Browsing by Author "Verma, Praveen K."

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An alternative approach in Gateway ® cloning when the bacterial antibiotic selection cassettes of the entry clone and destination vector are the same
(Springer, 2013) Kumar, Kamal; Yadav, Saurabh; Purayannur, Savithri; Verma, Praveen K.
The Gateway(®) recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.
Analysis of a salinity induced BjSOS3 protein from Brassica indicate it to be structurally and functionally related to its ortholog from Arabidopsis
(Elsevier, 2011) Kushwaha, Hemant R.; Kumar, Gautam; Verma, Praveen K.; Singla-Pareek, Sneh L.; Pareek, Ashwani
Arabidopsis has been a favorite model system for plant biologist. It is anticipated that comparative analysis of this plant with other members of Brassicaceae may aid in identification of orthologs playing role as key genetic determinants for salinity response. In this endeavor, we have recently identified SOS family members from Brassica juncea in our laboratory and reported their salinity responsive transcriptional induction in seedlings of various diploid and amphidiploids species. In the present study, we have carried out detailed time kinetics for BjSOS3 expression in a salinity tolerant B. juncea var. CS52. Transcript analysis at the sensitive growth stages of plants viz. seedling and reproductive stage indicated clear differential transcriptional regulation of BjSOS3 under non-induced as well as salinity induced conditions in a time and organ specific manner, mirroring their respective tolerance physiology. Similar to its ortholog from Arabidopsis thaliana, the modeled BjSOS3 protein show typical features of a Ca(2+) binding protein with four conserved EF-hands. We have also attempted to study the binding of SOS3 protein with the modeled SOS2 protein. It has been established that SOS3 protein senses Ca(2+) though the binding is very weak; we show the down regulation of BjSOS3 mRNA in presence of calcium chelator - EGTA under the various stress conditions including ABA. In situ localization of BjSOS3-GFP fusion protein in onion peel has shown its presence strongly in plasma membrane as well as cytosol. The leads presented in the paper will assist in understanding and establishing the SOS signaling machinery in B. juncea.
Ascochyta rabiei: A threat to global chickpea production
(John Wiley & Sons, 2022) Singh, Ritu; Kumar, Kamal; Purayannur, Savithri; Chen, Weidong; Verma, Praveen K.
The necrotrophic fungus Ascochyta rabiei causes Ascochyta blight (AB) disease in chickpea. A. rabiei infects all aerial parts of the plant, which results in severe yield loss. At present, AB disease occurs in most chickpea-growing countries. Globally increased incidences of A. rabiei infection and the emergence of new aggressive isolates directed the interest of researchers toward understanding the evolution of pathogenic determinants in this fungus. In this review, we summarize the molecular and genetic studies of the pathogen along with approaches that are helping in combating the disease. Possible areas of future research are also suggested.
BAR domain is essential for early endosomal trafficking and dynamics in Ascochyta rabiei
(Springer Nature Publishing AG, 2023) Shree, Ankita; Sinha, Manisha; Verma, Praveen K.
Ascochyta blight disease is a devastating disease caused by the fungal pathogen Ascochyta rabiei that threatens chickpea production around the globe. Endocytic mechanism has a significant role in fungal growth and virulence. The underlying biology of biogenesis of central component of endocytosis viz Rab5 vesicles, is not completely understood. The involvement of F-BAR domain containing protein (ArF-BAR) in various cellular processes that collectively make ArF-BAR as an important virulence determinant. Here, we report that ArF-BAR is involved in biogenesis and motility of early endosome. In the absence of ArF-BAR gene (Δarf-bar), fungal mutants exhibited reduced number of EGFP coated ArRab5 vesicles, along with the considerable reduction in their dynamics. Here, we show that ArF-BAR interacts with clathrin light chain (ArCLC), specifically with its F-BAR domain. These findings suggests the novel role of ArF-BAR in biogenesis and dynamics of early endosome. Additionally, ArF-BAR is involved in clathrin-mediated mechanism of endocytosis which is required for host infection and disease development. Identification of this pathway offers new impending targets for disease intervention in plants.
Biotechnological interventions for production of therapeutic secondary metabolites using hairy root cultures of medicinal plants
(Elsevier B.V., 2016) Srivastava, V.; Mehrotra, S.; Verma, Praveen K.
