Papers by Diwaker Tripathi
<p>(a) Schematic diagram of the constructs tested for leucine independent growth and β-gala... more <p>(a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; blue boxes, IYSV N (N1-full length, amino acids 1–273; N2, amino acids 1–90; N3, amino acids 91–180; N4, amino acids 181–220; N5, amino acids 221–273); yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).</p
<p><sup>a</sup> Primer names with the suffix ‘F’ are forward primers, while tho... more <p><sup>a</sup> Primer names with the suffix ‘F’ are forward primers, while those with ‘R’ are reverse primers.</p><p><sup>b</sup> Sequence of primer, where the underlined nucleotides are <i>att</i> sequence for the Gateway cloning.</p><p>List of primers used for bimolecular fluorescence complementation and pull down assays.</p
<p>(a) Schematic diagram of the constructs tested for leucine independent growth and β-gala... more <p>(a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; blue boxes, IYSV N (N1-full length, amino acids 1–273; N2, amino acids 1–90; N3, amino acids 91–180; N4, amino acids 181–220; N5, amino acids 221–273), white box, full length <i>Cauliflower mosaic virus</i> P6 (amino acids 1–520). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).</p
<p>Self-interaction of nucleocapsid; N (1) and movement; NSm (2) proteins, and cross-intera... more <p>Self-interaction of nucleocapsid; N (1) and movement; NSm (2) proteins, and cross-interaction of IYSV N and NSm proteins (3). ‘Load’ above the column represents the protein content initially loaded to the MBP column; ‘wash’ above the column comprises of the protein not adhering to the column; ‘elution’ was the material that attached to the column and was desorbed by the addition of maltose. Proteins were subjected to protein gel blot analysis and probed with anti-GST primary antibody. (1.a) MBP-tagged N mixed with GST-tagged N. (1.b) MBP alone mixed with GST-tagged N. (1.c) MBP-tagged N mixed with GST alone. (1.d) GST alone mixed with MBP alone. (1.e) The same combination as 1.a, except that proteins were first subjected to RNase treatment. (2.a) MBP-tagged NSm mixed with GST-tagged NSm. (2.b) MBP alone mixed with GST-tagged NSm. (2.c) MBP-tagged NSm mixed with GST alone. (2.d) The same combination as 2.a, except that proteins were first subjected to RNase treatment. (3.a) MBP-tagged N mixed with GST-tagged NSm.</p
BACKGROUND: Localization and interaction studies of viral proteins provide important information ... more BACKGROUND: Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners. PRINCIPAL FINDINGS: Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and β-galactosidase assay. The N protein self-association was f...
<p>Interaction assays were performed in leaf epidermal cells of IYSV-infected transgenic &l... more <p>Interaction assays were performed in leaf epidermal cells of IYSV-infected transgenic <i>Nicotiana benthamiana</i> plants expressing cyan fluorescent protein fused to the nuclear marker histone 2B (CFP-H2B), and cyan endoplasmic reticulum (ER-CFP) marker. Column 1 shows BiFC, column 2 shows localization of CFP-H2B and ER-CFP (nucleus and ER), and column 3 shows a merge of all panels (overlay). The first and second proteins mentioned in each pair of interactors were expressed as C-terminal fusions to the amino-terminal half of YFP and as C-terminal fusions to the carboxy-terminal half of YFP respectively. A set of positive interactions is shown here after testing interactions in all pairwise combinations: (a-c) N/N, (d-f) NSm/ NSm, (g-i) NSm/N. Each micrograph represents a minimum of 50 cells that were examined for interaction. Scale bar = 20μm.</p
<p>Confocal micrographs represent IYSV fusion proteins to the C-terminus of green fluoresce... more <p>Confocal micrographs represent IYSV fusion proteins to the C-terminus of green fluorescent protein (GFP). Columns from left to right show GFP-gene fusion or free GFP (1), RFP-H2B (2), and the overlay of the images (3). (a-c). IYSV N fusion with GFP in RFP-H2B plants; (d-f). Free GFP in RFP-H2B plants. Each micrograph represents minimum 50 cells that were examined for localization. Scale bar = 20μm.</p
<p>(a) Schematic diagram of the constructs tested for leucine independent growth and β-gala... more <p>(a) Schematic diagram of the constructs tested for leucine independent growth and β-galactosidase activity. Black box, LexA DBD in pEG202; hatched box, B42 TAD in pJG4-5; yellow boxes, IYSV NSm (NSm1-full length, amino acids 1–312; NSm2, amino acids 1–160; NSm3, amino acids 100–200; NSm4, amino acids 201–312); white box, full length <i>Cauliflower mosaic virus</i> P6 (amino acids 1–520). Numbers to the left of each pair of constructions correspond to the β-galactosidase assays shown in (b). (b) β-galactosidase activity of yeast transformants expressing constructs as shown in (a).</p
