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Plants recognize molecules related to a variety of biotic stresses through pattern recognition receptors to activate plant immunity. In the interactions between plants and chewing herbivores, such as lepidopteran larvae, oral secretions (OS) are deposited on wounded sites, which results in the elicitation of plant immune responses. The widely conserved receptor-like kinase CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) has been broadly associated with the recognition of microbial components, such as fungal chitin, but its relevance to herbivory remained unclear. In this study, we used receptor-knockout rice (Oryza sativa) and larvae of the lepidopteran pest Mythimna loreyi to demonstrate that the induction of immune responses triggered by larval OS in rice cells largely depends on CERK1 (OsCERK1). CHITIN ELICITOR-BINDING PROTEIN (CEBiP), an OsCERK1-interacting receptor-like protein that was proposed as the main chitin receptor, also contributed to the responses of rice cells to OS collected from three different lepidopteran species. Furthermore, CEBiP knockout rice seedlings showed lower OS-triggered accumulation of jasmonic acid. These results strongly suggest that the OsCERK1 and CEBiP recognize a particular OS component in chewing lepidopteran herbivores, and point toward the presence of chitooligosaccharides in the OS. Targeted perturbation to chitin recognition, through the use of fungal effector proteins, confirmed the presence of chitooligosaccharides in the OS. Treatments of wounds on rice plants with chitooligosaccharides enhanced a set of immune responses, leading to resistance against an herbivorous insect. Our data show that rice recognizes chitooligosaccharides during larval herbivory to activate resistance, and identifies chitin as a novel herbivore-associated molecular pattern., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Silicon (Si) uptake is generally beneficial for plants that need protection from insect herbivores. In pursue of mechanisms involved in Si-mediated defense, we comprehensively explored the impact of Si on several defensive and metabolic traits in rice exposed to simulated and real herbivory of Mythimna loreyi Duponchel larvae. Hydroponic experiments showed that Si-deprived rice supplemented with Si 72 h prior to insect infestation were similarly resistant to larvae as plants continuously grown in Si-containing media. Both Si and herbivory altered primary metabolism in rice, including the levels of several sugars, amino acids, and organic acids. While the accumulation of sugars was generally positively correlated with Si presence, multiple amino acids showed a negative correlation trend with Si supplementation. The levels of secondary metabolites, including isopentylamine, p -coumaroylputrescine and feruloylputrescine, were typically higher in the leaves of Si-supplemented plants exposed to herbivory stress compared to Si-deprived plants. In addition, simulated herbivory treatment in Si-supplemented plants induced more volatile emissions relative to Si-deprived plants, which was consistent with the increased transcripts of key genes involved in volatile biosynthesis. In ecological interactions, Si alone did not affect the oviposition choice of M. loreyi but gravid females showed a significant preference for simulated herbivory-treated/Si-deprived compared to Si-supplemented plants. Our data suggest that apart from mechanical defense, Si may affect rice metabolism in multiple ways that might enhance/modulate defense responses of rice under herbivory stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Osibe, Hojo, Shinya, Mitani-Ueno and Galis.)

Acute stress responses include release of defensive volatiles from herbivore-attacked plants. Here we used two closely related monocot species, rice as a representative C3 plant, and sorghum as a representative C4 plant, and compared their basal and stress-induced headspace volatile organic compounds (VOCs). Although both plants emitted similar types of constitutive and induced VOCs, in agreement with the close phylogenetic relationship of the species, several mono- and sesquiterpenes have been significantly less abundant in headspace of sorghum relative to rice. Furthermore, in spite of generally lower VOC levels, some compounds, such as the green leaf volatile ( Z )-3-hexenyl acetate and homoterpene DMNT, remained relatively high in the sorghum headspace, suggesting that a separate mechanism for dispersal of these compounds may have evolved in this plant. Finally, a variable amount of several VOCs among three sorghum cultivars of different geographical origins suggested that release of VOCs could be used as a valuable resource for the increase of sorghum resistance against herbivores.

