OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity

Microbe‐associated molecular pattern (MAMP)‐triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif‐containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor‐like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN‐triggered immunity in rice. Silencing of OsCERK1 suppressed PGN‐induced (and chitin‐induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor‐binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand‐induced heterooligomeric receptor complexes. Furthermore, the receptor‐like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.     

Herbivore perception decreases photosynthetic carbon assimilation and reduces stomatal conductance by engaging 12‐oxo‐phytodienoic acid,mitogen‐activated protein kinase 4 and cytokinin perception

Herbivory‐induced changes in photosynthesis have been documented in many plant species; however, the complexity of photosynthetic regulation and analysis has thwarted progress in understanding the mechanism involved, particularly those elicited by herbivore‐specific elicitors. Here, we analysed the early photosynthetic gas exchange responses in Nicotiana attenuata plants after wounding and elicitation with Manduca sexta oral secretions and the pathways regulating these responses. Elicitation with M. sexta oral secretions rapidly decreased photosynthetic carbon assimilation (A_C) in treated and systemic (untreated, vascularly connected) leaves, which were associated with changes in stomatal conductance, rather than with changes in Rubisco activity and 1‐5 ribulose‐1,5‐bisphosphate turnover. Phytohormone profiling and gas exchange analysis of oral secretion‐elicited transgenic plants altered in phytohormone regulation, biosynthesis and perception, combined with micrografting techniques, revealed that the local photosynthetic responses were mediated by 12‐oxo‐phytodienoic acid, while the systemic responses involved interactions among jasmonates, cytokinins and abscisic acid signalling mediated by mitogen‐activated protein kinase 4. The analysis also revealed a role for cytokinins interacting with mitogen‐activated protein kinase 4 in CO₂‐mediated stomatal regulation. Hence, oral secretions, while eliciting jasmonic acid‐mediated defence responses, also elicit 12‐oxo‐phytodienoic acid‐mediated changes in stomatal conductance and A_C, an observation illustrating the complexity and economy of the signalling that regulates defence and carbon assimilation pathways in response to herbivore attack.     

Silicon amendment to rice plants contributes to reduced feeding in a phloem‐sucking insect through modulation of callose deposition

Silicon (Si) uptake by Poaceae plants has beneficial effects on herbivore defense. Increased plant physical barrier and altered herbivorous feeding behaviors are documented to reduce herbivorous arthropod feeding and contribute to enhanced plant defense. Here, we show that Si amendment to rice (Oryza sativa) plants contributes to reduced feeding in a phloem feeder, the brown planthopper (Nilaparvata lugens, BPH), through modulation of callose deposition. We associated the temporal dynamics of BPH feeding with callose deposition on sieve plates and further with callose synthase and hydrolase gene expression in plants amended with Si. Biological assays revealed that BPH feeding was lower in Si‐amended than in nonamended plants in the early stages post‐BPH infestation. Histological observation showed that BPH infestation triggered fast and strong callose deposition in Si‐amended plants compared with nonamended plants. Analysis using qRT‐PCR revealed that expression of the callose synthase gene OsGSL1 was up‐regulated more and that the callose hydrolase (β‐1,3‐glucanase) gene Gns5 was up‐regulated less in Si‐amended than in nonamended plants during the initial stages of BPH infestation. These dynamic expression levels of OsGSL1 and Gns5 in response to BPH infestation correspond to callose deposition patterns in Si‐amended versus nonamended plants. It is demonstrated here that BPH infestation triggers differential gene expression associated with callose synthesis and hydrolysis in Si‐amended and nonamended rice plants, which allows callose to be deposited more on sieve tubes and sieve tube occlusions to be maintained more thus contributing to reduced BPH feeding on Si‐amended plants.     

The maize lipoxygenase,ZmLOX10, mediates green leaf volatile,jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro‐economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13‐LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10‐derived signaling is required for LOX8‐mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound‐induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore‐induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV‐, JA‐ and HIPV‐deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10‐dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro‐ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.     

