Browsing by Author "Singh, Yeshveer"

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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.
Establishment of Agrobacterium rhizogenes-mediated hairy root transformation of Crocus sativus L
(Springer Nature Publishing AG, 2021) Sharma, Shilpi; Singh, Yeshveer; Verma, Praveen K.
Efficient transformation system for genetic improvement is essential in Crocus sativus, as it lacks sexual reproduction. This is the first report wherein an efficient protocol is developed for the transformation of Crocus sativus L. by Agrobacterium rhizogenes strain ARqua1 with a transformation efficiency of 78.51%. The ARqua1 strain harboring both Ri plasmid and binary vector plasmid pSITE-4NB, and marker genes for red fluorescent protein (RFP) and a β-glucuronidase (GUS) reporter gene were used for selection. Transformation was confirmed by RFP signal, GUS reporter assay and polymerase chain reaction (PCR) analysis of the test samples after 21 days post inoculation. These results confirm the establishment of protocol for hairy root transformation in C. sativus that can be further used for gene transfer or gene editing in Crocus for its genetic improvement.
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.
Guiding the guards: MPK3/6-VLN3 module regulating stomatal defense
(Elsevier B.V., 2022) Singh, Yeshveer; Verma, Praveen K.
Stomata offer an effortless opportunity for pathogens to enter host plants and exploit that resource. Upon pathogen attack, stomatal closure is a commonly observed response to prevent microbial invasion. A recent study by Zou et al. shows that stomatal closure following exposure to microbe-associated molecular patterns (MAMPs) is mediated by altered actin dynamics in an MPK3/6 phosphorylation- and VLN3-dependent manner.
The R2R3-MYB-SG7 transcription factor CaMYB39 orchestrates surface phenylpropanoid metabolism and pathogen resistance in chickpea
(John Wiley & Sons, 2023) Saxena, Samiksha; Pal, Lalita; Naik, Jogindra; Singh, Yeshveer; Verma, Praveen K.; Chattopadhyay, Debasis; Pandey, Ashutosh
Flavonoids are important plant pigments and defense compounds; understanding the transcriptional regulation of flavonoid biosynthesis may enable engineering crops with improved nutrition and stress tolerance. Here, we characterize R2R3-MYB domain subgroup 7 transcription factor CaMYB39, which regulates flavonol biosynthesis primarily in chickpea trichomes. CaMYB39 overexpression in chickpea was accompanied by a change in flux availability for the phenylpropanoid pathway, particularly flavonol biosynthesis. Lines overexpressing CaMYB39 showed higher isoflavonoid levels, suggesting its role in regulating isoflavonoid pathway. CaMYB39 transactivates the transcription of early flavonoid biosynthetic genes (EBG). FLAVONOL SYNTHASE2, an EBG, encodes an enzyme with higher substrate specificity for dihydrokaempferol than other dihydroflavonols explaining the preferential accumulation of kaempferol derivatives as prominent flavonols in chickpea. Interestingly, CaMYB39 overexpression increased trichome density and enhanced accumulation of diverse flavonol derivatives in trichome-rich tissues. Moreover, CaMYB39 overexpression reduced ROS levels and induced defense gene expression which aids in partially blocking the penetration efficiency of the fungal pathogen, Ascochyta rabiei, resulting in lesser symptoms, thus establishing its role against deadly Ascochyta blight(AB) disease. Overall, our study reports an instance where R2R3-MYB-SG7 member, CaMYB39, besides regulating flavonol biosynthesis, modulates diverse pathways like general phenylpropanoid, isoflavonoid, trichome density and defense against necrotrophic fungal infection in chickpea.
Surviving the odds: from perception to survival of fungal phytopathogens under host-generated oxidative burst
(Elsevier B.V., 2021) Singh, Yeshveer; Nair, Athira Mohandas; Verma, Praveen K.
Fungal phytopathogens pose a serious threat to global crop production. Only a handful of strategies are available to combat these fungal infections, and the increasing incidence of fungicide resistance is making the situation worse. Hence, the molecular understanding of plant–fungus interactions remains a primary focus of plant pathology. One of the hallmarks of host–pathogen interactions is the overproduction of reactive oxygen species (ROS) as a plant defense mechanism, collectively termed the oxidative burst. In general, high accumulation of ROS restricts the growth of pathogenic organisms by causing localized cell death around the site of infection. To survive the oxidative burst and achieve successful host colonization, fungal phytopathogens employ intricate mechanisms for ROS perception, ROS neutralization, and protection from ROS-mediated damage. Together, these countermeasures maintain the physiological redox homeostasis that is essential for cell viability. In addition to intracellular antioxidant systems, phytopathogenic fungi also deploy interesting effector-mediated mechanisms for extracellular ROS modulation. This aspect of plant–pathogen interactions is significantly under-studied and provides enormous scope for future research. These adaptive responses, broadly categorized into ‘‘escape’’ and ‘‘exploitation’’ mechanisms, are poorly understood. In this review, we discuss the oxidative stress response of filamentous fungi, their perception signaling, and recent insights that provide a comprehensive understanding of the distinct survival mechanisms of fungal pathogens in response to the host-generated oxidative burst.
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.