Crops experience herbivory by arthropods and microbial infections. In the interaction between plants and chewing herbivores, lepidopteran larval oral secretions (OS) and plant-derived damage-associated molecular patterns (DAMPs) trigger plant defense responses. However, the mechanisms underlying anti-herbivore defense, especially in monocots, have not been elucidated. The receptor-like cytoplasmic kinase Broad-Spectrum Resistance 1 (BSR1) of Oryza sativa L. (rice) mediates cytoplasmic defense signaling in response to microbial pathogens and enhances disease resistance when overexpressed. Here, we investigated whether BSR1 contributes to anti-herbivore defense responses. BSR1 knockout suppressed rice responses triggered by OS from the chewing herbivore Mythimna loreyi Duponchel (Lepidoptera: Noctuidae) and peptidic DAMPs OsPeps, including the activation of genes required for biosynthesis of diterpenoid phytoalexins (DPs). BSR1 -overexpressing rice plants exhibited hyperactivation of DP accumulation and ethylene signaling after treatment with simulated herbivory and acquired enhanced resistance to larval feeding. As the biological significance of herbivory-induced accumulation of rice DPs remains unexplained, their physiological activities in M. loreyi were analyzed. The addition of momilactone B, a rice DP, to the artificial diet suppressed the growth of M. loreyi larvae. Altogether, this study revealed that BSR1 and herbivory-induced rice DPs are involved in the defense against chewing insects, in addition to pathogens.

Key Message: This study describes biological functions of the bHLH transcription factor RERJ1 involved in the jasmonate response and the related defense-associated metabolic pathways in rice, with particular focus on deciphering the regulatory mechanisms underlying stress-induced volatile emission and herbivory resistance. RERJ1 is rapidly and drastically induced by wounding and jasmonate treatment but its biological function remains unknown as yet. Here we provide evidence of the biological function of RERJ1 in plant defense, specifically in response to herbivory and pathogen attack, and offer insights into the RERJ1-mediated regulation of metabolic pathways of specialized defense compounds, such as monoterpene linalool, in possible collaboration with OsMYC2-a well-known master regulator in jasmonate signaling. In rice (Oryza sativa L.), the basic helix-loop-helix (bHLH) family transcription factor RERJ1 is induced under environmental stresses, such as wounding and drought, which are closely linked to jasmonate (JA) accumulation. Here, we investigated the biological function of RERJ1 in response to biotic stresses, such as herbivory and pathogen infection, using an RERJ1-defective mutant. Transcriptome analysis of the rerj1-Tos17 mutant revealed that RERJ1 regulated the expression of a typical family of conserved JA-responsive genes (e.g., terpene synthases, proteinase inhibitors, and jasmonate ZIM domain proteins). Upon exposure to armyworm attack, the rerj1-Tos17 mutant exhibited more severe damage than the wildtype, and significant weight gain of the larvae fed on the mutant was observed. Upon Xanthomonas oryzae infection, the rerj1-Tos17 mutant developed more severe symptoms than the wildtype. Among RERJ1-regulated terpene synthases, linalool synthase expression was markedly disrupted and linalool emission after wounding was significantly decreased in the rerj1-Tos17 mutant. RERJ1 appears to interact with OsMYC2-a master regulator of JA signaling-and many OsJAZ proteins, although no obvious epistatic interaction was detected between them at the transcriptional level. These results indicate that RERJ1 is involved in the transcriptional induction of JA-mediated stress-responsive genes via physical association with OsMYC2 and mediates defense against herbivory and bacterial infection through JA signaling., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: We show that in rice, the amino acid-conjugates of JA precursor, OPDA, may function as a non-canonical signal for the production of phytoalexins in coordination with the innate chitin signaling. The core oxylipins, jasmonic acid (JA) and JA-Ile, are well-known as potent regulators of plant defense against necrotrophic pathogens and/or herbivores. However, recent studies also suggest that other oxylipins, including 12-oxo-phytodienoic acid (OPDA), may contribute to plant defense. Here, we used a previously characterized metabolic defense marker, p-coumaroylputrescine (CoP), and fungal elicitor, chitooligosaccharide, to specifically test defense role of various oxylipins in rice (Oryza sativa). While fungal elicitor triggered a rapid production of JA, JA-Ile, and their precursor OPDA, rice cells exogenously treated with the compounds revealed that OPDA, rather than JA-Ile, can stimulate the CoP production. Next, reverse genetic approach and oxylipin-deficient rice mutant (hebiba) were used to uncouple oxylipins from other elicitor-triggered signals. It appeared that, without oxylipins, residual elicitor signaling