Non‐pathogenic rhizobacteria interfere with the attraction of parasitoids to aphid‐induced plant volatiles via jasmonic acid signalling

Beneficial soil‐borne microbes, such as mycorrhizal fungi or rhizobacteria, can affect the interactions of plants with aboveground insects at several trophic levels. While the mechanisms of interactions with herbivorous insects, that is, the second trophic level, are starting to be understood, it remains unknown how plants mediate the interactions between soil microbes and carnivorous insects, that is, the third trophic level. Using Arabidopsis thaliana Col‐0 and the aphid Myzus persicae, we evaluate here the underlying mechanisms involved in the plant‐mediated interaction between the non‐pathogenic rhizobacterium Pseudomonas fluorescens and the parasitoid Diaeretiella rapae, by combining ecological, chemical and molecular approaches. Rhizobacterial colonization modifies the composition of the blend of herbivore‐induced plant volatiles. The volatile blend from rhizobacteria‐treated aphid‐infested plants is less attractive to an aphid parasitoid, in terms of both olfactory preference behaviour and oviposition, than the volatile blend from aphid‐infested plants without rhizobacteria. Importantly, the effect of rhizobacteria on both the emission of herbivore‐induced volatiles and parasitoid response to aphid‐infested plants is lost in an Arabidopsis mutant (aos/dde2‐2) that is impaired in jasmonic acid production. By modifying the blend of herbivore‐induced plant volatiles that depend on the jasmonic acid‐signalling pathway, root‐colonizing microbes interfere with the attraction of parasitoids of leaf herbivores.     

Jasmonoyl‐l‐isoleucine hydrolase 1 (JIH1) regulates jasmonoyl‐l‐isoleucine levels and attenuates plant defenses against herbivores

For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA‐Ile permanently inactivates JA‐Ile‐mediated signaling in plants, the alternative deactivation pathway of JA‐Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl‐l ‐isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA‐Ile and IAA‐Ala in vitro. When the herbivory‐inducible NaJIH1 gene was silenced by RNA interference, JA‐Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild‐type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild‐type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA‐Ile‐dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory‐elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA‐Ile‐hydroxylation and carboxylation of JA‐Ile, rapidly attenuates the JA‐Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature.     

The tyrosine‐sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner

The tyrosine‐sulfated peptides PSKα and PSY1 bind to specific leucine‐rich repeat surface receptor kinases and control cell proliferation in plants. In a reverse genetic screen, we identified the phytosulfokine (PSK) receptor PSKR1 as an important component of plant defense. Multiple independent loss‐of‐function mutants in PSKR1 are more resistant to biotrophic bacteria, show enhanced pathogen‐associated molecular pattern responses and less lesion formation after infection with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. By contrast, pskr1 mutants are more susceptible to necrotrophic fungal infection with Alternaria brassicicola, show more lesion formation and fungal growth which is not observed on wild‐type plants. The antagonistic effect on biotrophic and necrotrophic pathogen resistance is reflected by enhanced salicylate and reduced jasmonate responses in the mutants, suggesting that PSKR1 suppresses salicylate‐dependent defense responses. Detailed analysis of single and multiple mutations in the three paralogous genes PSKR1, ‐2 and PSY1‐receptor (PSY1R) determined that PSKR1 and PSY1R, but not PSKR2, have a partially redundant effect on plant immunity. In animals and plants, peptide sulfation is catalyzed by a tyrosylprotein sulfotransferase (TPST). Mutants lacking TPST show increased resistance to bacterial infection and increased susceptibility to fungal infection, mimicking the triple receptor mutant phenotypes. Feeding experiments with PSKα in tpst‐1 mutants partially restore the defense‐related phenotypes, indicating that perception of the PSKα peptide has a direct effect on plant defense. These results suggest that the PSKR subfamily integrates growth‐promoting and defense signals mediated by sulfated peptides and modulates cellular plasticity to allow flexible adjustment to environmental changes.     

Molecular and ecological plant defense responses along an elevational gradient in a boreal ecosystem

