6000) during seedling stage of wheat showed positive correlation between the expression of TaHSFA6e, putative targets (HSP70, HSP90, HSP17) and TAC. Halna (thermotolerant) performed better, as compared to other contrasting cvs. TaHSFA6e TF can be used as promising candidate gene for manipulating the heat stress-tolerance network.

Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity

Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H2O2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H2O2-responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSsS189R) and the other in a non-Trp/GH3 motif (NSsL172R). Tobacco rattle virus (TRV) expressing NSsS189R enhanced the PCD response, but not TRV-NSsL172R, while RNA silencing suppression activity was lost in TRV-NSsL172R, but not in TRV-NSsS189R. Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs.