Whole genome sequencing of a novel Bacillus thuringiensis isolated from Assam soil.

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.

Bruchid beetle ovipositioning mediated defense responses in black gram pods.

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.

Multilocus sequence typing for phylogenetic view and vip gene diversity of Bacillus thuringiensis strains of the Assam soil of North East India.

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes' crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.

Activity of Arabidopsis Rubisco small subunit promoter in various tissues of chickpea.

The activity of a green tissue-specific promoter of the Rubisco small subunit gene from Arabidopsis (AraSSU) was studied using transgenic chickpea lines. We generated transgenic chickpea lines expressing an AraSSU promoter-driven cry2Aa gene through the Agrobacterium-mediated transformation method. Lines with AraSSU expressed the gene in all green tissues at high levels (> 90 ng/mg of fresh weight tissue) compared to lines generated using CaMV35S (< 10 ng/mg FW). We used vertical cross sections of various tissues of homozygous progeny using microtome for immunolocalization. The immunolocalization showed the expression of the cry2Aa gene in the green mesophyll cells of the leaves of both AraSSU and CaMV35 chickpea lines. Moreover, the accumulation of AraSSU-regulated Cry2Aa protein was also observed in vascular tissues, including enucleate sieve elements and their companion cells. However, no expression was observed in the roots of AraSSU lines. In the case of CaMV35 lines, the transgene expression was observed in all the tissues. Since our data indicated that the AraSSU promoter is active in non-green tissues such as vascular bundles. Therefore, we validated this by RT-PCR. We found Cry2Aa RNA transcripts in leaves, stems without epidermis (for vascular tissues), and roots with and without epidermis. Thus, the AraSSU promoter is active in all above-ground tissues of the chickpea plant.

Comparative transcriptome profiling reveals differential defense responses among Alternaria brassicicola resistant Sinapis alba and susceptible Brassica rapa.

Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba, a close relative of Brassicas, and the highly susceptible Brassica rapa. The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa. Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola, which will aid in devising strategies for breeding resistant varieties of oilseed Brassica.

Deciphering the antimicrobial activity of multifaceted rhizospheric biocontrol agents of solanaceous crops viz., Trichoderma harzianum MC2, and Trichoderma harzianum NBG.

The Solanaceae family is generally known to be the third most economically important plant taxon, but also harbors a host of plant pathogens. Diseases like wilt and fruit rot of solanaceous crops cause huge yield losses in the field as well as in storage. In the present study, eight isolates of Trichoderma spp. were obtained from rhizospheric micro-flora of three solanaceous crops: tomato, brinjal, and chili plants, and were subsequently screened for pre-eminent biocontrol activity against three fungal (Fusarium oxysporum f. sp. lycopersicum, Colletotrichum gloeosporioides, and Rhizoctonia solani) and one bacterial (Ralstonia solanacearum) pathogen. Morphological, ITS, and tef1α marker-based molecular identification revealed eight isolates were different strains of Trichoderma. Seven isolates were distinguished as T. harzianum while one was identified as T. asperellum. In vitro antagonistic and biochemical assays indicated significant biocontrol activity governed by all eight isolates. Two fungal isolates, T. harzianum MC2 and T. harzianum NBG were further evaluated to decipher their best biological control activity. Preliminary insights into the secondary metabolic profile of both isolates were retrieved by liquid chromatography-mass spectrometry (LC-MS). Further, a field experiment was conducted with the isolates T. harzianum MC2 and T. harzianum NBG which successfully resulted in suppression of bacterial wilt disease in tomato. Which possibly confer biocontrol properties to the identified isolates. The efficacy of these two strains in suppressing bacterial wilt and promoting plant growth in the tomato crop was also tested in the field. The disease incidence was significantly reduced by 47.50% and yield incremented by 54.49% in plants treated in combination with both the bioagents. The results of scanning electron microscopy were also in consensus with the in planta results. The results altogether prove that T. harzianum MC2 and T. harzianum NBG are promising microbes for their prospective use in agricultural biopesticide formulations.

