Regulation of Arabidopsis defense responses against Spodoptera littoralisby CPK-mediated calcium signaling

Abstract

BackgroundPlant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable.ResultsTo investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response.ConclusionsThese results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses.

Mechanisms of plant defense against insect herbivores

“…35,141 Induction of H 2 O 2 has been studied in oat, wheat, barley and groundnut against D. noxia, R. padi, Schizaphis graminum Rond., H. armigera and S. litura. 4,5,132,139 Argandona et al 144 observed induction of H 2 O 2 in barley infested with S. graminum after 20 min of infestation, indicating that H 2 O 2 could be the beginning of a cascade of physiological and molecular events leading to production of further defensive components, and protection of plants from subsequent damage. ROS mediate the defensive gene activation and establish additional defenses by regulating the transcription and/or by interacting with other signal components like phosphorylation in plant systems in response to a variety of stresses.…”

“…35 Herbivore induced signals rapidly spread over the leaf and leads to a strong Ca 2+ -dependent transmembrane potential (Vm) depolarization in the damage zone, and is followed by a transient Vm hyperpolarization in the surrounding area, and a constant depolarization at distances greater than 6-7 mm. 35,139 Organelle and apoplastic fluid Ca 2+ concentration is generally higher (about 10 4 to 10 5 times) as compared with that in the cytosol (100 and 200 nM.). However, upon insect attack, the cytosolic Ca 2+ increases, which in turn activates the calcium-sensing proteins such as calmodulin, calmodulin-binding proteins, and calcium-dependent protein kinases (CDPKs) that promote the signaling events such as, phosphorylation and transcriptional change.…”

“…However, upon insect attack, the cytosolic Ca 2+ increases, which in turn activates the calcium-sensing proteins such as calmodulin, calmodulin-binding proteins, and calcium-dependent protein kinases (CDPKs) that promote the signaling events such as, phosphorylation and transcriptional change. 139,140 However, CDPKs are the important proteins against biotic and abiotic stresses, which form Ca 2+ sensors that contain a protein kinase domain and a calmodulin like domain (including an EF-hand calcium-binding site) in a single polypeptide. 130,141 NtCDPK2 regulates the activation of stress-induced MAP kinases in tobacco.…”

“…142 Involvement of two Arabidopsis CPKs (CPK3 and CPK13) in herbivory-induced signaling network through HsfB2a-mediated regulation of the defense-related transcriptional machinery has been observed in tobacco. 139 Damage by S. littoralis larvae on Phaseolus lunatus L. induced Ca 2+ not only in cells adjacent to the feeding site, but throughout the leaf. 35 Expression of calmodulin binding proteins involved in plant defense signaling increased considerably in wheat damaged by D. noxia and Arabidopsis by M. persicae.…”

Biological Activity of Insecticidal Toxins: Structural Basis, Site-Directed Mutagenesis and Perspectives

“…Plant defensins are antimicrobial proteins with eight conserved cysteines and four disulfide bridges. Defensins attack lepidopteran -amylases, causing feeding inhibition (Kanchiswamy et al, 2010;Rayapuram & Baldwin, 2008). Plant glucanases, chitinases, lectins and dehydrins are induced after attack by lepidopteran and coleopteran pests (Ralph et al, 2006).…”