The repository of molecules (secondary metabolites) in plants have been identified as an effective candidate to cure human diseases. This immense potential is continuously being utilized by exploiting natural plant resources. Such practices led to environmental imbalances and many natural sites were deserted. This chapter provides current perspectives on plant hairy root cultures as an important model for sustainable therapeutic secondary metabolite production. Here, the imperative interventions of biotechnology to enhance the accumulation of such molecules are discussed. Current research trends for metabolite enhancement through media optimization, elicitation, precursor feeding, metabolic engineering, and upscaling are taken into consideration.
Biotic and abiotic stress signaling in plants
(Springer, 2013) Verma, Sandhya; Nizam, Shadab; Verma, Praveen K.
In the environment, plants are constantly being exposed to a number of adverse conditions. Being immobile and deprived of highly specialized immune system, they have developed intricate mechanisms to adapt and survive under various types of abiotic and biotic stresses. On the perception of certain stimuli various signaling cascades are stimulated generating appropriate responses. This result in massive transcriptional reprogramming that makes the plant tolerant against the stress. Recent advances in the field of genomics and proteomics approach have widened our view regarding plant signal transduction and gene regulation. The cDNA and GeneChip microarrays are tremendously helpful in identifying novel signaling determinants on genome wide scale. Therefore, the role of various genes can be elucidated in response to the stress conditions. Post-translational modifications of the proteins can be well understood by proteomics analyses. Several recent studies using these techniques have added to our understanding of stress signaling mechanisms in plants. In this chapter, we have emphasized on the contribution made by genomics and proteomics approaches to gain further insights in understanding the molecular mechanisms of plants responses against stress.
Bipartite molecular approach for species delimitation and resolving cryptic speciation of Exobasidium vexans within the Exobasidium genus
(Elsevier B.V., 2021) Chaliha, Chayanika; Kaladhar, V. Chandra; Doley, Robin; Verma, Praveen K.; Kumar, Aditya; Kalita, Eeshan
Exobasidium vexans, a basidiomycete pathogen, is the causal organism of blister blight disease in tea. The molecular identification of the pathogen remains a challenge due to the limited availability of genomic data in sequence repositories and cryptic speciation within its genus Exobasidium. In this study, the nuclear internal transcribed spacer rDNA region (ITS) based DNA barcode was developed for E. vexans, to address the problem of molecular identification within the background of cryptic speciation. The isolation of E. vexans strain was confirmed through morphological studies followed by molecular identification utilizing the developed ITS barcode. Phylogenetic analysis based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) confirmed the molecular identification of the pathogen as E. vexans strain. Further, BI analysis using BEAST mediated the estimation of the divergence time and evolutionary relationship of E. vexans within genus Exobasidium. The speciation process followed the Yule diversification model wherein the genus Exobasidium is approximated to have diverged in the Paleozoic era. The study thus sheds light on the molecular barcode-based species delimitation and evolutionary relationship of E. vexans within its genus Exobasidium.
Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228
(BioMed Central Ltd, 2021) Randhawa, Anmoldeep; Pasari, Nandita; Sinha, Tulika; Gupta, Mayank; Nair, Anju M.; Ogunyewo, Olusola A.; Verma, Sandhya; Verma, Praveen K.; Yazdani, Syed Shams
Background: Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results: Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions: In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.
Broadening the horizon of crop research: a decade of advancements in plant molecular genetics to divulge phenotype governing genes
(Springer Nature Publishing AG, 2022) Singh, Ritu; Kumar, Kamal; Bharadwaj, Chellapilla; Verma, Praveen K.