Rhizospheric Bacillus spp. Exhibit Miticidal Efficacy against Oligonychus coffeae (Acari: Tetranychidae) of Tea.

Oligonychus coffeae (Acari: Tetranychidae), popularly known as red spider mite (RSM) is one of the major pests of commercial tea (Camellia sinensis (L.) O. Kuntze) plantation world over. Many attempts have been made in the past to control this devastating pest using a variety of microbial bioagents, however, area-wise field success is very limited. We carried out an in vitro study to explore the potential of rhizospheric Bacillus spp. (B. amyloliquefaciens BAC1, B. subtilis LB22, and B. velezensis AB22) against O. coffeae through adulticidal and ovicidal activity. The 100% adult and egg mortality was observed with bacterial suspension (1 × 109 CFU/mL) by B. velezensis AB22, showing the lowest LC50 values for both adults and eggs of O. coffeae, i.e., 0.28 × 105 and 0.29 × 105, respectively. The study also throws some insights into the underlying mechanism through electron microscopy study and identification of some putative pesticidal metabolites from all the species. The three Bacillus species were observed to have four commonly secreted putative bioactive secondary metabolites, brevianamide A, heptadecanoic acid, thiolutin, and versimide responsible for their bio-efficacy against O. coffeae. The outcome of our study provides a strong possibility of introducing Bacillus spp. as a biological miticide and developing synthetic metabolites mimicking the mechanistic pathway involved in microbial bioefficacy.

Introgressing cry1Ac for Pod Borer Resistance in Chickpea Through Marker-Assisted Backcross Breeding.

The gram pod borer Helicoverpa armigera is a major constraint to chickpea (Cicer arietinum L.) production worldwide, reducing crop yield by up to 90%. The constraint is difficult to overcome as chickpea germplasm including wild species either lacks pod borer resistance or if possessing resistance is cross-incompatible. This study describes conversion of elite but pod borer-susceptible commercial chickpea cultivars into resistant cultivars through introgression of cry1Ac using marker-assisted backcross breeding. The chickpea cultivars (PBG7 and L552) were crossed with pod borer-resistant transgenic lines (BS 100B and BS 100E) carrying cry1Ac that led to the development of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2, and BC2F3 populations from three cross combinations. The foreground selection revealed that 35.38% BC1F1 and 8.4% BC1F2 plants obtained from Cross A (PBG7 × BS 100B), 50% BC1F1 and 76.5% BC1F2 plants from Cross B (L552 × BS 100E), and 12.05% BC2F2 and 82.81% (average) BC2F3 plants derived from Cross C (PBG7 × BS 100E) carried the cry1Ac gene. The bioassay of backcross populations for toxicity to H. armigera displayed up to 100% larval mortality. BC1F1 and BC1F2 populations derived from Cross B and BC2F3 population from Cross C segregated in the Mendelian ratio for cry1Ac confirmed inheritance of a single copy of transgene, whereas BC1F1 and BC1F2 populations obtained from Cross A and BC2F2 population from Cross C exhibited distorted segregation ratios. BC1F1 plants of Cross A and Cross B accumulated Cry1Ac protein ranging from 11.03 to 11.71 µgg-1 in leaf tissue. Cry1Ac-positive BC2F2 plants from Cross C demonstrated high recurrent parent genome recovery (91.3%) through background selection using SSR markers and phenome recovery of 90.94%, amongst these 30% plants, were homozygous for transgene. The performance of BC2F3 progenies derived from homozygous plants was similar to that of the recurrent parent for main agronomic traits, such as number of pods and seed yield per plant. These progenies are a valuable source for H. armigera resistance in chickpea breeding programs.

Compositional analysis of transgenic Bt-chickpea resistant to Helicoverpa armigera.