had only a minimal effect but, in synergy with OPDA, exerted a strong stimulatory activity towards CoP production. Furthermore, as CoP levels were compromised in the OPDA-treated Osjar1 mutant cells impaired in the oxylipin-amino acid conjugation, putative OPDA-amino acid conjugates emerged as hypothetical regulators of CoP biosynthesis. Accordingly, we found several OPDA-amino acid conjugates in rice cells treated with exogenous OPDA, and OPDA-Asp was detected, although in small amounts, in the chitooligosaccharide-treated rice. However, as synthetic OPDA-Asp and OPDA-Ile, so far, failed to induce CoP in cells, it suggests that yet another presumed OPDA-amino acid form(s) could be acting as novel regulator(s) of phytoalexins in rice., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Success of plants largely depends on their ability to defend against herbivores. Since emergence of the first voracious consumers, plants maintained adapting their structures and chemistry to escape from extinction. The constant pressure was further accelerated by adaptation of herbivores to plant defenses, which all together sparked the rise of a chemical empire comprised of thousands of specialized metabolites currently found in plants. Metabolic diversity in the plant kingdom is truly amazing, and although many plant metabolites have already been identified, a large number of potentially useful chemicals remain unexplored in plant bio-resources. Similarly, biosynthetic routes for plant metabolites involve many enzymes, some of which still wait for identification and biochemical characterization. Moreover, regulatory mechanisms that control gene expression and enzyme activities in specialized metabolism of plants are scarcely known. Finally, understanding of how plant defense chemicals exert their toxicity and/or repellency against herbivores remains limited to typical examples, such as proteinase inhibitors, cyanogenic compounds and nicotine. In this review, we attempt summarizing the current status quo in metabolic defense of plants that is predominantly based on the survey of ubiquitous examples of plant interactions with chewing herbivores., (© 2021 Institute of Botany, Chinese Academy of Sciences.)

A vast array of herbivorous arthropods live with symbiotic microorganisms. However, little is known about the nature and functional mechanism of bacterial effects on plant defense responses towards herbivores. We explored the role of microbes present in extracts of oral secretion (OS) isolated from larvae of Spodoptera litura, a generalist herbivore, in phytohormone signaling-dependent defense responses in Arabidopsis thaliana (Arabidopsis). In response to mechanical damage (MD) with application of bacteria-free OS (OS - ) prepared by sterilization or filtration of OS, Arabidopsis leaves exhibited enhanced de novo synthesis of oxylipins, and induction of transcript abundance of the responsible genes, in comparison to those in leaves with MD + nonsterilized OS (OS + ), indicating that OS bacteria serve as suppressors of these genes. By contrast, de novo synthesis/signaling of salicylic acid and signaling of abscisic acid were enhanced by OS bacteria. These signaling networks were cross-regulated by each other. Meta-analysis of OS bacteria identified 70 bacterial strains. Among them was Staphylococcus epidermidis, an anaerobic staphylococcus that was shown to contribute to the suppression/manipulation of phytohormone-dependent plant defense signaling. The presence of OS bacteria was consequently beneficial for S. litura larvae hosted by Brassicaceae., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Despite the importance of volatile organic compounds (VOCs) for plants, control mechanisms for their basal and stress-induced biosynthesis and release remain unclear. We sampled and characterized headspace and internal leaf volatile pools in rice (Oryza sativa), after a simulated herbivory treatment, which triggers an endogenous jasmonate burst. Certain volatiles, such as linalool, were strongly upregulated by simulated herbivory stress. In contrast, other volatiles, such as β-caryophyllene, were constitutively emitted and fluctuated according to time of day. Transcripts of the linalool synthase gene transiently increased 1-3 h after exposure of rice to simulated herbivory, whereas transcripts of caryophyllene synthase peaked independently at dawn. Unexpectedly, although emission and accumulation patterns of rice inducible and constitutive VOCs were substantially different, both groups of volatiles were compromised in jasmonate-deficient hebiba mutants, which lack the allene oxide cyclase (AOC) gene. This suggests that rice employs at least two distinct oxylipin-dependent mechanisms downstream of AOC to control production of constitutive and herbivore-induced volatiles. Levels of the JA precursor, 12-oxo-phytodienoic acid (OPDA), were correlated with constitutive volatile levels suggesting that OPDA or its derivatives could be involved in control of volatile emission in rice., (© 2021 John Wiley & Sons Ltd.)

