External jasmonic acid isoleucine mediates amplification of plant elicitor peptide receptor (PEPR) and jasmonate-based immune signalling.

Jasmonic acid-isoleucine (JA-Ile) is a plant defence hormone whose cellular levels are elevated upon herbivory and regulate defence signalling. Despite their pivotal role, our understanding of the rapid cellular perception of bioactive JA-Ile is limited. This study identifies cell type-specific JA-Ile-induced Ca2+ signal and its role in self-amplification and plant elicitor peptide receptor (PEPR)-mediated signalling. Using the Ca2+ reporter, R-GECO1 in Arabidopsis, we have characterized a monophasic and sustained JA-Ile-dependent Ca2+ signature in leaf epidermal cells. The rapid Ca2+ signal is independent of positive feedback by the JA-Ile receptor, COI1 and the transporter, JAT1. Microarray analysis identified up-regulation of receptors, PEPR1 and PEPR2 upon JA-Ile treatment. The pepr1 pepr2 double mutant in R-GECO1 background exhibits impaired external JA-Ile induced Ca2+ cyt elevation and impacts the canonical JA-Ile responsive genes. JA responsive transcription factor, MYC2 binds to the G-Box motif of PEPR1 and PEPR2 promoter and activates their expression upon JA-Ile treatment and in myc2 mutant, this is reduced. External JA-Ile amplifies AtPep-PEPR pathway by increasing the AtPep precursor, PROPEP expression. Our work shows a previously unknown non-canonical PEPR-JA-Ile-Ca2+ -MYC2 signalling module through which plants sense JA-Ile rapidly to amplify both AtPep-PEPR and jasmonate signalling in undamaged cells.

AtSWEET11 and AtSWEET12 transporters function in tandem to modulate sugar flux in plants.

The sugar will eventually be exported transporter (SWEET) members in Arabidopsis, AtSWEET11 and AtSWEET12 are the important sucrose efflux transporters that act synergistically to perform distinct physiological roles. These two transporters are involved in apoplasmic phloem loading, seed filling, and sugar level alteration at the site of pathogen infection. Here, we performed the structural analysis of the sucrose binding pocket of AtSWEET11 and AtSWEET12 using molecular docking followed by rigorous molecular dynamics (MD) simulations. We observed that the sucrose molecule binds inside the central cavity and in the middle of the transmembrane (TM) region of AtSWEET11 and AtSWEET12, that allows the alternate access to the sucrose molecule from either side of the membrane during transport. Both AtSWEET11 and AtSWEET12, shares the similar amino acid residues that interact with sucrose molecule. Further, to achieve more insights on the role of these two transporters in other plant species, we did the phylogenetic and the in-silico analyses of AtSWEET11 and AtSWEET12 orthologs from 39 economically important plants. We reported the extensive information on the gene structure, protein domain and cis-acting regulatory elements of AtSWEET11 and AtSWEET12 orthologs from different plants. The cis-elements analysis indicates the involvement of AtSWEET11 and AtSWEET12 orthologs in plant development and also during abiotic and biotic stresses. Both in silico and in planta expression analysis indicated AtSWEET11 and AtSWEET12 are well-expressed in the Arabidopsis leaf tissues. However, the orthologs of AtSWEET11 and AtSWEET12 showed the differential expression pattern with high or no transcript expression in the leaf tissues of different plants. Overall, these results offer the new insights into the functions and regulation of AtSWEET11 and AtSWEET12 orthologs from different plant species. This might be helpful in conducting the future studies to understand the role of these two crucial transporters in Arabidopsis and other crop plants.

Molecular mechanisms of Piriformospora indica mediated growth promotion in plants.

Piriformospora indica is a root endophyte having a vast host range in plants. Plant growth promotion is a hallmark of the symbiotic interaction of P. indica with its hosts. As a plant growth-promoting microorganism, it is important to know the mechanisms involved in growth induction. Hitherto, multiple reports have demonstrated various molecular mechanisms of P. indica-mediated growth promotion, including protein kinase-mediated pathway, enhanced nutrient uptake and polyamine-mediated growth phytohormone elevation. Here, we briefly present a discussion on the state-of-the-art molecular mechanisms of P. indica-mediated growth promotion in host plants, in order to obtain a future prospect on utilization of this microorganism for sustainable agriculture.

Piriformospora indica recruits host-derived putrescine for growth promotion in plants.