<p>Confocal micrographs represent IYSV fusion proteins to the C-terminus of green fluoresce... more <p>Confocal micrographs represent IYSV fusion proteins to the C-terminus of green fluorescent protein (GFP). Columns from left to right show GFP-gene fusion (1), RFP-H2B (2), ER-RFP (3), and the overlay of the images (4). (a-d) GFP-IYSV N co-expression with RFP-H2B and ER-RFP; (e-h) GFP-IYSV NSm co-expression with RFP-H2B and ER-RFP; (i-l) GFP-IYSV NSs co-expression with RFP-H2B and ER-RFP; (m-p) GFP-IYSV G<sub>N</sub> co-expression with RFP-H2B and ER-RFP; (q-t) GFP-IYSV G<sub>C</sub> co-expression with RFP-H2B and ER-RFP. Each micrograph represents a minimum of 50 cells that were examined for localization. Scale bar = 20μm.</p
Frontiers in Plant Science, 2020
The purinoceptor P2K1/DORN1 recognizes extracellular ATP, a damage-associated molecular pattern (... more The purinoceptor P2K1/DORN1 recognizes extracellular ATP, a damage-associated molecular pattern (DAMP) released upon cellular disruption by wounding and necrosis, which in turn, boost plant innate immunity. P2K1 is known to confer plant resistance to foliar biotrophic, hemi-biotrophic, and necrotrophic pathogens. However, until now, no information was available on its function in defense against root pathogens. In this report, we describe the contribution of P2K1 to resistance in Arabidopsis against Rhizoctonia solani, a broad host range, necrotrophic soilborne fungal pathogen. In pot assays, the Arabidopsis P2K1 overexpression line OxP2K1 showed longer root length and a greater rosette surface area than wild type in the presence of the pathogen. In contrast, the knockout mutant dorn1-3 and the double mutant rbohd/f, defective in two subunits of the respiratory burst complex NADPH oxidase, exhibited significant reductions in shoot and root lengths and rosette surface area compared to wild type when the pathogen was present. Expression of PR1, PDF1.2, and JAZ5 in the roots was reduced in dorn1-3 and rbohd/f and elevated in OxP2K1 relative to wild type, indicating that the salicylate and jasmonate defense signaling pathways functioned in resistance. These results indicated that a DAMP-mediated defense system confers basal resistance against an important root necrotrophic fungal pathogen.
PLOS ONE, 2019
Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting ... more Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting with plant proteins. In the old world, this group of viruses are usually associated with betasatellite that induces severe disease symptoms by encoding a protein, βC1, which is a pathogenicity determinant. Here, we show that βC1 encoded by Cotton leaf curl Multan betasatellite (CLCuMB) requires Gossypium hirsutum calmodulin-like protein 11 (Gh-CML11) to infect cotton. First, we used the in silico approach to predict the interaction of CLCuMB-βC1 with Gh-CML11. A number of sequence-and structure-based in-silico interaction prediction techniques suggested a strong putative binding of CLCuMB-βC1 with Gh-CML11 in a Ca +2-dependent manner. In-silico interaction prediction was then confirmed by three different experimental approaches: The Gh-CML11 interaction was confirmed using CLCuMB-βC1 in a yeast two hybrid system and pull down assay. These results were further validated using bimolecular fluorescence complementation system showing the interaction in cytoplasmic veins of Nicotiana benthamiana. Bioinformatics and molecular studies suggested that CLCuMB-βC1 induces the overexpression of Gh-CML11 protein and ultimately provides calcium as a nutrient source for virus movement and transmission. This is the first comprehensive study on the interaction between CLCuMB-βC1 and Gh-CML11 proteins which provided insights into our understating of the role of βC1 in cotton leaf curl disease.
Frontiers in Plant Science, 2019
Cotton leaf curl disease (CLCuD) caused by viruses of genus Begomovirus is a major constraint to ... more Cotton leaf curl disease (CLCuD) caused by viruses of genus Begomovirus is a major constraint to cotton (Gossypium hirsutum) production in many cotton-growing regions of the world. Symptoms of the disease are caused by Cotton leaf curl Multan betasatellite (CLCuMB) that encodes a pathogenicity determinant protein, βC1. Here, we report the identification of interacting regions in βC1 protein by using computational approaches including sequence recognition, and binding site and interface prediction methods. We show the domain-level interactions based on the structural analysis of G. hirsutum SnRK1 protein and its domains with CLCuMB-βC1. To verify and validate the in silico predictions, three different experimental approaches, yeast two hybrid, bimolecular fluorescence complementation and pull down assay were used. Our results showed that ubiquitin-associated domain (UBA) and autoinhibitory sequence (AIS) domains of G. hirsutum-encoded SnRK1 are involved in CLCuMB-βC1 interaction. This is the first comprehensive investigation that combined in silico interaction prediction followed by experimental validation of interaction between CLCuMB-βC1 and a host protein. We demonstrated that data from computational biology could provide binding site information between CLCuD-associated viruses/satellites and new hosts that lack known binding site information for protein-protein interaction studies. Implications of these findings are discussed.