The modern crop improvement programs rely heavily on two major steps—trait-associated QTL/gene/marker’s identification and molecular breeding. Thus, it is vital for basic and translational crop research to identify genomic regions that govern the phenotype of interest. Until the advent of next-generation sequencing, the forward-genetic techniques were laborious and time-consuming. Over the last 10 years, advancements in the area of genome assembly, genotyping, large-scale data analysis, and statistical algorithms have led faster identification of genomic variations regulating the complex agronomic traits and pathogen resistance. In this review, we describe the latest developments in genome sequencing and genotyping along with a comprehensive evaluation of the last 10-year headways in forward-genetic techniques that have shifted the focus of plant research from model plants to diverse crops. We have classified the available molecular genetic methods under bulk-segregant analysis-based (QTL-seq, GradedPool-Seq, QTG-Seq, Exome QTL-seq, and RapMap), target sequence enrichment-based (RenSeq, AgRenSeq, and TACCA), and mutation-based groups (MutMap, NIKS algorithm, MutRenSeq, MutChromSeq), alongside improvements in classical mapping and genome-wide association analyses. Newer methods for outcrossing, heterozygous, and polyploid plant genetics have also been discussed. The use of k-mers has enriched the nature of genetic variants which can be utilized to identify the phenotype-causing genes, independent of reference genomes. We envisage that the recent methods discussed herein will expand the repertoire of useful alleles and help in developing high-yielding and climate-resilient crops.
Cloning and expression of gamma carbonic anhydrase from Serratia sp. ISTD04 for sequestration of carbon dioxide and formation of calcite
(Elsevier B.V., 2015) Srivastava, Shaili; Bharti, Randhir Kumar; Verma, Praveen K.; Thakur, Indu shekhar
Bacterial strains isolated from marble mines rock and enriched in the chemostat culture with different concentrations of sodium bicarbonate. The enriched consortium had six bacterial isolates. One of bacterium isolate showed carbonic anhydrase (CA) activity by catalyzing the reversible hydration reaction of carbon dioxide to bicarbonate. The bacterium was identified as Serratia sp. by 16S rRNA sequence analysis. The carbonic anhydrase gene from Serratia sp. was found to be homologous with gamma carbonic anhydrase. The carbonic anhydrase gene was cloned in PET21b(+) and expressed it in recombinant Escherichia coli BL21 (DE3) with His-tag at the C-terminus. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. Expected size of carbonic anhydrase was approximately 29 kDa in SDS-PAGE gel. Recombinant carbonic anhydrase enzyme was used for biomineralization-based conversion of atmospheric CO2 into valuable calcite minerals. The calcification was confirmed by using XRD, FTIR, EDX and SEM analysis.
Comparative genomic analysis of GARP transcription factor family in legumes and identification of stress-responsive candidate genes
(Springer Nature Publishing AG, 2023) Singh, Ritu; Pandey, Ashutosh; Verma, Praveen K.
The GARP transcription factors have been identified for multiple biological functions throughout the life cycle of a plant. Despite of its involvement in crucial functions, systematic study of GARPs remains obscure in plants. In this study, we explored the genomic, molecular, and evolutionary perspectives of the GARP gene family in the three major leguminous plants, namely chickpea (Cicer arietinum), soybean (Glycine max), and barrel clover (Medicago truncatula). Here, we identified 53, 56, and 107 GARP genes in Cicer, Medicago, and Glycine, respectively. They were classified into four clades and two sub-clades as per phylogenetic analysis, and the result was supported by consensus motifs, domain organization, and exon–intron structures. Detailed comparative analysis indicates conservation of the GARP gene family in plants. Identification of paralogous and orthologous gene pairs revealed that the expansion of this family occurs mainly through genome duplication in legumes. Additionally, the three-dimensional structure and functional enrichment analysis indicated their major role in signaling, growth, development, and stress processes. The chickpea GARP genes were also characterized for their transcript modulation in diverse plant organs and during pathogenic stress. Differential regulation of 24 CaGARP genes was observed during Ascochyta Blight (AB) stress. Characterization of AB-responsive genes reveals an over-representation of stress and hormone-binding elements on the promoter of CaGARPs. Additionally, interactome analysis also confirms the role of GARPs in plant stress and development. Our findings not only provide a handful of stress-responsive genes but also lay the foundation for prospective functional studies of GARPs in legumes.
Comparative structural modeling of six old yellow enzymes (OYEs) from the necrotrophic fungus Ascochyta rabiei: Insight into novel OYE classes with differences in cofactor binding, organization of active site residues and stereopreferences
(PLOS, 2014) Nizam, Shadab; Gazara, Rajesh Kumar; Verma, Sandhya; Singh, Kunal; Verma, Praveen K.
Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.