Growth promotion induced by the endosymbiont Piriformospora indica has been observed in various plants; however, except growth phytohormones, specific functional metabolites involved in P. indica-mediated growth promotion are unknown. Here, we used a gas chromatography-mass spectrometry-based untargeted metabolite analysis to identify tomato (Solanum lycopersicum) metabolites whose levels were altered during P. indica-mediated growth promotion. Metabolomic multivariate analysis revealed several primary metabolites with altered levels, with putrescine (Put) induced most significantly in roots during the interaction. Further, our results indicated that P. indica modulates the arginine decarboxylase (ADC)-mediated Put biosynthesis pathway via induction of SlADC1 in tomato. Piriformospora indica did not promote growth in Sladc1-(virus-induced gene silencing of SlADC1) lines of tomato and showed less colonization. Furthermore, using LC-MS/MS we showed that Put promoted growth by elevation of auxin (indole-3-acetic acid) and gibberellin (GA4 and GA7) levels in tomato. In Arabidopsis (Arabidopsis thaliana) adc knockout mutants, P. indica colonization also decreased and showed no plant growth promotion, and this response was rescued upon exogenous application of Put. Put is also important for hyphal growth of P. indica, indicating that it is co-adapted by both host and microbe. Taken together, we conclude that Put is an essential metabolite and its biosynthesis in plants is crucial for P. indica-mediated plant growth promotion and fungal growth.

In silico identification of effector proteins from generalist herbivore Spodoptera litura.

BACKGROUND: The common cutworm, Spodoptera litura Fabricius is a leaf and fruit feeding generalist insect of the order Lepidoptera and a destructive agriculture pest. The broad host range of the herbivore is due to its ability to downregulate plant defense across different plants. The identity of Spodoptera litura released effectors that downregulate plant defense are largely unknown. The current study aims to identify genes encoding effector proteins from salivary glands of S. litura (Fab.). RESULTS: Head and salivary glands of Spodoptera litura were used for de-novo transcriptome analysis and effector prediction. Eight hundred ninety-nine proteins from the head and 330 from salivary gland were identified as secretory proteins. Eight hundred eight proteins from the head and 267 from salivary gland proteins were predicted to be potential effector proteins. CONCLUSIONS: This study is the first report on identification of potential effectors from Spodoptera litura salivary glands.

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling.

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.

Calcium channel CNGC19 mediates basal defense signaling to regulate colonization by Piriformospora indica in Arabidopsis roots.

The activation of calcium signaling is a crucial event for perceiving environmental stress. Colonization by Piriformospora indica, a growth-promoting root endosymbiont, activates cytosolic Ca2+ in Arabidopsis roots. In this study, we examined the role and functional relevance of calcium channels responsible for Ca2+ fluxes. Expression profiling revealed that CYCLIC NUCLEOTIDE GATED CHANNEL 19 (CNGC19) is an early-activated gene, induced by unidentified components in P. indica cell-wall extract. Functional analysis showed that loss-of-function of CNGC19 resulted in growth inhibition by P.indica, due to increased colonization and loss of controlled fungal growth. The cngc19 mutant showed reduced elevation of cytosolic Ca2+ in response to P. indica cell-wall extract in comparison to the wild-type. Microbe-associated molecular pattern-triggered immunity was compromised in the cngc19 lines, as evidenced by unaltered callose deposition, reduced cis-(+)-12-oxo-phytodienoic acid, jasmonate, and jasmonoyl isoleucine levels, and down-regulation of jasmonate and other defense-related genes, which contributed to a shift towards a pathogenic response. Loss-of-function of CNGC19 resulted in an inability to modulate indole glucosinolate content during P. indica colonization. CNGC19-mediated basal immunity was dependent on the AtPep receptor, PEPR. CNGC19 was also crucial for P. indica-mediated suppression of AtPep-induced immunity. Our results thus demonstrate that Arabidopsis CNGC19 is an important Ca2+ channel that maintains a robust innate immunity and is crucial for growth-promotion signaling upon colonization by P. indica.

Omega hydroxylated JA-Ile is an endogenous bioactive jasmonate that signals through the canonical jasmonate signaling pathway.

