Browsing by Author "Tilak Raj Sharma"

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Discovery and profiling of small RNAs from Puccinia triticina by deep sequencing and identification of their potential targets in wheat
(Springer Verlag, 2019) Himanshu Dubey; Kanti Kiran; Rajdeep Jaswal; Priyanka Jain; Arvind M Kayastha; Subhash C Bhardwaj; Tapan Kumar Mondal; Tilak Raj Sharma
Cross-kingdom RNAi is a well-documented phenomenon where sRNAs generated by host and pathogens may govern resistance or susceptible phenotypes during host-pathogen interaction. With the first example of the direct involvement of fungal generated sRNAs in virulence of plant pathogenic fungi Botrytis cinerea and recently from Puccinia striiformis f. sp. tritici, we attempted to identify sRNAs in Puccinia triticina (P. triticina). Four sRNA libraries were prepared and sequenced using Illumina sequencing technology and a total of ~ 1–1.28 million potential sRNAs and two microRNA-like small RNA (mil-RNAs) candidates were identified. Computational prediction of targets using a common set of sRNAs and P. triticina mil-RNAs (pt-mil-RNAs) within P. triticina and wheat revealed the majority of the targets as repetitive elements in P. triticina whereas in wheat, the target genes were identified to be involved in many biological processes including defense-related pathways. We found 9 receptor-like kinases (RLKs) and 14 target genes of each related to reactive oxygen species (ROS) pathway and transcription factors respectively, including significant numbers of target genes from various other categories. Expression analysis of twenty selected sRNAs, targeting host genes pertaining to ROS related, disease resistance, metabolic processes, transporter, apoptotic inhibitor, and transcription factors along with two pt-mil-RNAs by qRT-PCR showed distinct patterns of expression of the sRNAs in urediniospore-specific libraries. In this study, for the first time, we report identification of novel sRNAs identified in P. triticina including two pt-mil-RNAs that may play an important role in biotrophic growth and pathogenicity. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature.
Identification and characterization of Dicer-like genes in leaf rust pathogen (Puccinia triticina) of wheat
(Springer, 2020) Himanshu Dubey; Kanti Kiran; Rajdeep Jaswal; Subhash C. Bhardwaj; Tapan Kumar Mondal; Neha Jain; N.K. Singh; Arvind M. Kayastha; Tilak Raj Sharma
Puccinia triticina (P. triticina) is one of the most devastating fungal pathogens of wheat which causes significant annual yield loss to the crop. Understanding the gene regulatory mechanism of the biotrophic pathogen is one of the important aspects of host-pathogen interaction studies. Dicer-like genes are considered as important mediators of RNAi-based gene regulation. In this study, we report the presence of three Dicer-like genes (Pt-DCL1, Pt-DCL2, Pt-DCL3) in P. triticina genome identified through computational and biological analyses. Quantitative real-time PCR studies revealed an increase in the expression of these genes in germinating spore stages. Heterologous expression combined with mass spectrometry analysis of Pt-DCL2 confirmed the presence of a canonical Dicer-like gene in P. triticina. Phylogenetic analysis of the Pt-DCLs with the Dicer-like proteins from other organisms showed a distinct cluster of rust pathogens from the order Pucciniales. The results indicated a species-specific duplication of Dicer-like genes within the wheat rust pathogens. This study, for the first time, reports the presence of Dicer-dependent RNAi pathway in P. triticina that may play a role in gene regulatory mechanism of the pathogen during its development. Our study serves as a vital source of information for further RNAi-based molecular studies for better understanding and management of the wheat leaf rust disease. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature.
Magnaporthe oryzae encoded effector protein AvrPi54 interacts in vivo with rice encoded cognate resistance protein Pi54 at the host plasma membrane
(Springer, 2023) Banita Kumari Saklani; Soham Ray; Kirti Arora; Ravi Kumar Asthana; Tilak Raj Sharma
Plant pathogens deliver effector proteins which enter plant cells in order to establish the disease. The resistance (R) proteins in the plants can recognize some of these effectors and initiate a race-specific resistance response. These effectors which aid in triggering defense signaling in host upon R-protein mediated recognition are termed as Avirulence (Avr) proteins. AvrPi54 is an Avr protein from an ascomycete fungus, Magnaporthe oryzae, which causes hypersensitive response in rice cells containing broad-spectrum R-gene, Pi54. In this study, we have analysed several characteristics of AvrPi54 protein computationally and studied its subcellular localization in presence and absence of Pi54 protein. Our results suggest AvrPi54 is a small hydrophobic protein with no significant homology with any known proteins available in any publicly available database, till date. Transient expression of eYFP fused AvrPi54 protein (AvrPi54-eYFP) in epidermis of onion bulb suggested its translocation to nucleus and plasma membrane. Further, Bimolecular fluorescence complementation (BiFC) assay in the tobacco leaf cells depicted that Pi54 and AvrPi54 proteins interact at plasma membrane. Based on the obtained evidences, we infer that AvrPi54 probably is functional in nucleus of rice cells, either as a transcription factor or as a chromatin remodeler, thus playing important role in immunocompromization of the host. However, presence of Pi54 protein in the host cell might restrict the trespass of AvrPi54 into the nucleus by interacting directly with it at the plasma membrane and thus eliciting defense response. © 2022, The Author(s), under exclusive licence to Society for Plant Biochemistry and Biotechnology.