Plants have the capacity to alter their phenotype in response to environmental factors, such as herbivory, a phenomenon called phenotypic plasticity. However, little is known on how plant responses to herbivory are modulated by environmental variation along ecological gradients. To investigate this question, we used bilberry (Vaccinium myrtillus L.) plants and an experimental treatment to induce plant defenses (i.e., application of methyl jasmonate; MeJA), to observe ecological responses and gene expression changes along an elevational gradient in a boreal system in western Norway. The gradient included optimal growing conditions for bilberry in this region (ca. 500 m a.s.l.), and the plant's range limits at high (ca. 900 m a.s.l.) and low (100 m a.s.l.) elevations. Across all altitudinal sites, MeJA‐treated plants allocated more resources to herbivory resistance while reducing growth and reproduction than control plants, but this response was more pronounced at the lowest elevation. High‐elevation plants growing under less herbivory pressure but more resource‐limiting conditions exhibited consistently high expression levels of defense genes in both MeJA‐treated and untreated plants at all times, suggesting a constant state of “alert.” These results suggest that plant defense responses at both the molecular and ecological levels are modulated by the combination of climate and herbivory pressure, such that plants under different environmental conditions differentially direct the resources available to specific antiherbivore strategies. Our findings are important for understanding the complex impact of future climate changes on plant–herbivore interactions, as this is a major driver of ecosystem functioning and biodiversity.     

Host plant defense signaling in response to a coevolved herbivore combats introduced herbivore attack

Defense‐free space resulting from coevolutionarily naïve host plants recently has been implicated as a factor facilitating invasion success of some insect species. Host plants, however, may not be entirely defenseless against novel herbivore threats. Volatile chemical‐mediated defense signaling, which allows plants to mount specific, rapid, and intense responses, may play a role in systems experiencing novel threats. Here we investigate defense responses of host plants to a native and exotic herbivore and show that (1) host plants defend more effectively against the coevolved herbivore, (2) plants can be induced to defend against a newly‐associated herbivore when in proximity to plants actively defending against the coevolved species, and (3) these defenses affect larval performance. These findings highlight the importance of coevolved herbivore‐specific defenses and suggest that naïveté or defense limitations can be overcome via defense signaling. Determining how these findings apply across various host–herbivore systems is critical to understand mechanisms of successful herbivore invasion.     

OsNPR1 negatively regulates herbivore‐induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice

NPR1 (a non‐expressor of pathogenesis‐related genes1) has been reported to play an important role in plant defense by regulating signaling pathways. However, little to nothing is known about its function in herbivore‐induced defense in monocot plants. Here, using suppressive substrate hybridization, we identified a NPR1 gene from rice, OsNPR1, and found that its expression levels were upregulated in response to infestation by the rice striped stem borer (SSB) Chilo suppressalis and rice leaf folder (LF) Cnaphalocrocis medinalis, and to mechanical wounding and treatment with jasmonic acid (JA) and salicylic acid (SA). Moreover, mechanical wounding induced the expression of OsNPR1 quickly, whereas herbivore infestation induced the gene more slowly. The antisense expression of OsNPR1 (as‐npr1), which reduced the expression of the gene by 50%, increased elicited levels of JA and ethylene (ET) as well as of expression of a lipoxygenase gene OsHI‐LOX and an ACC synthase gene OsACS2. The enhanced JA and ET signaling in as‐npr1 plants increased the levels of herbivore‐induced trypsin proteinase inhibitors (TrypPIs) and volatiles, and reduced the performance of SSB. Our results suggest that OsNPR1 is an early responding gene in herbivore‐induced defense and that plants can use it to activate a specific and appropriate defense response against invaders by modulating signaling pathways.     

Four Arabidopsis berberine bridge enzyme‐like proteins are specific oxidases that inactivate the elicitor‐active oligogalacturonides

Recognition of endogenous molecules acting as ‘damage‐associated molecular patterns’ (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper‐accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth‐defence trade‐off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper‐accumulation. By combining affinity chromatography on acrylamide‐trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo‐protein, OGOX1 is a sulphite‐sensitive H₂O₂‐producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD‐binding proteins [Berberine Bridge Enzyme‐like (BBE‐like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE‐like enzymes in Arabidopsis are OG oxidases (OGOX1–4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity.     