Jasmonates are fatty acid derivatives that control several plant processes including growth, development and defense. Despite the chemical diversity of jasmonates, only jasmonoyl-L-isoleucine (JA-Ile) has been clearly characterized as the endogenous ligand of the jasmonate co-receptors (COI1-JAZs) in higher plants. Currently, it is accepted that ω-hydroxylation of JA-Ile leads to inactivation of the molecule. This study shows that ω-hydroxylated JA-Ile (12-OH-JA-Ile) retains bioactivity and signals through the canonical JA-pathway. The results suggest that 12-OH-JA-Ile differentially activates a subset of JA-Ile co-receptors that may control and/or modulate particular jasmonate dependent responses. It is proposed that after a strong immune response mediated by JA-Ile, the ω-hydroxylated form modulates JA-Ile activated processes thereby improving plant resilience.

The Ca2+ Channel CNGC19 Regulates Arabidopsis Defense Against Spodoptera Herbivory.

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 (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 intravascular Ca2+ fluxes. CNGC19 is a Ca2+-permeable channel, as hyperpolarization of CNGC19-expressing Xenopus oocytes in the presence of both cyclic adenosine monophosphate and Ca2+ results in Ca2+ influx. Breakdown of Ca2+-based defense in cngc19 mutants leads to a decrease in herbivory-induced jasmonoyl-l-isoleucine biosynthesis and expression of JA responsive genes. The 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, which is involved in the chain elongation pathway of Met-derived glucosinolates. Furthermore, CNGC19 interacts with herbivory-induced CALMODULIN2 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.

Spodoptera litura-mediated chemical defense is differentially modulated in older and younger systemic leaves of Solanum lycopersicum.

MAIN CONCLUSION: Metabolite profiling, biochemical assays, and transcript analysis revealed differential modulation of specific induced defense responses in local, older, and younger systemic leaves in Solanum lycopersicum upon Spodoptera litura herbivory. Plants reconfigure their metabolome upon herbivory to induce production of defense metabolites involved in both direct and indirect defenses against insect herbivores. Herbivory mediated leaf-to-leaf systemic induction pattern of primary and non-volatile secondary metabolites is not well studied in tomato. Here, we show that, in cultivated tomato Solanum lycopersicum herbivory by generalist insect, Spodoptera litura results in differential alteration of primary metabolites, majorly sugars and amino acids and specific secondary metabolites in local, younger, and older systemic leaves. Cluster analysis of 55 metabolites identified by GC-MS showed correlation between local and younger systemic leaves. Re-allocation of primary metabolites like glucose and amino acids from the local to systemic leaf was observed. Secondary metabolites chlorogenic acid, caffeic acid, and catechin were significantly induced during herbivory in systemic leaves. Among specific secondary metabolites, chlorogenic acid and catechin significantly inhibits S. litura larval growth in all stages. Local leaf exhibited increased lignin accumulation upon herbivory. Differential alteration of induced defense responses like reactive oxygen species, polyphenol oxidase activity, proteinase inhibitor, cell wall metabolites, and lignin accumulation was observed in systemic leaves. The metabolite alteration also resulted in increased defense in systemic leaves. Thus, comparative analysis of metabolites in local and systemic leaves of tomato revealed a constant re-allocation of primary metabolites to systemic leaves and differential induction of secondary metabolites and induced defenses upon herbivory.

PiHOG1, a stress regulator MAP kinase from the root endophyte fungus Piriformospora indica, confers salinity stress tolerance in rice plants.

In this study, yeast HOG1 homologue from the root endophyte Piriformospora indica (PiHOG1) was isolated and functionally characterized. Functional expression of PiHOG1 in S. cerevisiae ∆hog1 mutant restored osmotolerance under high osmotic stress. Knockdown (KD) transformants of PiHOG1 generated by RNA interference in P. indica showed that genes for the HOG pathway, osmoresponse and salinity tolerance were less stimulated in KD-PiHOG1 compared to the wild-type under salinity stress. Furthermore, KD lines are impaired in the colonization of rice roots under salinity stress of 200 mM NaCl, and the biomass of the host plants, their shoot and root lengths, root number, photosynthetic pigment and proline contents were reduced as compared to rice plants colonized by WT P. indica. Therefore, PiHOG1 is critical for root colonisation, salinity tolerance and the performance of the host plant under salinity stress. Moreover, downregulation of PiHOG1 resulted not only in reduced and delayed phosphorylation of the remaining PiHOG1 protein in colonized salinity-stressed rice roots, but also in the downregulation of the upstream MAP kinase genes PiPBS2 and PiSSK2 involved in salinity tolerance signalling in the fungus. Our data demonstrate that PiHOG1 is not only involved in the salinity response of P. indica, but also helping host plant to overcome salinity stress.

