Browsing by Author "Verma, Sandhya"

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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.
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.
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.
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.
Draft Genome Sequence of Bacillus altitudinis Lc5, a biocontrol and plant growth-promoting endophyte strain isolated from indigenous black rice of Manipur
(American Society for Microbiology, 2018) Potshangbam, Momota; Sahoo, Dinabandhu; Verma, Praveen K.; Verma, Sandhya; Kalita, Mohan Chandra; Devi, Sarangthem Indira
We report here the 3.6-Mb draft genome of Bacillus altitudinis Lc5, a potential plant growth promoter and an active antagonistic endophyte of black rice. This genome study will provide better insights into the strain’s mechanisms for plant growth promotion and biocontrol, thus facilitating its application in organic agriculture.
Draft genome sequencing and secretome analysis of fungal phytopathogen Ascochyta rabiei provides insight into the necrotrophic effector repertoire
(Nature Publishing Group, 2016) Verma, Sandhya; Gazara, Rajesh Kumar; Nizam, Shadab; Parween, Sabiha; Chattopadhyay, Debasis; Verma, Praveen K.
Constant evolutionary pressure acting on pathogens refines their molecular strategies to attain successful pathogenesis. Recent studies have shown that pathogenicity mechanisms of necrotrophic fungi are far more intricate than earlier evaluated. However, only a few studies have explored necrotrophic fungal pathogens. Ascochyta rabiei is a necrotrophic fungus that causes devastating blight disease of chickpea (Cicer arietinum). Here, we report a 34.6 megabase draft genome assembly of A. rabiei. The genome assembly covered more than 99% of the gene space and 4,259 simple sequence repeats were identified in the assembly. A total of 10,596 high confidence protein-coding genes were predicted which includes a large and diverse inventory of secretory proteins, transporters and primary and secondary metabolism enzymes reflecting the necrotrophic lifestyle of A. rabiei. A wide range of genes encoding carbohydrate-active enzymes capable for degradation of complex polysaccharides were also identified. Comprehensive analysis predicted a set of 758 secretory proteins including both classical and non-classical secreted proteins. Several of these predicted secretory proteins showed high cysteine content and numerous tandem repeats. Together, our analyses would broadly expand our knowledge and offer insights into the pathogenesis and necrotrophic lifestyle of fungal phytopathogens.
Functional analysis of SCD1 gene involved in pathogenicity of spot blotch disease of wheat causing fungus Bipolaris sorokiniana
(Springer Nature Publishing AG, 2022) Singh, Kartar; Aggarwal, Rashmi; Verma, Praveen K.; Verma, Sandhya; Sharma, Sapna; Manjunatha, C.; Choudhary, Manoj; Kulshreshtha, Deepika; Rawat, Kirti
Spot blotch of wheat is an important disease of wheat mainly of warm and humid areas. Melanin produced by various fungi has an important role to play in survival and longevity of fungal propagules against various environmental stresses through providing structural strength. Melanin production is positively correlated with virulence and conidiogenesis that varies in different isolates of plant pathogenic fungus Bipolaris sorokiniana. Scytalone dehydratase gene (SCD1) plays an important role in synthesis of fungal dihydroxynaphthalene melanin. In this study, the SCD1 gene was isolated, characterized and its DNA sequence along with flanking regions was determined. Structural analysis of gene revealed that 516 bp un-interpreted open reading frame. Sequence analysis showed that SCD1 gene encodes a 180 amino acids putative protein. A comparison of the predicted amino acid sequence and actual nucleotide sequence of the SCD1 gene was made with other fungal reductase genes involved in melanin biosynthesis which indicates 56–98% sequence identity. Targeted disruption of SCD1gene revealed that this gene is necessary for melanin biosynthesis in B. sorokiniana. SCD1 gene mutant isolate lack melanin synthesis and showed significant reduction in pathogenicity on wheat plants. This study expands our knowledge and provides comprehensive understanding of the pathogenesis of B. sorokiniana and insight of fungal phytopathogens.
