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Plants and insects have co-existed for almost 400 million years and their interactions can be beneficial or harmful, thus reflecting their intricate co-evolutionary dynamics. Many herbivorous arthropods cause tremendous crop loss, impacting the agro-economy worldwide. Plants possess an arsenal of chemical defenses that comprise diverse secondary metabolites that help protect against harmful herbivorous arthropods. In response, the strategies that herbivores use to cope with plant defenses can be behavioral, or molecular and/or biochemical of which salivary secretions are a key determinant. Insect salivary secretions/oral secretions (OSs) play a crucial role in plant immunity as they contain several biologically active elicitors and effector proteins that modulate plants' defense responses. Using this oral secretion cocktail, insects overcome plant natural defenses to allow successful feeding. However, a lack of knowledge of the nature of the signals present in oral secretion cocktails has resulted in reduced mechanistic knowledge of their cellular perception. In this review, we discuss the latest knowledge on herbivore oral secretion derived elicitors and effectors and various mechanisms involved in plant defense modulation. Identification of novel herbivore-released molecules and their plant targets should pave the way for understanding the intricate strategies employed by both herbivorous arthropods and plants in their interactions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

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 Ca 2+ signal and its role in self-amplification and plant elicitor peptide receptor (PEPR)-mediated signalling. Using the Ca 2+ reporter, R-GECO1 in Arabidopsis, we have characterized a monophasic and sustained JA-Ile-dependent Ca 2+ signature in leaf epidermal cells. The rapid Ca 2+ 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 Ca 2+ 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-Ca 2+ -MYC2 signalling module through which plants sense JA-Ile rapidly to amplify both AtPep-PEPR and jasmonate signalling in undamaged cells., (© 2024 John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

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.

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., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 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 (Ca 2+ ) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca 2+ 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 Ca 2+ 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 Ca 2+ reporter background and identify novel Ca 2+ regulated targets.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 Ca 2+ elevation, which triggers downstream defense. However, not all the Ca 2+ 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 Ca 2+ flux and plant defense. Loss of CNGC19 function results in decreased herbivory defense. The cngc19 mutant shows aberrant and attenuated intravascular Ca 2+ fluxes. CNGC19 is a Ca 2+ -permeable channel, as hyperpolarization of CNGC19-expressing Xenopus oocytes in the presence of both cyclic adenosine monophosphate and Ca 2+ results in Ca 2+ influx. Breakdown of Ca 2+ -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 Ca 2+ channel CNGC19 in the recognition of herbivory and the activation of defense signaling., (© 2019 American Society of Plant Biologists. All rights reserved.)

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.

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.

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., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

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.

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.

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.

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., (© The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.)

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.

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.

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.

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.

In the interaction between Arabidopsis (Arabidopsis thaliana) and the generalist herbivorous insect Spodoptera littoralis, little is known about early events in defense signaling and their link to downstream phytohormone pathways. S. littoralis oral secretions induced both Ca²⁺ and phytohormone elevation in Arabidopsis. Plant gene expression induced by oral secretions revealed up-regulation of a gene encoding a calmodulin-like protein, CML42. Functional analysis of cml42 plants revealed more resistance to herbivory than in the wild type, because caterpillars gain less weight on the mutant, indicating that CML42 negatively regulates plant defense; cml42 also showed increased aliphatic glucosinolate content and hyperactivated transcript accumulation of the jasmonic acid (JA)-responsive genes VSP2 and Thi2.1 upon herbivory, which might contribute to increased resistance. CML42 up-regulation is negatively regulated by the jasmonate receptor Coronatine Insensitive1 (COI1), as loss of functional COI1 resulted in prolonged CML42 activation. CML42 thus acts as a negative regulator of plant defense by decreasing COI1-mediated JA sensitivity and the expression of JA-responsive genes and is independent of herbivory-induced JA biosynthesis. JA-induced Ca²⁺ elevation and root growth inhibition were more sensitive in cml42, also indicating higher JA perception. Our results indicate that CML42 acts as a crucial signaling component connecting Ca²⁺ and JA signaling. CML42 is localized to cytosol and nucleus. CML42 is also involved in abiotic stress responses, as kaempferol glycosides were down-regulated in cml42, and impaired in ultraviolet B resistance. Under drought stress, the level of abscisic acid accumulation was higher in cml42 plants. Thus, CML42 might serve as a Ca²⁺ sensor having multiple functions in insect herbivory defense and abiotic stress responses.

Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H₂O₂ and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H₂O₂ formation is even reduced by the fungus. Importantly, phospholipase D (PLD)α1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

Elevation of intracellular calcium levels in a plant cell is an early signaling event in many mutualistic and pathogenic plant/microbe interactions. In pathogenic plant/fungus interactions, receptor-mediated cytoplasmic calcium elevations induce defense genes via the activation of ion fluxes at the plasma membrane, an oxidative burst and MAPK activation. Mycorrhizal and beneficial endophytic plant/fungus interactions result in a better plant performance through sequencial cytoplasmic and nuclear calcium elevations. The specificity of the calcium responses depends on the calcium signature, its amplitude, duration, frequency and location, a selective activation of calcium channels in the diverse cellular membranes and the stimulation of calcium-dependent signaling components. Arabidopsis contains more than 100 genes for calcium-binding proteins and channels and the response to pathogens and beneficial fungi relies on a highly specific activation of individual members of these protein families. Genetic tools are required to understand this complex response patterns and the cross talks between the individual calcium-dependent signaling pathways. The beneficial interaction of Arabidopsis with the growth-promoting endophyte Piriformospora indica provides a nice model system to unravel signaling events leading to mutualistic or pathogenic plant/fungus interactions.

Ascorbate is a major antioxidant and radical scavenger in plants. Monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR) are two enzymes of the ascorbate-glutathione cycle that maintain ascorbate in its reduced state. MDAR2 (At3g09940) and DHAR5 (At1g19570) expression was upregulated in the roots and shoots of Arabidopsis seedlings co-cultivated with the root-colonizing endophytic fungus Piriformospora indica, or that were exposed to a cell wall extract or a culture filtrate from the fungus. Growth and seed production were not promoted by Piriformospora indica in mdar2 (SALK_0776335C) and dhar5 (SALK_029966C) T-DNA insertion lines, while colonized wild-type plants were larger and produced more seeds compared to the uncolonized controls. After 3 weeks of drought stress, growth and seed production were reduced in Piriformospora indica-colonized plants compared to the uncolonized control, and the roots of the drought-stressed insertion lines were colonized more heavily by the fungus than were wild-type plants. Upregulation of the message for the antimicrobial PDF1.2 protein in drought-stressed insertion lines indicated that MDAR2 and DHAR5 are crucial for producing sufficient ascorbate to maintain the interaction between Piriformospora indica and Arabidopsis in a mutualistic state.

Calcium (Ca2+), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca2+] elevations are early events in the interaction between the plant growth-promoting fungus Piriformospora indica and Arabidopsis thaliana. A cell wall extract (CWE) from the fungus promotes the growth of wild-type seedlings but not of seedlings from P. indica-insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca2+ signalling-related functions. The CWE induces a transient cytosolic Ca2+ ([Ca2+](cyt)) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum) plants, as well as in BY-2 suspension cultures expressing the Ca2+ bioluminescent indicator aequorin. Nuclear Ca2+ transients were also observed in tobacco BY-2 cells. The Ca2+ response was more pronounced in roots than in shoots and involved Ca2+ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca2+ response by staurosporine and the refractory nature of the Ca2+ elevation suggest that a receptor may be involved. The CWE does not stimulate H2O2 production and the activation of defence gene expression, although it led to phosphorylation of mitogen-activated protein kinases (MAPKs) in a Ca2+-dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica. Thus, Ca2+ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco.

Arabidopsis growth and reproduction are stimulated by the endophytic fungus Piriformospora indica. The fungus produces low amounts of auxins, but the auxin levels and the expression of auxin-regulated genes are not altered in colonized roots. Also, mutants with reduced auxin levels (ilr1-1, nit1-3, tfl2, cyp79 b2b3) respond to P. indica. However, the fungus rescues the dwarf phenotype of the auxin overproducer sur1-1 by converting free auxin into conjugates, which also results in the downregulation of the auxin-induced IAA6 and the upregulation of the P. indica-induced LRR1 gene. The fungus produces relatively high levels of cytokinins, and the cytokinin levels are higher in colonized roots compared with the uncolonized controls. trans-Zeatin cytokinin biosynthesis and the CRE1/AHK2 receptor combination are crucial for P. indica-mediated growth stimulation, while mutants lacking cis-zeatin, impaired in other cytokinin receptor combinations, or containing reduced cytokinin levels respond to the fungus. Since root colonization is not affected in the cytokinin mutants, we propose that cytokinins are required for P. indica-induced growth promotion. Finally, a comparative analysis of the phytohormone mutants allows the conclusion that the response to P. indica is independent of the architecture and size of the roots.

Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single-copy gene DMI-1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica-insensitive-2; pii-2), and a corresponding insertion line, are impaired in a leucine-rich repeat protein (At1g13230). The protein pii-2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X-100-insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine-rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii-2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica-induced growth promotion and enhanced seed production in Arabidopsis thaliana.