Browsing by Author "Nizam, Shadab"

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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.
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.
Comparative transcriptome analysis of the necrotrophic fungus Ascochyta rabiei during oxidative stress: insight for fungal survival in the host plant
(PLOS, 2012) Singh, Kunal; Nizam, Shadab; Sinha, Manisha; Verma, Praveen K.
Localized cell death, known as the hypersensitive response (HR), is an important defense mechanism for neutralizing phytopathogens. The hallmark of the HR is an oxidative burst produced by the host plant. We aimed to identify genes of the necrotrophic chickpea blight fungus Ascochyta rabiei that are involved in counteracting oxidative stress. A subtractive cDNA library was constructed after menadione treatment, which resulted in the isolation of 128 unigenes. A reverse northern blot was used to compare transcript profiles after H2O2, menadione and sodium nitroprusside treatments. A total of 70 unigenes were found to be upregulated by more than two-fold following menadione treatment at different time intervals. A large number of genes not previously associated with oxidative stress were identified, along with many stress-responsive genes. Differential expression patterns of several genes were validated by quantitative real-time PCR (qRT-PCR) and northern blotting. In planta qRT-PCR of several selected genes also showed differential expression patterns during infection and disease progression. These data shed light on the molecular responses of the phytopathogen A. rabiei to overcome oxidative and nitrosative stresses and advance the understanding of necrotrophic fungal pathogen survival mechanisms.
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 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.
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.
Genome sequencing and its reference to plant pathogens
(Indian Phytopathological Society, 2011) Verma, Praveen K.; Nizam, Shadab
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.
A highly efficient Agrobacterium mediated transformation system of chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri using DsRed-Express to follow root colonization
(Elsevier, 2012) Islam, Md. Nazrul; Nizam, Shadab; Verma, Praveen K.
The soil-borne fungus Fusarium oxysporum f. sp. ciceri (Foc) causes vascular wilt of chickpea (Cicer arietinum L.), resulting in substantial yield losses worldwide. Agrobacterium tumefaciens mediated transformation (ATMT) has served as a resourceful tool for plant-pathogen interaction studies and offers a number of advantages over conventional transformation systems. Here, we developed a highly efficient A. tumefaciens mediated transformation system for Foc. In addition, a binary vector for constitutive expression of red fluorescent protein (DsRed-Express) was used to study developmental stages and host-pathogen interactions. Southern hybridisation was performed to confirm the transformation event and the presence of T-DNA in selected hygromycin resistant transformants. Most of the transformants showed single copy integrations at random positions. Microscopic studies revealed significant levels of fluorescent protein, both in conidia and mycelia. Confocal microscopy of chickpea roots infected with the transformed Foc showed rapid colonisation. These studies will allow us to develop strategies to determine the mechanisms of Foc-chickpea interaction in greater detail and to apply functional genomics for the characterisation of involved genes at the molecular level either by insertional mutagenesis or gene knock-out.