Calmodulin-like protein CML37 is a positive regulator of ABA during drought stress in Arabidopsis.

Plants need to adapt to various stress factors originating from the environment. Signal transduction pathways connecting the recognition of environmental cues and the initiation of appropriate downstream responses in plants often involve intracellular Ca(2+) concentration changes. These changes must be deciphered into specific cellular signals. Calmodulin-like proteins, CMLs, act as Ca(2+) sensors in plants and are known to be involved in various stress reactions. Here, we show that in Arabidopsis 2 different CMLs, AtCML37 and AtCML42 are antagonistically involved in drought stress response. Whereas a CML37 knock-out line, cml37, was highly susceptible to drought stress, CML42 knockout line, cml42, showed no obvious effect compared to wild type (WT) plants. Accordingly, the analysis of the phytohormone abscisic acid (ABA) revealed a significant reduction of ABA upon drought stress in cml37 plants, while in cml42 plants an increase of ABA was detected. Summarizing, our results show that both CML37 and CML42 are involved in drought stress response but show antagonistic effects.

Overexpression of an AP2/ERF Type Transcription Factor OsEREBP1 Confers Biotic and Abiotic Stress Tolerance in Rice.

AP2/ERF-type transcription factors regulate important functions of plant growth and development as well as responses to environmental stimuli. A rice AP2/ERF transcription factor, OsEREBP1 is a downstream component of a signal transduction pathway in a specific interaction between rice (Oryza sativa) and its bacterial pathogen, Xoo (Xanthomonas oryzae pv. oryzae). Constitutive expression of OsEREBP1 in rice driven by maize ubiquitin promoter did not affect normal plant growth. Microarray analysis revealed that over expression of OsEREBP1 caused increased expression of lipid metabolism related genes such as lipase and chloroplastic lipoxygenase as well as several genes related to jasmonate and abscisic acid biosynthesis. PR genes, transcription regulators and Aldhs (alcohol dehydrogenases) implicated in abiotic stress and submergence tolerance were also upregulated in transgenic plants. Transgenic plants showed increase in endogenous levels of α-linolenate, several jasmonate derivatives and abscisic acid but not salicylic acid. Soluble modified GFP (SmGFP)-tagged OsEREBP1 was localized to plastid nucleoids. Comparative analysis of non-transgenic and OsEREBP1 overexpressing genotypes revealed that OsEREBP1 attenuates disease caused by Xoo and confers drought and submergence tolerance in transgenic rice. Our results suggest that constitutive expression of OsEREBP1 activates the jasmonate and abscisic acid signalling pathways thereby priming the rice plants for enhanced survival under abiotic or biotic stress conditions. OsEREBP1 is thus, a good candidate gene for engineering plants for multiple stress tolerance.

Systemic cytosolic Ca(2+) elevation is activated upon wounding and herbivory in Arabidopsis.

Calcium ion (Ca(2+) ) signalling triggered by insect herbivory is an intricate network with multiple components, involving positive and negative regulators. Real-time, noninvasive imaging of entire Arabidopsis thaliana rosettes was employed to monitor cytosolic free calcium ([Ca(2+) ]cyt ) elevations in local and systemic leaves in response to wounding and Spodoptera littoralis feeding. Luminescence emitted by the cytosol-localized Ca(2+) reporter aequorin was imaged using a high-resolution photon-counting camera system. Spodoptera littoralis feeding on Arabidopsis induced both local and systemic [Ca(2+) ]cyt elevations. Systemic [Ca(2+) ]cyt signals were found predominantly in adjacent leaves with direct vascular connections to the treated leaf and appeared with a delay of 1 to 2 min. Simulated herbivory by wounding always induced a local [Ca(2+) ]cyt response, but a systemic one only when the midrib was wounded. This systemic [Ca(2+) ]cyt response was suppressed by the presence of insect-derived oral secretions as well as in a mutant of the vacuolar cation channel, Two Pore Channel 1 (TPC1). Our results provide evidence that in Arabidopsis insect herbivory induces both local and systemic [Ca(2+) ]cyt signals that distribute within the vascular system. The systemic [Ca(2+) ]cyt signal could play an important signalling role in systemic plant defence.

