Background: Bacillus thuringiensis (Bt) is a gram-positive ubiquitous saprophytic bacterium that produces proteins (Crystal protein, Vegetative insecticidal protein, and Secreted insecticidal protein) toxic to insects during its growth cycle. In the present study, the whole genome of a locally isolated B. thuringiensis strain BA04 was sequenced to explore the genetic makeup and to identify the genes responsible to produce insecticidal proteins including the virulence factors. The strain was isolated from the soil sample of the Kaziranga National Park, Assam, North-Eastern part of India (Latitude: 26°34′39.11''N and Longitude: 93°10′16.04''E). Results: The whole genome sequencing (WGS) of the BA04 strain revealed that it has a circular genome of size 6,113,005 bp with four numbers of plasmids. A total of 6,111 genes including two novel crystal protein-encoding genes (MH753362.1 and MH753363.1) were identified. The BLASTn analysis of MH753362.1 showed 84% similarities (maximum identity) with Cry1Ia (KJ710646.1) gene, whereas MH753363.1 exhibited 66% identity with Insecticidal Crystal Protein (ICP)-6 gene (KM053257.1). At the protein level, MH753362.1 and MH753363.1 shared 79% identity with Cry1Ia (AIW52613.1) and 40% identity with Insecticidal Crystal Protein (ICP)-6 (AJW76687.1) respectively. Three-dimensional structures of these two novel protein sequences revealed that MH753362.1 have 48% structural similarity with Cry8ea1 protein, whereas MH753363.1 showed only 20% structural similarity with Cry4Aa protein. Apart from these insecticidal genes, the strain was also found to contain virulence and virulence-associated factors including the antibiotic resistance genes and Clustered regularly interspaced short palindromic repeat (CRISPR) sequences. Conclusion: This is the first report on the whole genome sequence of Bt strain BA04 isolated from Assam, a North-Eastern state of India. The WGS of strain BA04 unveils the presence of two novel types of insecticidal crystal protein-encoding genes which can be used for the development of insect-resistant transgenic crops. Additionally, the strain could be used for the formulations of effective biopesticides. The WGS provides the fastest and cheapest platform for a better understanding of the genetic makeup of a strain and helps to explore the role of virulence genes in pathogenicity against the insect host.

Background: Black gram [Vigna mungo (L)] seeds are a rich source of digestible protein and dietary fibre, both for human and animal consumption. However, the quality and quantity of the Vigna seeds are severely affected by bruchid beetles during storage. Therefore, analyses of the expression of the bruchid induced transcript dynamics in black gram pods would be helpful to understand the underlying defense mechanism against bruchid oviposition. Results: We used the RNAseq approach to survey the changes in transcript profile in the developing seeds of a moderately resistant cultivar IC-8219 against bruchid oviposition using a susceptible cultivar T-9 as a control. A total of 96,084,600 and 99,532,488 clean reads were generated from eight (4 each) samples of IC-8219 and T-9 cultivar, respectively. Based on the BLASTX search against the NR database, 32,584 CDSs were generated of which 31,817 CDSs were significantly similar to Vigna radiata, a close relative of Vigna mungo. The IC-8219 cultivar had 630 significantly differentially expressed genes (DEGs) of which 304 and 326 genes up and down-regulated, respectively. However, in the T-9 cultivar, only 168 DEGs were identified of which 142 and 26 genes up and down-regulated, respectively. The expression analyses of 10 DEGs by qPCR confirmed the accuracy of the RNA-Seq data. Gene Ontology and KEGG pathway analyses helped us to better understand the role of these DEGs in oviposition mediated defense response of black gram. In both the cultivars, the most significant transcriptomic changes in response to the oviposition were related to the induction of defense response genes, transcription factors, secondary metabolites, enzyme inhibitors, and signal transduction pathways. It appears that the bruchid ovipositioning mediated defense response in black gram is induced by SA signaling pathways and defense genes such as defensin, genes for secondary metabolites, and enzyme inhibitors could be potential candidates for resistance to bruchids. Conclusion: We generated a transcript profile of immature black gram pods upon bruchid ovipositioning by de novo assembly and studied the underlying defense mechanism of a moderately resistant cultivar.

Black gram (Vigna mungo) seeds are a rich source of digestible proteins, however, during storage these seeds are severely damaged by bruchids (Callosobruchus spp.), reducing seed quality and yield losses. Most of the cultivated genotypes of black gram are susceptible to bruchids, however, few tolerant genotypes have also been identified but the mechanism of tolerance is poorly understood. We employed Suppression Subtractive Hybridization (SSH) to identify specifically, but rarely expressed bruchid egg induced genes in black gram. In this study, Suppression Subtractive Hybridization (SSH) library was constructed to study the genes involved in defense response in black gram against bruchid infestation. An EST library of 277 clones was obtained for further analyses. Based on CAP3 assembly, 134 unigenes were computationally annotated using Blast2GOPRO software. In all, 20 defense related genes were subject to quantitative PCR analysis (qPCR) out of which 12 genes showed up-regulation in developing seeds of the pods oviposited by bruchids. Few major defense genes like defensin, pathogenesis related protein (PR), lipoxygenase (LOX) showed high expression levels in the oviposited population when compared with the non-oviposited plants. This is the first report on defense related gene transcript dynamics during the bruchid-black gram interaction using SSH library. This library would be useful to clone defense related gene(s) such as defensin as represented in our library for crop improvement.