Plant Signaling & Behavior, 2018
Damage-associated molecular patterns (DAMPs), such as extracellular ATP, act as danger signals in... more Damage-associated molecular patterns (DAMPs), such as extracellular ATP, act as danger signals in response to biotic and abiotic stresses. Extracellular ATP is perceived by a plant purinoceptor, P2 receptor kinase 1 (P2K1), inducing downstream signaling for defense responses. How ATP induces these defense responses has not been well studied. A recent study by Tripathi et al. (Plant Physiology, 176: 511-523, 2018) revealed a synergistic interaction between extracellular ATP and jasmonate (JA) signaling during plant defense responses. This signaling crosstalk requires the formation of secondary messengers, i.e., cytosolic calcium, reactive oxygen species, and nitric oxide. This finding has given a new direction towards understanding the defense signals activated by DAMPs. In this addendum, we discuss possible insights into how extracellular ATP signaling interacts with the JA signaling pathway for plant defense responses.
Current Plant Biology, 2019
Any interaction of plants with phytopathogens involves the generation of various chemical molecul... more Any interaction of plants with phytopathogens involves the generation of various chemical molecules that are critical for activation of their defense machinery. One of the chemicals, salicylic acid (SA) induces systemic acquired resistance (SAR) in plants. The activation of SAR provides a broad-spectrum resistance against a wide range of related or unrelated pathogens. There has been considerable progress in the biochemical and molecular understanding of SAR activation in various plants. In addition, several chemicals including SA and its analogs are known to provide a direct or indirect defense against pathogens when applied to plants. Molecular mechanism of plant defense induced by synthetic chemical inducers is not very well understood. This review highlights the importance of salicylic acid and its most studied analog, Acibenzolar-S-methyl in inducing SAR and it also provides a description of other major chemical elicitors of plant defenses and their possible molecular mechanism.
Plant physiology, Jan 27, 2017
Damaged cells send various signals to stimulate defense responses. Recent identification and gene... more Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea. The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1-3. Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those e...
Mechanism of Plant Hormone Signaling under Stress, 2017
The Formation, Structure and Activity of Phytochemicals, 2015
Salicylic acid (SA) is an important plant hormone with a wide range of effects on plant growth an... more Salicylic acid (SA) is an important plant hormone with a wide range of effects on plant growth and metabolism. Plants lacking SA exhibit enhanced susceptibility to pathogens. SA plays important signaling roles in resistance against biotrophic and hemi-biotrophic phytopathogens. It is synthesized in plastids along two pathways, one involving phenylalanine ammonia lyase (PAL) and the other isochorismate synthase (ICS). In Arabidopsis, during immune response most SA is synthesized through the ICS-dependent pathway, but clearly an ICS-independent pathway also exists. Several SA effector proteins have been identified and characterized which mediate downstream SA signaling. This includes SABP, a catalase, SABP2, a methyl salicylate esterase, SABP3, a carbonic anhydrase, NPR1 (non-expressor of pathogenesis-related 1), NPR3 (a NPR1 paralog), and NPR4 (another NPR1 paralog). NPR3 and NPR4 regulate the turnover of NPR1, a process which plays a key role in activating defense gene expression. The role of SA in abiotic stress signaling is gradually becoming clearer. Various components of SA signaling in biotic stress also appear to impact abiotic stress signaling.
Salicylic acid (SA) is an important signal in various plant processes. It is well known and widel... more Salicylic acid (SA) is an important signal in various plant processes. It is well known and widely studied for its role in plant disease resistance. Several proteins, which physically interact with SA has been identified and characterized for their possible role in disease resistance signaling. These plant proteins bind to SA with varying affinity and they differ considerably in their structure and activity. The protein, which binds to SA with highest affinity amongst all the characterized SA-binding proteins, is SABP2. It is a 29-kDa protein and has esterase like enzymatic activity. It is able to use plant synthesized methyl salicylate as a substrate and convert it into SA, which triggers disease resistance in plants. Silencing of SABP2 makes plants more susceptible to pathogens and their capacity to induce SAR is severely compromised. The esterase activity of SABP2 is required to process the phloem mobile signal, MeSA in distal uninoculated tissues to induce resistance. The bindin...
PloS one, 2015
Localization and interaction studies of viral proteins provide important information about their ... more Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners. Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and β-galactosidase assay. The N protein self-association was found to be mediated through the ...
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Papers by Diwaker Tripathi