Upregulation of jasmonate biosynthesis and jasmonate-responsive genes in rice leaves in response to a bacterial pathogen mimic.

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, secretes several cell wall degrading enzymes including cellulase (ClsA) and lipase/esterase (LipA). Prior treatment of rice leaves with purified cell wall degrading enzymes such as LipA can confer enhanced resistance against subsequent X. oryzae pv. oryzae infection. To understand LipA-induced rice defense responses, microarray analysis was performed 12 h after enzyme treatment of rice leaves. This reveals that 867 (720 upregulated and 147 downregulated) genes are differentially regulated (≥2-fold). A number of genes involved in defense, stress, signal transduction, and catabolic processes were upregulated while a number of genes involved in photosynthesis and anabolic processes were downregulated. The microarray data also suggested upregulation of jasmonic acid (JA) biosynthetic and JA-responsive genes. Estimation of various phytohormones in LipA-treated rice leaves demonstrated a significant increase in the level of JA-Ile (a known active form of JA) while the levels of other phytohormones were not changed significantly with respect to buffer-treated control. This suggests a role for JA-Ile in cell wall damage induced innate immunity. Furthermore, a comparative analysis of ClsA- and LipA-induced rice genes has identified key rice functions that might be involved in elaboration of damage-associated molecular pattern (DAMP)-induced innate immunity.

Mutation of the Arabidopsis calmodulin-like protein CML37 deregulates the jasmonate pathway and enhances susceptibility to herbivory.

Throughout their life, plants are challenged by various abiotic and biotic stress factors. Among those are attacks from herbivorous insects. The molecular mechanisms underlying the detection of herbivores and the subsequent signal transduction are not well understood. As a second messenger, fluxes in intracellular Ca(2+) levels play a key role in mediating stress response pathways. Ca(2+) signals are decoded by Ca(2+) sensor proteins such as calmodulin-like proteins (CMLs). Here, we demonstrate that recombinant CML37 behaves like a Ca(2+) sensor in vitro and, in Arabidopsis, AtCML37 is induced by mechanical wounding as well as by infestation with larvae of the generalist lepidopteran herbivore Spodoptera littoralis. Loss of function of CML37 led to a better feeding performance of larvae suggesting that CML37 is a positive defense regulator. No herbivory-induced changes in secondary metabolites such as glucosinolates or flavonoids were detected in cml37 plants, although a significant reduction in the accumulation of jasmonates was observed, due to reduced expression of JAR1 mRNA and cellular enzyme activity. Consequently, the expression of jasmonate-responsive genes was reduced as well. Summarizing, our results suggest that the Ca(2+) sensor protein, CML37, functions as a positive regulator in Ca(2+) signaling during herbivory, connecting Ca(2+) and jasmonate signaling.

An Arabidopsis mutant impaired in intracellular calcium elevation is sensitive to biotic and abiotic stress.

BACKGROUND: Ca2+, a versatile intracellular second messenger in various signaling pathways, initiates many responses involved in growth, defense and tolerance to biotic and abiotic stress. Endogenous and exogenous signals induce cytoplasmic Ca2+ ([Ca2+]cyt) elevation, which are responsible for the appropriate downstream responses. RESULTS: Here we report on an ethyl-methane sulfonate-mediated Arabidopsis mutant that fails to induce [Ca2+]cyt elevation in response to exudate preparations from the pathogenic mibrobes Alternaria brassicae, Rhizoctonia solani, Phytophthora parasitica var. nicotianae and Agrobacterium tumefaciens. The cytoplasmic Ca2+elevation mutant1 (cycam1) is susceptible to infections by A. brassicae, its toxin preparation and sensitive to abiotic stress such as drought and salt. It accumulates high levels of reactive oxygen species and contains elevated salicylic acid, abscisic acid and bioactive jasmonic acid iso-leucine levels. Reactive oxygen species- and phytohormone-related genes are higher in A. brassicae-treated wild-type and mutant seedlings. Depending on the analysed response, the elevated levels of defense-related compounds are either caused by the cycam mutation and are promoted by the pathogen, or they are mainly due to the pathogen infection or application of pathogen-associated molecular patterns. Furthermore, cycam1 shows altered responses to abscisic acid treatments: the hormone inhibits germination and growth of the mutant. CONCLUSIONS: We isolated an Arabidopsis mutant which fails to induce [Ca2+]cyt elevation in response to exudate preparations from various microbes. The higher susceptibility of the mutant to pathogen infections correlates with the higher accumulation of defense-related compounds, such as phytohormones, reactive oxygen-species, defense-related mRNA levels and secondary metabolites. Therefore, CYCAM1 couples [Ca2+]cyt elevation to biotic, abiotic and oxidative stress responses.