SpeedFlower: a comprehensive speed breeding protocol for indica and japonica rice
(John Wiley and Sons Inc, 2024) Pramod Gorakhanath Kabade; Shilpi Dixit; Uma Maheshwar Singh; Shamshad Alam; Sankalp Bhosale; Sanjay Kumar; Shravan Kumar Singh; Jyothi Badri; Nadimpalli Rama Gopala Varma; Sanjay Chetia; Rakesh Singh; Sharat Kumar Pradhan; Shubha Banerjee; Rupesh Deshmukh; Suresh Prasad Singh; Sanjay Kalia; Tilak Raj Sharma; Sudhanshu Singh; Hans Bhardwaj; Ajay Kohli; Arvind Kumar; Pallavi Sinha; Vikas Kumar Singh
To increase rice yields and feed billions of people, it is essential to enhance genetic gains. However, the development of new varieties is hindered by longer generation times and seasonal constraints. To address these limitations, a speed breeding facility has been established and a robust speed breeding protocol, SpeedFlower is developed that allows growing 4–5 generations of indica and/or japonica rice in a year. Our findings reveal that a high red-to-blue (2R > 1B) spectrum ratio, followed by green, yellow and far-red (FR) light, along with a 24-h long day (LD) photoperiod for the initial 15 days of the vegetative phase, facilitated early flowering. This is further enhanced by 10-h short day (SD) photoperiod in the later stage and day and night temperatures of 32/30 °C, along with 65% humidity facilitated early flowering ranging from 52 to 60 days at high light intensity (800 μmol m−2 s−1). Additionally, the use of prematurely harvested seeds and gibberellic acid treatment reduced the maturity duration by 50%. Further, SpeedFlower was validated on a diverse subset of 198 rice accessions from 3K RGP panel encompassing all 12 distinct groups of Oryza sativa L. classes. Our results confirmed that using SpeedFlower one generation can be achieved within 58–71 days resulting in 5.1–6.3 generations per year across the 12 sub-groups. This breakthrough enables us to enhance genetic gain, which could feed half of the world's population dependent on rice. © 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Variation in the LRR region of Pi54 protein alters its interaction with the AvrPi54 protein revealed by in silico analysis
(Public Library of Science, 2019) Chiranjib Sarkar; Banita Kumari Saklani; Pankaj Kumar Singh; Ravi Kumar Asthana; Tilak Raj Sharma
Rice blast, caused by the ascomycete fungus Magnaporthe oryzae is a destructive disease of rice and responsible for causing extensive damage to the crop. Pi54, a dominant blast resistance gene cloned from rice line Tetep, imparts a broad spectrum resistance against various M. oryzae isolates. Many of its alleles have been explored from wild Oryza species and landraces whose sequences are available in the public domain. Its cognate effector gene AvrPi54 has also been cloned from M. oryzae. Complying with the Flor’s gene-for-gene system, Pi54 protein interacts with AvrPi54 protein following fungal invasion leading to the resistance responses in rice cell that prevents the disease development. In the present study Pi54 alleles from 72 rice lines were used to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. The physiochemical properties of these proteins varied due to the nucleotide level polymorphism. The ab initio tertiary structures of these R- and Avr- proteins were generated and subjected to the in silico interaction. In this interaction, the residues in the LRR region of R- proteins were shown to interact with the Avr protein. These R proteins were found to have variable strengths of binding due to the differential spatial arrangements of their amino acid residues. Additionally, molecular dynamic simulations were performed for the protein pairs that showed stronger interaction than Pi54tetep (original Pi54 from Tetep) protein. We found these proteins were forming h-bond during simulation which indicated an effective binding. The root mean square deviation values and potential energy values were stable during simulation which validated the docking results. From the interaction studies and the molecular dynamics simulations, we concluded that the AvrPi54 protein interacts directly with the resistant Pi54 proteins through the LRR region of Pi54 proteins. Some of the Pi54 proteins from the landraces namely Casebatta, Tadukan, Varun dhan, Govind, Acharmita, HPR-2083, Budda, Jatto, MTU-4870, Dobeja-1, CN-1789, Indira sona, Kulanji pille and Motebangarkaddi cultivars show stronger binding with the AvrPi54 protein, thus these alleles can be effectively used for the rice blast resistance breeding program in future. © 2019 Sarkar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.