Comparative structural modelling of a monothiol GRX from chickpea: insight in iron-sulfur cluster assembly
(Elsevier, 2012) Yadav, Saurabh; Kushwaha, Hemant Ritturaj; Kumar, Kamal; Verma, Praveen K.
Glutaredoxins (GRXs) are small, ubiquitous, multifunctional, heat-stable and glutathione-dependent thiol-disulphide oxidoreductases, classified under thioredoxin-fold superfamily. In the green lineage, GRXs constitute a complex family of proteins. Based on their active site, GRXs are classified into two subfamilies: dithiol and monothiol. Monothiol GRXs contain 'CGFS' as a redox active motif and assist in maintaining redox state and iron homeostasis within the cell. Using RACE strategy, a full length cDNA of chickpea (Cicer arietinum) glutaredoxin 3 (CarGRX3) was cloned and sequenced. The cDNA contains open reading frame of 537 bp encoding 178 amino acids and exhibits features of other known 'CGFS' type GRXs. Based on the multiple sequence alignment among CarGRX3 and monothiol GRXs of other photosynthetic organisms, the characteristic motif (KGX4PXCGFSX([29/30/32])KX4WPTXPQX4GX3GGXDI) with 18 invariant residues was observed. The proposed structure of CarGRX3 was compared with structurally resolved monothiol GRXs of other organisms. The CarGRX3 and nearest Arabidopsis homolog (AtGRXcp) shares 76% sequence identity which was reflected by their 3D-structure conservation. The structure of chickpea monothiol GRX (CarGRX3) coordinates glutathione ligated [2Fe-2S] cluster in a homodimeric form, highlighting the structural basis for iron-sulfur cluster (ISC) assembly and delivery to acceptor proteins. The present study on CarGRX3 model highlighted the utility of the theoretical approaches to understand complex biological phenomena such as glutathione docking and incorporation of GSH-ligated [2Fe-2S] cluster.
Comparative transcriptome analysis of the necrotrophic fungus Ascochyta rabiei during oxidative stress: insight for fungal survival in the host plant
(PLOS, 2012) Singh, Kunal; Nizam, Shadab; Sinha, Manisha; Verma, Praveen K.
Localized cell death, known as the hypersensitive response (HR), is an important defense mechanism for neutralizing phytopathogens. The hallmark of the HR is an oxidative burst produced by the host plant. We aimed to identify genes of the necrotrophic chickpea blight fungus Ascochyta rabiei that are involved in counteracting oxidative stress. A subtractive cDNA library was constructed after menadione treatment, which resulted in the isolation of 128 unigenes. A reverse northern blot was used to compare transcript profiles after H2O2, menadione and sodium nitroprusside treatments. A total of 70 unigenes were found to be upregulated by more than two-fold following menadione treatment at different time intervals. A large number of genes not previously associated with oxidative stress were identified, along with many stress-responsive genes. Differential expression patterns of several genes were validated by quantitative real-time PCR (qRT-PCR) and northern blotting. In planta qRT-PCR of several selected genes also showed differential expression patterns during infection and disease progression. These data shed light on the molecular responses of the phytopathogen A. rabiei to overcome oxidative and nitrosative stresses and advance the understanding of necrotrophic fungal pathogen survival mechanisms.
Comprehensive genome-wide analysis reveals different classes of enigmatic old yellow enzyme in fungi
(Nature Publishing Group, 2014) Nizam, Shadab; Verma, Sandhya; Borah, Nilam Nayan; Gazara, Rajesh Kumar; Verma, Praveen K.
In this study, we systematically identify Old Yellow Enzymes (OYEs) from a diverse range of economically important fungi representing different ecology and lifestyle. Using active site residues and sequence alignments, we present a classification for these proteins into three distinct classes including a novel class (Class III) and assign names to sequences. Our in-depth phylogenetic analysis suggests a complex history of lineage-specific expansion and contraction for the OYE gene family in fungi. Comparative analyses reveal remarkable diversity in the number and classes of OYE among fungi. Quantitative real-time PCR (qRT-PCR) of Ascochyta rabiei OYEs indicates differential expression of OYE genes during oxidative stress and plant infection. This study shows relationship of OYE with fungal ecology and lifestyle, and provides a foundation for future functional analysis and characterization of OYE gene family.
