Publications of NIPGR Scientists
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Item 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.Item 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, JyotiThe 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.Item 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, KirtiSpot 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.Item 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 ShamsBackground: 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.Item 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.Item 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 IndiraWe 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.Item 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 ShekharBackground: 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.Item 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.Item 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 ShekharWe 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.Item 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 ShekharThe 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.