Jasmonic acid (JA) and its biologically active form jasmonoyl-L-isoleucine (JA-Ile) regulate defense responses to various environmental stresses and developmental processes in plants. JA and JA-Ile are synthesized from α-linolenic acids derived from membrane lipids via 12-oxo-phytodienoic acid (OPDA). In the presence of JA-Ile, the COI1 receptor physically interacts with JAZ repressors, leading to their degradation, resulting in the transcription of JA-responsive genes by MYC transcription factors. Although the biosynthesis of JA-Ile is conserved in vascular plants, it is not recognized by COI1 in bryophytes and is not biologically active. In the liverwort Marchantia polymorpha , dinor-OPDA (dn-OPDA), a homolog of OPDA with two fewer carbons, and its isomer dn- iso -OPDA accumulate after wounding and are recognized by COI1 to activate downstream signaling. The moss Calohypnum plumiforme produces the antimicrobial-specialized metabolites, momilactones. It has been reported that JA and JA-Ile are not detected in C. plumiforme and that OPDA, but not JA, can induce momilactone accumulation and the expression of these biosynthetic genes, suggesting that OPDA or its derivative is a biologically active molecule in C. plumiforme that induces chemical defense. In the present study, we investigated the biological functions of OPDA and its derivatives in C. plumiforme . Searching for the components potentially involving oxylipin signaling from transcriptomic and genomic data revealed that two COI1 , three JAZ , and two MYC genes were present. Quantification analyses revealed that OPDA and its isomer iso -OPDA accumulated in larger amounts than dn-OPDA and dn- iso -OPDA after wounding. Moreover, exogenously applied OPDA, dn-OPDA, or dn- iso -OPDA induced the transcription of JAZ genes. These results imply that OPDA, dn-OPDA, and/or their isomers potentially act as biologically active molecules to induce the signaling downstream of COI1-JAZ. Furthermore, co-immunoprecipitation analysis showed the physical interaction between JAZs and MYCs, indicating the functional conservation of JAZs in C. plumiforme with other plants. These results suggest that COI1-JAZ-MYC mediated signaling is conserved and functional in C. plumiforme ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Inagaki, Miyamoto, Ando, Murakami, Sugisawa, Morita, Yumoto, Teruya, Uchida, Kato, Kaji, Takaoka, Hojo, Shinya, Galis, Nozawa, Sawasaki, Nojiri, Ueda and Okada.)