Neomycin inhibition of (+)-7-iso-jasmonoyl-L-isoleucine accumulation and signaling.

The majority of plant defenses against insect herbivores are coordinated by jasmonate (jasmonic acid, JA; (+)-7-iso-jasmonoyl-L-isoleucine, JA-Ile)-dependent signaling cascades. Insect feeding and mimicking herbivory by application of oral secretions (OS) from the insect induced both cytosolic Ca(2+) and jasmonate-phytohormone elevation in plants. Here it is shown that in Arabidopsis thaliana upon treatment with OS from lepidopteran Spodoptera littoralis larvae, the antibiotic neomycin selectively blocked the accumulation of OS-induced Ca(2+) elevation and level of the bioactive JA-Ile, in contrast to JA level. Furthermore, neomycin treatment affected the downstream expression of JA-Ile-responsive genes, VSP2 and LOX2, in Arabidopsis. The neomycin-dependent reduced JA-Ile level is partially due to increased CYP94B3 expression and subsequent JA-Ile turn-over to12-hydroxy-JA-Ile. It is neither due to the inhibition of the enzymatic conjugation process nor to substrate availability. Thus, blocking Ca(2+) elevation specifically controls JA-Ile accumulation and signaling, offering an insight into role of calcium in defense against insect herbivory.

Multiple calmodulin-like proteins in Arabidopsis are induced by insect-derived (Spodoptera littoralis) oral secretion.

In plant cells, diverse environmental changes often induce transient elevation in the intracellular calcium concentrations, which are involved in signaling pathways leading to the respective cellular reactions. Therefore, these calcium elevations need to be deciphered into specific downstream responses. Calmodulin-like-proteins (CMLs) are calcium-sensing proteins present only in higher plants. They are involved in signaling processes induced by both abiotic as well as biotic stress factors. However, the role of CMLs in the interaction of plants with herbivorous insects is almost unknown. Here we show that in Arabidopsis thaliana a number of CMLs genes (CML9, 11,12,16,17 and 23) are upregulated due to treatments with oral secretion of larvae of the herbivorous insect Spodoptera littoralis. We identified that these genes belong to two groups that respond with different kinetics to the treatment with oral secretion. Our data indicate that signaling networks involving multiple CMLs very likely have important functions in plant defense against insect herbivores, in addition to their involvement in many other stress-induced processes in plants.

Direct proof of ingested food regurgitation by Spodoptera littoralis caterpillars during feeding on Arabidopsis.

Oral secretions of herbivorous lepidopteran larvae contain a mixture of saliva and regurgitant from the insect gut. Different compounds from the oral secretions can be recognized by the host plants and, thus, represent elicitors that induce plant defenses against feeding herbivores. Exogenously applied oral secretions can initiate the biosynthesis of jasmonates, phytohormones involved in the regulation of plant defense. However, it is not known (a) whether or not non-manipulated insects indeed release oral secretions including gut-derived compounds into a leaf wound during the natural feeding process, or (b) whether they adjust the release of gut components to the state of plant defense. We addressed these questions by using Arabidopsis thaliana as host plant and larvae of the generalist herbivorous insect Spodoptera littoralis. We investigated the conversion of the plant-derived jasmonate precursor, cis-12-oxophytodienoic acid (cis-OPDA), to iso-OPDA by the larvae. This enzymatic reaction is mediated by a specific glutathione-S-transferase in the insect gut, but not in the plant. Any presence of iso-OPDA in plant tissue, thus, indicated that gut content had been regurgitated into the plant wound. Our study demonstrates that the plant is the only source for the substrate cis-OPDA by using aos (allene oxide synthase) mutants that are unable to synthesize OPDA. The fact that iso-OPDA accumulated over time on feeding-damaged leaves shows that the feeding larvae are constantly regurgitating on leaves. Although the larvae provided the signaling compounds that were recognized by the plant and elicited defense reactions, the larval regurgitation behavior did not depend on whether they fed on a defensive wild type plant or on a non defensive coi1-16 plant. This suggests that S. littoralis larvae do not adjust regurgitation to the state of plant defense.