Genome sequence of carbon dioxide-sequestering serratia sp. strain ISTD04 isolated from marble mining rocks
(American Society for Microbiology, 2016) Kumar, Manish; Gazara, Rajesh Kumar; Verma, Sandhya; Kumar, Madan; Verma, Praveen K.; Thakur, Indu Shekhar
The Serratia sp. strain ISTD04 has been identified as a carbon dioxide (CO2)-sequestering bacterium isolated from marble mining rocks in the Umra area, Rajasthan, India. This strain grows chemolithotrophically on media that contain sodium bicarbonate (NaHCO3) as the sole carbon source. Here, we report the genome sequence of 5.07 Mb Serratia sp. ISTD04.
Genome sequence of pandoraea sp. ISTKB, a lignin-degrading betaproteobacterium, isolated from rhizospheric soil
(American Society for Microbiology, 2016) Kumar, Madan; Gazara, Rajesh Kumar; Verma, Sandhya; Kumar, Manish; Verma, Praveen K.; Thakur, Indu Shekhar
We report here the genome sequence of Pandoraea sp. ISTKB, a betaproteobacterium isolated from rhizospheric soil in the backwaters of Alappuzha, Kerala, India. The strain is alkalotolerant and grows on medium containing lignin as a sole carbon source. Genes and pathways related to lignin degradation were complemented by genomic analysis.
Genomic and proteomic analysis of lignin degrading and polyhydroxyalkanoate accumulating beta-proteobacterium Pandoraea sp ISTKB
(BioMed Central Ltd, 2018) Kumar, Madan; Verma, Sandhya; Gazara, Rajesh Kumar; Kumar, Manish; Pandey, Ashok; Verma, Praveen K.; Thakur, Indu Shekhar
Background: Lignin is a major component of plant biomass and is recalcitrant to degradation due to its complex and heterogeneous aromatic structure. The biomass-based research mainly focuses on polysaccharides component of biomass and lignin is discarded as waste with very limited usage. The sustainability and success of plant polysaccharide-based biorefinery can be possible if lignin is utilized in improved ways and with minimal waste generation. Discovering new microbial strains and understanding their enzyme system for lignin degradation are necessary for its conversion into fuel and chemicals. The Pandoraea sp. ISTKB was previously characterized for lignin degradation and successfully applied for pretreatment of sugarcane bagasse and polyhydroxyalkanoate (PHA) production. In this study, genomic analysis and proteomics on aromatic polymer kraft lignin and vanillic acid are performed to find the important enzymes for polymer utilization. Results: Genomic analysis of Pandoraea sp. ISTKB revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin degradation and PHA production. We also applied label-free quantitative proteomic approach to identify the expression profile on monoaromatic compound vanillic acid (VA) and polyaromatic kraft lignin (KL). Genomic and proteomic analysis simultaneously discovered Dyp-type peroxidase, peroxidases, glycolate oxidase, aldehyde oxidase, GMC oxidoreductase, laccases, quinone oxidoreductase, dioxygenases, monooxygenases, glutathione-dependent etherases, dehydrogenases, reductases, and methyltransferases and various other recently reported enzyme systems such as superoxide dismutases or catalase-peroxidase for lignin degradation. A strong stress response and detoxification mechanism was discovered. The two important gene clusters for lignin degradation and three PHA polymerase spanning gene clusters were identified and all the clusters were functionally active on KL-VA Conclusions: The unusual aerobic'-CoA'-mediated degradation pathway of phenylacetate and benzoate (reported only in 16 and 4-5% of total seguenced bacterial genomes), peroxidase-accessory enzyme system, and fenton chemistry based are the major pathways observed for lignin degradation. Both ortho and meta ring cleavage pathways for aromatic compound degradation were observed in expression profile. Genomic and proteomic approaches providedvalidation to this strain's robust machinery for the metabolism of recalcitrant compounds and PHA production and provide an opportunity to target important enzymes for lignin valorization in future.
High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens
(Elsevier, 2012) Nizam, Shadab; Verma, Sandhya; Singh, Kunal; Aggarwal, Rashmi; Srivastava, Krishna Dutt; Verma, Praveen K.
