Browsing by Author "Sinha, Manisha"

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BAR domain is essential for early endosomal trafficking and dynamics in Ascochyta rabiei
(Springer Nature Publishing AG, 2023) Shree, Ankita; Sinha, Manisha; Verma, Praveen K.
Ascochyta blight disease is a devastating disease caused by the fungal pathogen Ascochyta rabiei that threatens chickpea production around the globe. Endocytic mechanism has a significant role in fungal growth and virulence. The underlying biology of biogenesis of central component of endocytosis viz Rab5 vesicles, is not completely understood. The involvement of F-BAR domain containing protein (ArF-BAR) in various cellular processes that collectively make ArF-BAR as an important virulence determinant. Here, we report that ArF-BAR is involved in biogenesis and motility of early endosome. In the absence of ArF-BAR gene (Δarf-bar), fungal mutants exhibited reduced number of EGFP coated ArRab5 vesicles, along with the considerable reduction in their dynamics. Here, we show that ArF-BAR interacts with clathrin light chain (ArCLC), specifically with its F-BAR domain. These findings suggests the novel role of ArF-BAR in biogenesis and dynamics of early endosome. Additionally, ArF-BAR is involved in clathrin-mediated mechanism of endocytosis which is required for host infection and disease development. Identification of this pathway offers new impending targets for disease intervention in plants.
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.
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.
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.