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Abstract Background Plant 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. Results To 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. Conclusions These 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.

Microbes and enzymes play essential roles in soil and plant rhizosphere ecosystem functioning. However, fungicides and plant root secretions may impact the diversity and abundance of microbiota structure and enzymatic activities in the plant rhizosphere. In this study, we analyzed soil samples from the rhizosphere of four cannabinoid-rich hemp ( Cannabis sativa ) cultivars (Otto II, BaOx, Cherry Citrus, and Wife) subjected to three different treatments (natural infection, fungal inoculation, and fungicide treatment). DNA was extracted from the soil samples, 16S rDNA was sequenced, and data were analyzed for diversity and abundance among different fungicide treatments and hemp cultivars. Fungicide treatment significantly impacted the diversity and abundance of the hemp rhizosphere microbiota structure, and it substantially increased the abundance of the phyla Archaea and Rokubacteria. However, the abundances of the phyla Pseudomonadota and Gemmatimonadetes were substantially decreased in treatments with fungicides compared to those without fungicides in the four hemp cultivars. In addition, the diversity and abundance of the rhizosphere microbiota structure were influenced by hemp cultivars. The influence of Cherry Citrus on the diversity and abundance of the hemp rhizosphere microbiota structure was less compared to the other three hemp cultivars (Otto II, BaOx, and Wife). Moreover, fungicide treatment affected enzymatic activities in the hemp rhizosphere. The application of fungicides significantly decreased enzyme abundance in the rhizosphere of all four hemp cultivars. Enzymes such as dehydrogenase, dioxygenase, hydrolase, transferase, oxidase, carboxylase, and peptidase significantly decreased in all the four hemp rhizosphere treated with fungicides compared to those not treated. These enzymes may be involved in the function of metabolizing organic matter and degrading xenobiotics. The ecological significance of these findings lies in the recognition that fungicides impact enzymes, microbiota structure, and the overall ecosystem within the hemp rhizosphere.

Gossypol is an important allelochemical produced by the subepidermal glands of some cotton varieties and important for their ability to respond to changing biotic stress by exhibiting antibiosis against some cotton pests. Plant growth-promoting rhizobacteria (PGPR) are root-colonizing bacteria that increase plant growth and often elicit defence against plant pathogens and insect pests. Little is known about the effect of PGPR on cotton plant-insect interactions and the potential biochemical and molecular mechanisms by which PGPR enhance cotton plant defence. Here, we report that PGPR (Bacillus spp.) treated cotton plants showed significantly higher levels of gossypol compared with untreated plants. Similarly, the transcript levels of the genes (i.e. (+)-δ-cadinene synthase gene family) involved in the biosynthesis of gossypol were higher in PGPR-treated plants than in untreated plants. Furthermore, the levels of jasmonic acid, an octadecanoid-derived defence-related phytohormone and the transcript level of jasmonic acid responsive genes were higher in PGPR-treated plants than in untreated plants. Most intriguingly, Spodoptera exigua showed reduced larval feeding and development on PGPR-treated plants. These findings demonstrate that treatment of plants with rhizobacteria may induce significant biochemical and molecular changes with potential ramifications for plant-insect interactions., (© 2015 John Wiley & Sons Ltd.)

Background: Plant induced defense against herbivory are generally associated with metabolic costs that result in the allocation of photosynthates from growth and reproduction to the synthesis of defense compounds. Therefore, it is essential that plants are capable of sensing and differentiating mechanical injury from herbivore injury. Studies have shown that oral secretions (OS) from caterpillars contain elicitors of induced plant responses. However, studies that shows whether these elicitors originated from salivary glands or from other organs associated with feeding, such as the ventral eversible gland (VEG) are limited. Here, we tested the hypothesis that the secretions from the VEG gland of Spodoptera exigua caterpillars contain elicitors that induce plant defenses by regulating the expression of genes involved in the biosynthesis of volatile organic compounds (VOCs) and other defense-related genes. To test this hypothesis, we quantified and compared the activity of defense-related enzymes, transcript levels of defense-related genes and VOC emission in tomato plants damaged by S. exigua caterpillars with the VEG intact (VEGI) versus plants damaged by caterpillars with the VEG ablated (VEGA)., Results: The quantified defense-related enzymes (i.e. peroxidase, polyphenol oxidase, and lipoxigenase) were expressed in significantly higher amounts in plants damaged by VEGI caterpillars than in plants damaged by VEGA caterpillars. Similarly, the genes that encode for the key enzymes involved in the biosynthesis of jasmonic acid and terpene synthase genes that regulate production of terpene VOCs, were up-regulated in plants damaged by VEGI caterpillars. Moreover, the OS of VEGA caterpillars were less active in inducing the expression of defense genes in tomato plants. Increased emissions of VOCs were detected in the headspace of plants damaged by VEGI caterpillars compared to plants damaged by VEGA caterpillars., Conclusion: These results suggest that the VEG of S. exigua caterpillars contains elicitors of late plant defense signaling in tomato which trigger defense-related enzymatic activity, regulate expression of defense-related genes, and induce emission of plant VOCs. These signaling cascades may have important ramifications for plant-insect and tritrophic interactions.

Background: Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses., Methodology/principal Findings: Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca(2+)](cyt)) and H(2)O(2) production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca(2+)](cyt) and H(2)O(2). Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides., Conclusions/significance: By studying early and late responses of G. biloba to herbivory, we provided the first evidence that this "living fossil" plant responds to herbivory with the same defense mechanisms adopted by the most recent angiosperms.

Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plant's response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insect's response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants, mechanically damaged leaves, and leaves damaged by herbivores. The terpenoid quantitative gene expressions (qPCR) were then assayed. Upon herbivory, M. aquatica synthesizes and emits (+)-menthofuran, which acts as a deterrent to C. herbacea. Herbivory was found to up-regulate the expression of genes involved in terpenoid biosynthesis. The increased emission of (+)-menthofuran was correlated with the upregulation of (+)-menthofuran synthase.