Constructing synthetic pathways in plants: Strategies and tools
(Elsevier B.V., 2020) Dwivedi, Anuj; Kumar, Kamal; Verma, Praveen K.
Plants, being primary producers, are the ultimate choice for the synthetic biology and metabolite engineering. For thousands of years, conventional methods allowed plants to meet various human requirements. Synthetic biology is a combination of engineering and biological science that facilitates with large number of new opportunities toward generation of reprogrammed cell with new biological behavior. The application of this technology can enhance traditional crop production and maximize the desired biomass production in required plants. Efforts in establishments of such individual plant systems will result in pioneering in various applications, such as generation of food, fuel, fiber, and biomedical therapies. The primary goal of this chapter is to discuss the advancement in strategies and tools form the core of synthetic pathway construction. However, synthetic biology in plant system is still at infancy stage and thus implications of synthetic biology strategies deployed in crops for biomass production have been discussed.
Crystal structure of ArOYE6 reveals a novel C-terminal helical extension and mechanistic insights into the distinct class III OYEs from pathogenic fungi
(John Wiley & Sons, 2022) Singh, Yeshveer; Sharma, Ruby; Mishra, Manasi; Verma, Praveen K.; Saxena, Ajay Kumar
Old Yellow Enzymes (OYEs) play critical role in antioxidation, detoxification and ergot alkaloid biosynthesis processes in various organisms. The yeast- and bacteria-like OYEs have been structurally characterized earlier, however, filamentous fungal pathogens possess a novel OYE class i.e. class III, whose biochemical and structural intricacies remain unexplored to date. Here, we present the 1.6 Å X-ray structure of a class III member, old yellow enzyme 6 from necrotrophic fungus Ascochyta rabiei (ArOYE6), in FMN-bound form (PDB ID-7FEV) and provide mechanistic insights into their catalytic capability. We demonstrate that ArOYE6 exists as a monomer in solution, forms (β/α)8 barrel structure harboring non-covalently bound FMN at cofactor binding site, and utilizes reduced nicotinamide adenine dinucleotide phosphate as its preferred reductant. The large hydrophobic cavity situated above FMN, specifically accommodates 12-oxo-phytodienoic acid and N-ethylmaleimide substrates as observed in ArOYE6-FMN-substrate ternary complex models. Site-directed mutations in the conserved catalytic (His196, His199, Tyr201) and FMN-binding (Lys249, Arg348) residues render the enzyme inactive. Intriguingly, ArOYE6 structure contains a novel C-terminus (369-445 residues) made of three α-helices, which stabilizes the FMN binding pocket as its mutation/truncation lead to complete loss of FMN binding. Moreover, the loss of extended C-terminus does not alter the monomeric nature of ArOYE6. In this study, for the first time, we provide the structural and biochemical insights for a fungi-specific class III OYE homolog and dissect the oxidoreductase mechanism. Our findings hold broad biological significance during host-fungus interactions owing to conservation of this class among pathogenic fungi, and would have potential implications in the pharmacochemical industry.
Differential transcript accumulation in chickpea during early phases of compatible interaction with a necrotrophic fungus Ascochyta rabiei
(Springer Science, 2012) Jaiswal, Purnima; Cheruku, Jyothi Reddy; Kumar, Kamal; Yadav, Saurabh; Singh, Archana; Kumari, Pragati; Dube, Sunil Chandra; Upadhyaya, Kailash C.; Verma, Praveen K.
The initial phases of the disease establishment are very crucial for the compatible interactions. Pathogens must overcome the responses generated by the host for the onset of disease invasion. The compatible interaction is inadequately represented in plant-pathogen interaction studies. To gain broader insight into the early responses elicited by chickpea blight fungus Ascochyta rabiei during compatible interaction; we isolated early responsive genes of chickpea using PCR based suppression subtractive hybridization (SSH) strategy. We obtained ~250 unique genes after homology search and redundancy elimination. Based on their potential cellular functions, these genes were broadly classified into eleven different categories viz. stress, signaling, gene regulation, cellular metabolism and genes of unknown functions. Present study revealed few unexpected genes which have a possible role in induced immunity and disease progression. We employed macroarray, northern blot, real-time PCR and cluster analysis to develop transcript profiles. Most of the genes analyzed were early induced and were transcriptionally upregulated upon 24 h post inoculation. Our approach has rendered the isolation of early responsive genes involved in signaling and regulation of metabolic changes upon fungal infection. The information obtained will help to dissect the molecular mechanisms during compatible chickpea-Ascochyta interactions.