Tetranychus evansi spider mite populations suppress tomato defenses to varying degrees

Plant defense suppression is an offensive strategy of herbivores, in which they manipulate plant physiological processes to increase their performance. Paradoxically, defense suppression does not always benefit the defense‐suppressing herbivores, because lowered plant defenses can also enhance the performance of competing herbivores and can expose herbivores to increased predation. Suppression of plant defense may therefore entail considerable ecological costs depending on the presence of competitors and natural enemies in a community. Hence, we hypothesize that the optimal magnitude of suppression differs among locations. To investigate this, we studied defense suppression across populations of Tetranychus evansi spider mites, a herbivore from South America that is an invasive pest of solanaceous plants including cultivated tomato, Solanum lycopersicum, in other parts of the world. We measured the level of expression of defense marker genes in tomato plants after infestation with mites from eleven different T. evansi populations. These populations were chosen across a range of native (South American) and non‐native (other continents) environments and from different host plant species. We found significant variation at three out of four defense marker genes, demonstrating that T. evansi populations suppress jasmonic acid‐ and salicylic acid‐dependent plant signaling pathways to varying degrees. While we found no indication that this variation in defense suppression was explained by differences in host plant species, invasive populations tended to suppress plant defense to a smaller extent than native populations. This may reflect either the genetic lineage of T. evansi—as all invasive populations we studied belong to one linage and both native populations to another—or the absence of specialized natural enemies in invasive T. evansi populations.     

The rice hydroperoxide lyase OsHPL3 functions in defense responses by modulating the oxylipin pathway

As important signal molecules, jasmonates (JAs) and green leaf volatiles (GLVs) play diverse roles in plant defense responses against insect pests and pathogens. However, how plants employ their specific defense responses by modulating the levels of JA and GLVs remains unclear. Here, we describe identification of a role for the rice HPL3 gene, which encodes a hydroperoxide lyase (HPL), OsHPL3/CYP74B2, in mediating plant‐specific defense responses. The loss‐of‐function mutant hpl3‐1 produced disease‐resembling lesions spreading through the whole leaves. A biochemical assay revealed that OsHPL3 possesses intrinsic HPL activity, hydrolyzing hydroperoxylinolenic acid to produce GLVs. The hpl3‐1 plants exhibited enhanced induction of JA, trypsin proteinase inhibitors and other volatiles, but decreased levels of GLVs including (Z)‐3‐hexen‐1‐ol. OsHPL3 positively modulates resistance to the rice brown planthopper [BPH, Nilaparvata lugens (Stål)] but negatively modulates resistance to the rice striped stem borer [SSB, Chilo suppressalis (Walker)]. Moreover, hpl3‐1 plants were more attractive to a BPH egg parasitoid, Anagrus nilaparvatae, than the wild‐type, most likely as a result of increased release of BPH‐induced volatiles. Interestingly, hpl3‐1 plants also showed increased resistance to bacterial blight (Xanthomonas oryzae pv. oryzae). Collectively, these results indicate that OsHPL3, by affecting the levels of JA, GLVs and other volatiles, modulates rice‐specific defense responses against different invaders.     

Ethylene production in Botrytis cinerea‐ and oligogalacturonide‐induced immunity requires calcium‐dependent protein kinases

Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe‐ or damage‐associated molecular patterns, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen‐secreted cell wall‐degrading enzymes, and flg22, a 22‐amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium‐binding proteins. Here we show that simultaneous loss of calcium‐dependent protein kinases CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor‐ induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen‐induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG‐triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG‐ and flg22‐induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways.     

Herbivore-specific plant volatiles prime neighboring plants for nonspecific defense responses

Plants produce species-specific herbivore-induced plant volatiles (HIPVs) after damage. We tested the hypothesis that herbivore-specific HIPVs prime neighboring plants to induce defenses specific to the priming herbivore. Since Manduca sexta (specialist) and Heliothis virescens (generalist) herbivory induced unique HIPV profiles in Nicotiana benthamiana, we used these HIPVs to prime receiver plants for defense responses to simulated herbivory (mechanical wounding and herbivore regurgitant application). Jasmonic acid (JA) accumulations and emitted volatile profiles were monitored as representative defense responses since JA is the major plant hormone involved in wound and defense signaling and HIPVs have been implicated as signals in tritrophic interactions. Herbivore species-specific HIPVs primed neighboring plants, which produced 2 to 4 times more volatiles and JA after simulated herbivory when compared to similarly treated constitutive volatile-exposed plants. However, HIPV-exposed plants accumulated similar amounts of volatiles and JA independent of the combination of priming or challenging herbivore. Furthermore, volatile profiles emitted by primed plants depended only on the challenging herbivore species but not on the species-specific HIPV profile of damaged emitter plants. This suggests that feeding by either herbivore species primed neighboring plants for increased HIPV emissions specific to the subsequently attacking herbivore and is probably controlled by JA.