Institutional Publications
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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 Global transcriptome and co-expression analysis reveals robust host defence pathway reprogramming and identifies key regulators of early phases of Cicer-Ascochyta interactions(American Phytopathological Society, 2022) Singh, Ritu; Dwivedi, Aditi; Singh, Yeshveer; Kumar, Kamal; Ranjan, Aashish; Verma, Praveen K.Ascochyta blight (AB) caused by a filamentous fungus Ascochyta rabiei is a major threat to global chickpea production. The mechanisms underlying chickpea response to A. rabiei remain elusive. Here, we investigated the comparative transcriptional dynamics of AB-resistant and susceptible chickpea genotypes upon A. rabiei infection to understand the early host defence response. Our findings revealed that AB-resistant plants underwent rapid and extensive transcriptional reprogramming compared to susceptible host. At early stage (24-hpi), mainly cell wall remodeling and secondary metabolite pathways were highly activated, while DEGs related with signaling components viz. protein kinases, transcription factors, and hormonal pathways show remarkable upsurge at 72-hpi, especially in resistant genotype. Notably, our data suggests imperative role of JA, ET, and ABA signaling in providing immunity against A. rabiei. Furthermore, gene co-expression networks and modules corroborated the importance of cell wall remodeling, signal transduction, and phytohormone pathways. The hub genes such as MYB14, PRE6, and MADS-SOC1 discovered in these modules might be the master regulators governing chickpea immunity. Overall, we not only provide novel insights for comprehensive understanding of immune signaling components mediating AB resistance/susceptibility at early Cicer-Ascochyta interactions, but also offer a valuable resource for developing AB-resistant chickpea.Item 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.Item 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.Item Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei(PLOS, 2021) Sinha, Manisha; Shree, Ankita; Singh, Kunal; Kumar, Kamal; Singh, Shreenivas Kumar; Kumar, Vimlesh; Verma, Praveen K.Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified a F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is necessary for virulence. The loss-of-function of ArF-BAR gene results in delayed formation of apical septum in fungal cells near growing hyphal tip that is crucial for host penetration, and impaired secretion of a candidate effector having secretory signal peptide for translocation across the endoplasmic reticulum membrane. The mRNA transcripts of ArF-BAR were induced in response to oxidative stress and infection. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1), as key transcriptional regulator of ArF-BAR expression. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate A. rabiei virulence through a mechanism that involves endocytosis, effector secretion, and actin cytoskeleton regulation.Item Functional characterization of genes involved in legume nodulation using hairy root cultures(Springer Nature Publishing AG, 2020) Singh, Jawahar; Kumar, Kamal; Verma, Praveen K.Legumes, the second most important crop to humans possess unique ability to fix atmospheric nitrogen, making them one of the major contributors to sustainable agriculture. In legumes, molecular characterization of genes by stable transformation is difficult due to their recalcitrant nature to the whole-plant regeneration in desired varieties. The Agrobacterium rhizogenes-mediated generation of transgenic hairy roots or composite plants may facilitate a rapid and convenient alternative to study nodule biology. Functional analysis of genes involved in legume nodulation has been proven as successful for model legumes, viz., Medicago truncatula and Lotus japonicus, using transgenic hairy roots. Besides sharing some common features of nodulation among legumes, the symbiotic signaling is a complex and specific process. Here, we describe an improved protocol for hairy root transformation of a legume crop chickpea (Cicer arietinum L.) and the method to study nodulation to uncover the signaling components. Using the described protocol, transgenic hairy roots were generated in chickpea and selected based on the red fluorescence protein (RFP) microscopy. This protocol can be extended to other underutilized legumes.Item 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.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 Phylogenomic analysis of MKKs and MAPKs from 16 legumes and detection of interacting pairs in chickpea divulge MAPK signalling modules(Nature Publishing Group, 2017) Purayannur, Savithri; Kumar, Kamal; Kaladhar, Vemula Chandra; Verma, Praveen K.The mitogen-activated protein kinase (MAPK)-mediated phosphorylation cascade is a vital component of plant cellular signalling. Despite this, MAPK signalling cascade is less characterized in crop legumes. To fill this void, we present here a comprehensive phylogeny of MAPK kinases (MKKs) and MAPKs identified from 16 legume species belonging to genistoid (Lupinus angustifolius), dalbergioid (Arachis spp.), phaseoloid (Glycine max, Cajanus cajan, Phaseolus vulgaris, and Vigna spp.), and galegoid (Cicer arietinum, Lotus japonicus, Medicago truncatula, Pisum sativum, Trifolium spp., and Vicia faba) clades. Using the genes of the diploid crop chickpea (C. arietinum), an exhaustive interaction analysis was performed between MKKs and MAPKs by split-ubiquitin based yeast two-hybrid (Y2H). Twenty seven interactions of varying strengths were identified between chickpea MKKs and MAPKs. These interactions were verified in planta by bimolecular fluorescence complementation (BiFC). As a first report in plants, four intra-molecular interactions of weak strength were identified within chickpea MKKs. Additionally; two TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors of class I were identified as novel down-stream interacting partners of seven MAPKs. We propose that this highly reliable MAPK interaction network, presented here for chickpea, can be utilized as a reference for legumes and thus will help in deciphering their role in legume-specific events.Item Genetic engineering to improve biotic stress tolerance in plants(Springer Nature, 2017) Purayannur, Savithri; Kumar, Kamal; Verma, Praveen K.Genetic engineering of plants for resistance is an effective method to counter pathogens and pests owing to the specificity and efficiency of the technology. The genes that have been used to genetically engineer resistance are as diverse as the diseases they act against. In cases where gene-for-gene resistance coded by resistance (R) genes exists, engineering resistance in plants becomes a straight path. Different classes of R genes have been engineered to provide resistance against viruses, bacteria, filamentous phytopathogens, and nematodes. Where the resistance mechanism is not R gene mediated, myriad of other mechanisms have been tried. These include the use of genes coding for antimicrobial compounds against bacterial and filamentous pathogens. The cloning of transcription factors, receptor genes, proteases, and genes involved in the systemic acquired resistance (SAR) has also been found to be effective. RNA silencing against specific genes involved in pathogenicity has proved to be an efficacious strategy against viruses and nematodes. Posttranscriptional silencing of genes coding for viral coat proteins has been successful, both scientifically and commercially. The most extensively used technology till date has been the introduction of cry genes from the bacterium Bacillus thuringiensis into plants to render them resistant against insect pests. Advances in molecular biology have paved the way for new strategies, the phenomenon of host-induced gene silencing (HIGS) being an interesting example. Amidst all the hue and cry raised against genetic modification of crops, it is necessary to highlight the scientific principles involved so as to make full use of a technology that could very well solve the problem of food shortage.