Browsing by Author "Prajapati, Ramgopal"

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The Ca2+ channel CNGC19 regulates Arabidopsis defense against spodoptera herbivory
(American Society of Plant Biologists, 2019) Meena, Mukesh Kumar; Prajapati, Ramgopal; Krishna, Deepthi; Divakaran, Keerthi; Pandey, Yogesh; Reichelt, Michael; Mathew, M.K.; Boland, Wilhelm; Mithöfer, Axel; Vadassery, Jyothilakshmi
Cellular calcium elevation is an important signal used by plants for recognition and signaling of environmental stress. Perception of the generalist insect, Spodoptera litura, by Arabidopsis thaliana activates cytosolic Ca2+ elevation, which triggers downstream defense. However, not all the Ca2+ channels generating the signal have been identified, nor are their modes of action known. We report on a rapidly activated, leaf vasculature- and plasma membrane-localized, CYCLIC NUCLEOTIDE GATED CHANNEL19 (CNGC19), which activates herbivory-induced Ca2+ flux and plant defense. Loss of CNGC19 function results in decreased herbivory defense. The cngc19 mutant shows aberrant and attenuated intra-vascular Ca2+ fluxes. CNGC19 is a Ca2+ permeable channel, as hyperpolarization of CNGC19-expressing Xenopus oocytes in the presence of both cAMP and Ca2+ results in Ca2+ influx. Breakdown of Ca2+-based defence in cngc19 mutants leads to a decrease in herbivory-induced JA-Ile biosynthesis and expression of JA responsive genes. cngc19 mutants are deficient in aliphatic glucosinolate accumulation and hyperaccumulate its precursor, methionine. CNGC19 modulates aliphatic glucosinolate biosynthesis in tandem with BRANCHED-CHAIN AMINO ACID TRANSAMINASE4 (BCAT4), which is involved in the chain elongation pathway of Met-derived glucosinolates. Furthermore, CNGC19 interacts with herbivory-induced CALMODULIN2 (CaM2) in planta. Together, our work reveals a key mechanistic role for the Ca2+ channel CNGC19 in the recognition of herbivory and the activation of defense signaling.
Forward genetic screen using transgenic calcium reporter aequorin to identify novel targets in calcium signaling
(Journal of Visualized Experiment, 2020) Mittal, Deepika; Mishra, Shruti; Prajapati, Ramgopal; Vadassery, Jyothilakshmi
Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets. The video component of this article can be found at https://www.jove.com/video/61259/
Jasmonic acid (JA) induced-calcium elevation in Arabidopsis is highly variable due to time of day and conversion to JA-Ile
(Springer Nature Publishing AG, 2020) Prajapati, Ramgopal; Mittal, Deepika; Meena, Mukesh Kumar; Vadassery, Jyothilakshmi
Plants have evolved mechanisms to effectively anticipate environmental changes via diurnal rhythmicity (day/night) maintained by the circadian clock. Jasmonic acid biosynthesis and signalling are known to be under the control of the circadian clock. Both JA and its bioactive form jasmonoyl-L-isoleucine (JA-Ile) when externally added can induce a cytosolic Ca2? influx in Arabidopsis thaliana. JA and JA-Ile induced Ca2? is poorly understood and often used interchangeably to study Ca2? regulation of jasmonates. We attempted to understand if they are similar and if diurnal rhythms or time of day regulate them. JA induced Cacyt 2? signature is variable according to time-of-day in Arabidopsis. JA is sensed in two ways according to the time-of-day (a) directly sensed as JA and induces Cacyt 2? elevation (b) JA gets converted into the JA-Ile by JAR1 and is sensed as JA-Ile, which we proved using jar1-1*aequorin. This twin sensing mode is responsible for variability in JA induced Cacyt 2? signature. We further suggest caution when using JA as a stimulant for Cacyt 2? elevation measurements to compare wild-type (Col-0 transformed with pMAQ2; transgenic aequorin) and effect of different mutations. On the other hand bioactive JA-Ile induced Cacyt 2? signature is constant diurnally with maximum amplitude at dawn which coincides with maximum sensitivity of JA-Ile receptor, COI1 and increased VSP2 expression. From the above study we conclude that JA-Ile induced Cacyt 2? elevation is a better read-out than the highly variable JA-induced Cacyt 2? elevations to study the output pathways.