Bipolaris sorokiniana, the causal agent of spot blotch of wheat, significantly reduces grain yield worldwide. In order to study pathogenic mechanisms of the fungus, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. To study different stages of hyphal fusion and pathogenic mechanisms of the fungus, two fluorescence markers viz. the red fluorescent protein (DsRed-Ex- press) and the green fluorescent protein (EGFP1) were constitutively expressed. Southern hybridizations confirmed the presence of T-DNA in all hygromycin B or geneticin resistant transformants, and also showed random and single copy integration. Fluorescence microscopy suggested the high level expression of both DsRed and EGFP fluorescent proteins in spores and mycelia. The results signify that DsRed and EGFP can be used as efficient reporter gene for monitoring B. sorokiniana hyphal fusion as well as colonization in the host tissues. This work will be useful to develop methodologies for understanding the mechanisms of Bipo- laris–wheat interaction and functional genomics of B. sorokiniana for various applications including insertion- al mutagenesis, targeted disruption of specific genes, ectopic complementation of loss-of-function strains and over-expression.
Invasion and colonization of pathogenic Fusarium oxysporum R1 in Crocus sativus L. during corm rot disease progression
(MDPI AG, 2022) Bhagat, Nancy; Magotra, Shanu; Gupta, Rikita; Sharma, Shikha; Verma, Sandhya; Verma, Praveen K.; Ali, Tahir; Shree, Ankita; Vakhlu, Jyoti
The corm rot of saffron caused by Fusarium oxysporum (Fox) has been reported to be the most destructive fungal disease of the herb globally. The pathogen, Fusarium oxysporum R1 (Fox R1) isolated by our group from Kashmir, India, was found to be different from Fusarium oxysporum f.sp. gladioli commonly reported corm rot agent of saffron. In the present study, Fox R1 was further characterized using housekeeping genes and pathogenicity tests, as Fusarium oxysporum R1 f.sp. iridacearum race 4. Though Fox R1 invaded the saffron plant through both corm and roots, the corm was found to be the preferred site of infection. In addition, the route of pathogen movement wastracked by monitoring visual symptoms, semi-quantitative PCR, quantitative-PCR (q-PCR), real-time imaging of egfp-tagged Fusarium oxysporum R1, and Fox R1 load quantification. This study is the first study of its kind on the bidirectional pathogenesis from corm to roots and vice-versa, as the literature only reports unidirectional upward movement from roots to other parts of the plant. In addition, the colonization pattern of Fox R1 in saffron corms and roots was studied. The present study involved a systematic elucidation of the mode and mechanism of pathogenesis in the saffron Fusarium oxysporum strain R1 pathosystem.
The nuclear effector ArPEC25 from the necrotrophic fungus Ascochyta rabiei targets the chickpea transcription factor CaβLIM1a and negatively modulates lignin biosynthesis, increasing host susceptibility
(Oxford University Press, 2023) Singh, Shreenivas Kumar; Shree, Ankita; Verma, Sandhya; Singh, Kunal; Kumar, Kamal; Srivastava, Vikas; Singh, Ritu; Saxena, Samiksha; Singh, Agam Prasad; Pandey, Ashutosh; Verma, Praveen K.
Fungal pathogens deploy a barrage of secreted effectors to subvert host immunity, often by evading, disrupting, or altering key components of transcription, defense signaling, and metabolic pathways. However, the underlying mechanisms of effectors and their host targets are largely unexplored in necrotrophic fungal pathogens. Here, we describe the effector protein Ascochyta rabiei PEXEL-like Effector Candidate 25 (ArPEC25), which is secreted by the necrotroph A. rabiei, the causal agent of Ascochyta blight disease in chickpea (Cicer arietinum), and is indispensable for virulence. After entering host cells, ArPEC25 localizes to the nucleus and targets the host LIM transcription factor CaβLIM1a. CaβLIM1a is a transcriptional regulator of CaPAL1, which encodes phenylalanine ammonia lyase, the regulatory, gatekeeping enzyme of the phenylpropanoid pathway. ArPEC25 inhibits the transactivation of CaβLIM1a by interfering with its DNA binding ability, resulting in negative regulation of the phenylpropanoid pathway and decreased levels of intermediates of lignin biosynthesis, thereby suppressing lignin production. Our findings illustrate the role of fungal effectors in enhancing virulence by targeting a key defense pathway that leads to the biosynthesis of various secondary metabolites and antifungal compounds. This study provides a template for the study of less explored necrotrophic effectors and their host target functions.
