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 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 KumarOld 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.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 mQTL-seq and classical mapping implicates the role of an AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family gene in ascochyta blight resistance of chickpea(John Wiley & Sons, 2018) Kumar, K; Purayannur, S; Kaladhar, VC; Parida, Swarup K.; Verma, Praveen K.Ascochyta blight (AB) caused by the fungal pathogen Ascochyta rabiei is a serious foliar disease of the legume crop chickpea (Cicer arietinum L.). Despite many genetic studies on chickpea-Ascochyta interaction, genome-wide scan of chickpea for the identification of AB associated QTLs and their gene(s) has not been accomplished. To elucidate narrow QTLs for AB resistance, here we report the use of multiple QTL-sequencing (mQTL-seq) approach on two sets of extreme AB phenotype bulks derived from Cicer intraspecific and interspecific crosses. Two major QTLs qABR4.1 and qABR4.2, and a minor QTL qABR4.3 were identified on assembled pseudomolecule 4 (Ca4). We narrowed qABR4.1 to a ‘robust ~50 kb region’ through mapping on a larger intraspecific RIL population and comparative analysis. Among four genes, the CaAHL18 gene showed higher expression under Ascochyta stress in AB resistant parent suggesting that it is the candidate gene under ‘robust qABR4.1’. Dual-luciferase assay with CaAHL18 polymorphic cis-regulatory sequences showed that higher expression is associated with an allelic variation. Thus, our findings on chickpea-Ascochyta interaction have narrowed down AB resistance associated QTLs on chickpea physical map and identified a novel QTL qABR4.3. The narrowed QTL and gene associated markers will help in biotechnological and breeding programs for chickpea improvement.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 WRKY domain-encoding genes of a crop legume chickpea (Cicer arietinum): comparative analysis with Medicago truncatula WRKY family and characterization of group-III gene(s)(Oxford University Press, 2016) Kumar, Kamal; Srivastava, Vikas; Purayannur, Savithri; Kaladhar, V. Chandra; Cheruvu, Purnima Jaiswal; Verma, Praveen K.The WRKY genes have been identified as important transcriptional modulators predominantly during the environmental stresses, but they also play critical role at various stages of plant life cycle. We report the identification of WRKY domain (WD)-encoding genes from galegoid clade legumes chickpea (Cicer arietinum L.) and barrel medic (Medicago truncatula). In total, 78 and 98 WD-encoding genes were found in chickpea and barrel medic, respectively. Comparative analysis suggests the presence of both conserved and unique WRKYs, and expansion of WRKY family in M. truncatula primarily by tandem duplication. Exclusively found in galegoid legumes, CaWRKY16 and its orthologues encode for a novel protein having a transmembrane and partial Exo70 domains flanking a group-III WD. Genomic region of galegoids, having CaWRKY16, is more dynamic when compared with millettioids. In onion cells, fused CaWRKY16-EYFP showed punctate fluorescent signals in cytoplasm. The chickpea WRKY group-III genes were further characterized for their transcript level modulation during pathogenic stress and treatments of abscisic acid, jasmonic acid, and salicylic acid (SA) by real-time PCR. Differential regulation of genes was observed during Ascochyta rabiei infection and SA treatment. Characterization of A. rabiei and SA inducible gene CaWRKY50 showed that it localizes to plant nucleus, binds to W-box, and have a C-terminal transactivation domain. Overexpression of CaWRKY50 in tobacco plants resulted in early flowering and senescence. The in-depth comparative account presented here for two legume WRKY genes will be of great utility in hastening functional characterization of crop legume WRKYs and will also help in characterization of Exo70Js.Item 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.Item 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.Item Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus Ascochyta rabiei(Springer, 2010) Nizam, Shadab; Singh, Kunal; Verma, Praveen K.Ascochyta blight caused by the ascomycete fungus Ascochyta rabiei, is a major biotic constraint of chickpea (Cicer arietinum L.), resulting in disastrous crop losses worldwide. To study early stages of development and pathogenic mechanisms of the fungus, two binary vectors for the constitutive expression of the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constructed. Furthermore, we have developed an improved and highly reproducible Agrobacterium tumefaciens-mediated transformation protocol for A. rabiei. Transformation events were confirmed through Southern hybridizations that suggest single-copy integration of reporter genes in majority of the transformants. High level expression of both DsRed and EGFP proteins was obtained both in spores and in mycelia as detected by fluorescence microscopy. Intense fluorescence was used as a highly efficient vital marker to visualize early developmental changes of the fungus. The formation of infection structures like appressoria and germ tubes were observed both in vitro and in planta. This work will be useful to develop methodologies for understanding the mechanisms of Ascochyta-chickpea interaction and functional genomics of A. rabiei towards the isolation of virulence genes.