Transgenic chickpeas expressing high levels of a truncated version of the cry1Ac (trcry1Ac) gene conferred complete protection to Helicoverpa armigera in the greenhouse. Homozygous progeny of two lines, Cry1Ac.1 and Cry1Ac.2, had similar growth pattern and other morphological characteristics, including seed yield, compared to the non-transgenic counterpart; therefore, seed compositional analysis was carried out. These selected homozygous chickpea lines were selfed for ten generations along with the non-transgenic parent under contained conditions. A comparative seed composition assessment, seed storage proteins profiling, and in vitro protein digestibility were performed to confirm that these lines do not have significant alterations in seed composition compared to the parent. Our analyses showed no significant difference in primary nutritional composition between transgenic and non-transgenic chickpeas. In addition, the seed storage protein profile also showed no variation between the transgenic chickpea lines. Seed protein digestibility assays using simulated gastric fluid revealed a similar rate of digestion of proteins from the transgenic trcry1Ac lines compared to the non-transgenic line. Thus, our data suggest no unintended changes in the seed composition of transgenic chickpea expressing a trcry1Ac gene.

Defense Response in Chickpea Pod Wall due to Simulated Herbivory Unfolds Differential Proteome Profile.

The pod wall of legumes is known to protect the developing seeds from pests and pathogens. However, the mechanism of conferring defense against insects has not yet been deciphered. Here, we have utilized 2-dimensional gel electrophoresis (2D-GE) coupled with mass spectrometry (MS/MS) to identify over expressed proteins in the pod wall of two different cultivars (commercial cultivar: JG 11 and tolerant cultivar: ICC 506-EB) of chickpea after 12 h of application of Helicoverpa armigera oral secretions (simulated herbivory). The assays were performed with a view that larvae are a voracious feeder and cause substantial damage to the pod within 12 h. A total of 600 reproducible protein spots were detected on gels, and the comparative analysis helped identify 35 (12 up-regulated, 23 down-regulated) and 20 (10 up-regulated, 10 down-regulated) differentially expressed proteins in JG 11 and ICC 506-EB, respectively. Functional classification of protein spots of each cultivar after MS/MS indicated that the differentially expressed proteins were associated with various metabolic activities. Also, stress-related proteins such as mannitol dehydrogenase (MADH), disease resistance-like protein-CSA1, serine/threonine kinase (D6PKL2), endoglucanase-19 etc. were up-regulated due to simulated herbivory. The proteins identified with a possible role in defense were further analyzed using the STRING database to advance our knowledge on their interacting partners. It decoded the involvement of several reactive oxygen species (ROS) scavengers and other proteins involved in cell wall reinforcement. The biochemical analysis also confirmed the active role of ROS scavengers during simulated herbivory. Thus, our study provides valuable new insights on chickpea-H.armigera interactions at the protein level.

Ethylene mediates repression of anthocyanin accumulation in black rice pericarps in the absence of light.

The antioxidant properties of black rice are attributed to the high anthocyanin content in the pericarp. Light-dependent regulation of anthocyanin biosynthesis and the associated regulatory genes have been extensively studied in many plant species, including rice. Light is considered indispensable for anthocyanin accumulation in plants. Here, we report that anthocyanin biosynthesis and accumulation in the dark is negatively regulated by ethylene, as the ethylene biosynthesis inhibitor aminoethoxyvinylglycine hydrochloride (AVG)-treated samples in the dark exhibited significantly higher transcript levels of biosynthesis genes, including CHI, DFR, ANS, ANR, F3H, F'3H, CHS and UGFT, compared to untreated controls. Upregulation of these biosynthesis genes was accompanied by simultaneous de-repression of the R2-R3 domain and bHLH-containing transcription factors Kala3 and Kala4 at the transcript level. Additionally, bHLH152, which shows high similarity to Arabidopsis PIF3, is involved in the regulation of anthocyanin. These findings highlight the role of ethylene in modulating tissue-specific regulation of anthocyanin biosynthesis genes in the dark.

Reference gene validation for normalization of RT-qPCR assay associated with germination and survival of rice under hypoxic condition.