Transcript profiling reveals potential regulators for oxidative stress response of a necrotrophic chickpea pathogen Ascochyta rabiei
(Springer Nature Publishing AG, 2020) Maurya, Ranjeet; Singh, Yeshveer; Sinha, Manisha; Singh, Kunal; Mishra, Pallavi; Singh, Shreenivas Kumar; Verma, Sandhya; Prabha, Kanchan; Kumar, Kamal; Verma, Praveen K.
Necrotrophic pathogens experience host-generated oxidative stress during pathogenesis. They overcome such hostile environment by intricate mechanisms which are largely understudied. In this article, reference-based transcriptome analysis of a devastating Ascochyta Blight (AB) disease causing chickpea pathogen Ascochyta rabiei was explored to get insights into survival mechanisms under oxidative stress. Here, expression profling of mock-treated and menadione-treated fungus was carried out by RNA-Seq approach. A signifcant number of genes in response to oxidative stress were overrepresented, suggestive of a robust and coordinated defense system of A. rabiei. A total 73 diferentially expressed genes were fltered out from both the transcriptomes, among them 64 were up-regulated and 9 were found down-regulated. The gene ontology and KEGG mapping were conducted to comprehend the possible regulatory roles of diferentially expressed genes in metabolic networks and biosynthetic pathways. Transcript profling, KEGG pathway and gene ontology-based enrichment analysis revealed 12 (16.43%) stress responsive factors, 25 (34.24%) virulence associated genes, 10 (13.69%) putative efectors and 28 (38.35%) important interacting proteins associated with various metabolic pathways. In addition, genes with diferential expression were further explored for underlying putative pathogenicity factors. We identifed fve genes ST47_g10291, ST47_g9396, ST47_g10294, ST47_g4395, and ST47_g7191 that were common to stress and fungal pathogenicity. The factors recognized in this work can be used to establish molecular tools to explain the regulatory gene networks engaged in stress response of fungal pathogens and disease management.
Transcription factor repertoire of necrotrophic fungal phytopathogen Ascochyta rabiei: predominance of MYB transcription factors as potential regulators of secretome
(Frontiers Media S.A., 2017) Verma, Sandhya; Gazara, Rajesh K.; Verma, Praveen K.
Transcription factors (TFs) are the key players in gene expression and their study is highly significant for shedding light on the molecular mechanisms and evolutionary history of organisms. During host–pathogen interaction, extensive reprogramming of gene expression facilitated by TFs is likely to occur in both host and pathogen. To date, the knowledge about TF repertoire in filamentous fungi is in infancy. The necrotrophic fungus Ascochyta rabiei, that causes destructive Ascochyta blight (AB) disease of chickpea (Cicer arietinum), demands more comprehensive study for better understanding of Ascochyta-legume pathosystem. In the present study, we performed the genome-wide identification and analysis of TFs in A. rabiei. Taking advantage of A. rabiei genome sequence, we used a bioinformatic approach to predict the TF repertoire of A. rabiei. For identification and classification of A. rabiei TFs, we designed a comprehensive pipeline using a combination of BLAST and InterProScan software. A total of 381 A. rabiei TFs were predicted and divided into 32 fungal specific families of TFs. The gene structure, domain organization and phylogenetic analysis of abundant families of A. rabiei TFs were also carried out. Comparative study of A. rabiei TFs with that of other necrotrophic, biotrophic, hemibiotrophic, symbiotic, and saprotrophic fungi was performed. It suggested presence of both conserved as well as unique features among them. Moreover, cis-acting elements on promoter sequences of earlier predicted A. rabiei secretome were also identified. With the help of published A. rabiei transcriptome data, the differential expression of TF and secretory protein coding genes was analyzed. Furthermore, comprehensive expression analysis of few selected A. rabiei TFs using quantitative real-time polymerase chain reaction revealed variety of expression patterns during host colonization. These genes were expressed in at least one of the time points tested post infection. Overall, this study illustrates the first genome-wide identification and analysis of TF repertoire of A. rabiei. This work would provide the basis for further studies to dissect role of TFs in the molecular mechanisms during A. rabiei–chickpea interactions.