Differential transcript accumulation in Cicer arietinum L. in response to a chewing Insect Helicoverpa armigera and defense regulators correlate with reduced insect performance
(Oxford University Press, 2008) Singh, Archana; Singh, Indrakant Kumar; Verma, Praveen K.
Monitoring transcriptional reorganization triggered in response to a particular stress is an essential first step for the functional analysis of genes involved in the process. To characterize Cicer arietinum L. defence responses against Helicoverpa armigera feeding, transcript patterns elicited by both herbivore and mechanical wounding were profiled and compared, and the application of defence regulators was assessed. A combination of approaches was employed to develop transcript profiles, including suppression subtractive hybridization (SSH), macroarray, northern blot, and cluster analysis. Of the 63 unique genes isolated, 29 genes expressed differentially when Helicoverpa feeding and wounding responses were compared. Comparative macroarray analyses revealed that most of the Helicoverpa-induced transcripts were methyl jasmonate (MeJA) and ethylene (ET) regulated. The effects of mild insect infestation and the exogenous application of signalling compounds on larval feeding behaviour were also monitored. Bioassays were performed to measure dispersal percentage and growth of larvae on elicited plants. Larvae released on elicited plants had decreased larval performance, demonstrating the central role of induced plant defence against herbivory. Similarly, wounding and exogenous application of MeJA and ET also affected larval growth and feeding behaviour. Our results demonstrated that Helicoverpa attack up-regulated large transcriptional changes and induced chickpea defence responses. Therefore, the results of this study advance the understanding of non-model plant–insect interactions on a broader scale.
Draft genome sequence and functional analysis of Lysinibacillus xylanilyticus t26, a plant growth-promoting bacterium isolated from Capsicum chinense rhizosphere
(Springer Nature Publishing AG, 2022) Phazna, T.A.; Ngashangva, Ng; Yentrembam, Randhir Babu Singh; Maurya, Ranjeet; Mukherjee, Pulok; Sharma, Chandradev; Verma, Praveen K.; Sarangthem, Indira
Capsicum chinense is the chilli species containing the highest amount of capsaicin, and is an important traditional spice crop of Northeast India. Capsaicinoids derived from C. chinense are used in anticancer and anti-obesity treatments, as temperature regulators, in pain therapy, and as antioxidants. The current production and yield are very low due to the lack of organized cultivation and scientific inputs, and various plant diseases. Synthetic pesticides are frequently applied to boost yields, which creates potential risks to the environment, crops, and humans. The use of plant growth-promoting rhizobacteria is an alternative strategy in crop disease management to reduce the dependency on agrochemicals, which have detrimental effects on the environment. Lysinibacillus xylanilyticus t26 isolated from the C. chinense rhizosphere has shown good prospects in plant growth promotion and biocontrol. It showed strong antagonistic activity against Pythium ultimum ITCC 1650, Rhizoctonia solani ITCC 6491, and Fusarium oxysporum ITCC 6246. The draft genome sequencing of L. xylanilyticus t26 yielded a total of 5.69 Mbp with a G+C content of 36.80%. Genome analysis revealed that L. xylanilyticus t26 is very similar to L. xylanilyticus MH683160.1, and is phylogenetically related to L. xylanilyticus IBBPo7. Bioinformatics analysis predicted that it harbored type III polyketides, non-ribosomal peptides, terpenes, and lantibiotics including cerecidin, bacteriocins, siderophores, and thiopeptides, which are important traits of rhizobacteria for the utilization of minerals and to compete with other microbes for food. The strain t26 is a potential biocontrol agent for soil-borne fungal diseases. In this study, we derived the possible siderophore production pathways through the analysis of L. xylanilyticus t26 draft genome and plant growth response bioassays. The availability of genome data provides information that this draft genome harbored a siderophore BGC, which is 33% similar to petrobactin.