Study on expression of genes for the traits associated with hypoxia tolerance during the germination demands robust choice of reference genes for transcript data normalization and gene validation through real-time quantitative polymerase chain reaction (RT-qPCR). However, reliability and stability of reference genes across different rice germplasms under hypoxic condition have not been accessed yet. Stability performance of reference genes such as eukaryotic elongation factor 1 α (eEF1α), ubiquitin 10 (UBQ10), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18SrRNA), 25S ribosomal RNA (25SrRNA), ß-tublin (ß-TUB), actin11 (ACT11), ubiquitin C (UBC), eukaryotic elongation factor 4 α (eIF4α), and ubiquitin5 (UBQ5) was accessed through statistical algorithms like geNorm, NormFinder, Comparative ΔCt method, BestKeeper, and RefFinder in three rice germplasms (KHO, RKB, and IR-64) with varied level of tolerance to hypoxic condition during germination. Among all genes used, OsGAPDH was found to be the most suitable reference gene under hypoxic condition. The performance of the highest-ranking reference gene (OsGAPDH) in terms of stability based on statistical algorithms was further validated for its reliability and stability through RT-qPCR with hypoxia-induced target gene OsTTP7. The identified stable housekeeping gene could be used as internal control for gene expression analysis in rice under hypoxia.

Isolation and characterization of Bacillus thuringiensis strains native to Assam soil of North East India.

We have identified both crystalliferous and acrystalliferous Bt isolates from the Assam soil of North East India for the first time. A total of 301 Bacillus type colonies were selected based on their appearance and colony morphology. Out of these colonies, 42 isolates had characteristics similar to Bt isolates on MYP (Mannitol Egg Yolk Polymyxin) agar base medium. The ERIC-PCR and 16S rDNA analyses confirmed that 42 isolates are Bacillus thuringiensis. Phase contrast microscopy showed that 37 isolates produced crystal endospore during the sporulation phase and 5 acrystalliferous isolates were also found. Amplification of cry gene was carried out using general Cry primers along with one cry2 gene specific primer. Out of 42 isolates, 50% of the isolates showed presence of cry2 gene followed by cry9 (40.47) and cry1 (40.47). Moreover, 21.42% of isolates showed the presence of more than one cry genes. We also screened these isolates for the possibility of having new Bt genes using universal primer and found two strains having a new type of Cry1I gene with 82 and 85% similarities with the available Cry1I gene sequences. Thus, these new types of Bt gene could be useful for Bt-based bioformulations and generation of transgenic plants.

Bruchid egg induced transcript dynamics in developing seeds of black gram (Vigna mungo).

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.

Biotechnologically generating 'super chickpea' for food and nutritional security.

Chickpea productivity is affected by various constraints that are biotic (Helicoverpa, Aphids, Callosobruchus, Bromus and Orobanche) and abiotic (drought and salinity). In addition, the grains of this legume are deficient in sulfur amino acids, methionine and cysteine. The possibilities for genetic improvement by marker-assisted breeding and selection approaches are limited in chickpeas due to their sexually incompatible gene pool. Transgenic chickpeas expressing either the cry1Ac/b or the cry2Aa gene and the bean α-amylase inhibitor gene are resistant to Helicoverpa and bruchids, respectively, but these chickpeas have yet to be commercialized. Unfortunately, attempts to generate transgenic chickpeas with increased tolerance to drought and salinity or with increased methionine content have been less successful. The commercialization of transgenic chickpeas containing a single transgene may not give adequate yield advantage, as chickpeas are affected by many production constraints in the field and in storage. Gene pyramiding by incorporating two or more genes may be useful because improving one trait at a time will be time-consuming, labor-intensive and costly. Use of modern multi-gene vectors that contain recognition sites for zinc finger nucleases (ZFNs) and homing endonucleases may simplify the incorporation of multiple genes into chickpeas. This approach necessitates a collaborative effort between individuals, public and private organizations to generate 'super chickpeas' that harbor multiple transgenic traits.