We examined the role of ethylene in the production of rice (Oryza sativa) volatile organic compounds (VOCs), which act as indirect defense signals against herbivores in tritrophic interactions. Rice plants were exposed to exogenous ethylene (1 ppm) after simulated herbivory, which consisted of mechanical wounding supplemented with oral secretions (WOS) from the generalist herbivore larva Mythimna loreyi. Ethylene treatment highly suppressed VOCs in WOS-treated rice leaves, which was further corroborated by the reduced transcript levels of major VOC biosynthesis genes in ethylene-treated rice. In contrast, the accumulation of jasmonates (JA), known to control VOCs in higher plants, and transcript levels of primary JA response genes, including OsMYC2, were not largely affected by ethylene application. At the functional level, flooding is known to promote internode elongation in young rice via ethylene signaling. Consistent with the negative role of ethylene on VOC genes, the accumulation of VOCs in water-submerged rice leaves was suppressed. Furthermore, in mature rice plants, which naturally produce less volatiles, VOCs could be rescued by the application of the ethylene perception inhibitor 1-methylcyclopropene. Our data suggest that ethylene acts as an endogenous suppressor of VOCs in rice plants during development and under stress., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Phosphatidic acid plays an important role in plant immune responses against phytopathogenic bacteria in Nicotiana benthamiana . Here we focused on phosphoinositide dependent protein kinases (PDKs) as a candidate required for phosphatidic acid signaling. Based on Arabidopsis PDK sequences, we identified four putative PDK orthologs in N. benthamiana genome. To address the role of PDKs in plant defense responses, we created all four NbPDKs -silenced plants by virus-induced gene silencing. the NbPDKs -silenced plants showed a moderately reduced growth phenotype. Induction of hypersensitive cell death was compromised in the NbPDKs -silenced plants challenged with Ralstonia solanacearum . The hypersensitive cell death induced by bacterial effectors was also reduced in the NbPDKs -silenced plants. the NbPDKs -silenced plants showed decreased production of salicylic acid, jasmonic acid and jasmonoyl-L-isoleucine, as well as hydrogen peroxide after inoculation with R. solanacearum . These results suggest that NbPDKs might have an important role in the regulation of the hypersensitive cell death via plant hormone signaling and oxidative burst., (© 2020 Japanese Society for Plant Biotechnology.)

Interspecific New Rice for Africa (NERICA) varieties have been recently developed and used in Sub-Saharan Africa but herbivore resistance properties of these plants remain poorly understood. Here we report that, compared to a local Japanese cultivar Nipponbare, NERICA 1, 4 and 10 are significantly more damaged by insect herbivores in the paddy fields. In contrast to high levels of leaf damage from rice skippers and grasshoppers, constitutive and induced volatile organic compounds for indirect plant defense were higher or similar in NERICAs and Nipponbare. Accumulation of direct defense secondary metabolites, momilactones A and B, and p-coumaroylputrescine (CoP) was reduced in NERICAs, while feruloylputrescine accumulated at similar levels in all varieties. Finally, we found that Nipponbare leaves were covered with sharp nonglandular trichomes impregnated with silicon but comparable defense structures were virtually absent in herbivory-prone NERICA plants. As damage to the larval gut membranes by Nipponbare silicified trichomes that pass intact through the insect digestive system, occurs, and larval performance is enhanced by trichome removal from otherwise chemically defended Nipponbare plants, we propose that silicified trichomes work as an important defense mechanism of rice against chewing insect herbivores., (© 2020 John Wiley & Sons Ltd.)

Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis , CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca 2+ , and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca 2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Phospholipid signaling plays an important role in plant immune responses against phytopathogenic bacteria in Nicotiana benthamiana. Here, we isolated two phospholipase C2 (PLC2) orthologs in the N. benthamiana genome, designated as PLC2-1 and 2-2. Both NbPLC2-1 and NbPLC2-2 were expressed in most tissues and were induced by infiltration with bacteria and flg22. NbPLC2-1 and NbPLC2-2 (NbPLC2s) double-silenced plants showed a moderately reduced growth phenotype. The induction of the hypersensitive response was not affected, but bacterial growth and the appearance of bacterial wilt were accelerated in NbPLC2s-silenced plants when they were challenged with a virulent strain of Ralstonia solanacearum that was compatible with N. benthamiana. NbPLC2s-silenced plants showed reduced expression levels of NbPR-4, a marker gene for jasmonic acid signaling, and decreased jasmonic acid and jasmonoyl-L-isoleucine contents after inoculation with R. solanacearum. The induction of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes was reduced in NbPLC2s-silenced plants after infiltration with R. solanacearum or Pseudomonas fluorescens. Accordingly, the resistance induced by flg22 was compromised in NbPLC2s-silenced plants. In addition, the expression of flg22-induced PTI marker genes, the oxidative burst, stomatal closure, and callose deposition were all reduced in the silenced plants. Thus, NbPLC2s might have important roles in pre- and post-invasive defenses, namely in the induction of PTI., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Lysin motif (LysM) is a carbohydrate-binding modules found in all kingdoms. LysM binds to N-acetylglucosamine-containing molecules such as peptidoglycan, chitin, Nod factor, and Myc factor and is found in peptidoglycan hydrolases, chitinases, and plant pathogen effectors and plant receptor/co-receptor for defense and symbiosis signaling. This chapter describes the synthesis of a nonradioactive chitin ligand, biotinylated chitin octasaccharide, (GlcNAc) 8 -Bio, and its application for the detection and characterization of chitin-binding LysM receptor CEBiP in the microsomal membrane fraction of rice suspension-cultured cells by affinity labeling. We also describe the purification of CEBiP from the plasma membrane of the rice cells by affinity chromatography with the synthesized (GlcNAc) 8 -APEA-CH-Sepharose as an affinity matrix.

Feeding of sucking insects, such as the rice brown planthopper (Nilaparvata lugens; BPH), causes only limited mechanical damage on plants that is otherwise essential for injury-triggered defense responses against herbivores. In pursuit of complementary BPH elicitors perceived by plants, we examined the potential effects of BPH honeydew secretions on the BPH monocot host, rice (Oryza sativa). We found that BPH honeydew strongly elicits direct and putative indirect defenses in rice, namely accumulation of phytoalexins in the leaves, and release of volatile organic compounds from the leaves that serve to attract natural enemies of herbivores, respectively. We then examined the elicitor active components in the honeydew and found that bacteria in the secretions are responsible for the activation of plant defense. Corroborating the importance of honeydew-associated microbiota for induced plant resistance, BPHs partially devoid of their microbiota via prolonged antibiotics ingestion induced significantly less defense in rice relative to antibiotic-free insects applied to similar groups of plants. Our data suggest that rice plants may additionally perceive herbivores via their honeydew-associated microbes, allowing them to discriminate between incompatible herbivores-that do not produce honeydew-and those that are compatible and therefore dangerous., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plants use many natural products to counter pests and diseases in nature. In rice, direct defense mechanisms include broad range of secondary metabolites, such as phenolamides (PA), diterpene phytoalexins, and flavonoid sakuranetin. Recently, accumulation of PAs in rice was shown to be under control of microbial symbionts in honeydew (HD), digestive waste from the rice brown planthopper ( Nilaparvata lugens ; BPH), but whether HD microbiota can also promote diterpene phytoalexins, momilactone A (MoA) and MoB, has not been reported. Here, we demonstrate that crude HD, but not a filtered one, induces MoA and MoB in rice, suggesting the involvement of BPH-HD endosymbionts. Consequently, microbial strains previously isolated from HD could promote MoA and MoB levels in wounded rice leaves, suggesting that rice indeed responds to BPH by cumulative chemical defense that involves both PA and diterpene phytoalexin pathways.

Plant defense against herbivores is modulated by herbivore-associated molecular patterns (HAMPs) from oral secretions (OS) and/or saliva of insects. Furthermore, feeding wounds initiate plant self-damage responses modulated by danger-associated molecular patterns (DAMPs) such as immune defense-promoting plant elicitor peptides (Peps). While temporal and spatial co-existence of both patterns during herbivory implies a possibility of their close interaction, the molecular mechanisms remain undetermined. Here we report that exogenous application of rice (Oryza sativa) peptides (OsPeps) can elicit multiple defense responses in rice cell cultures. Specific activation of OsPROPEP3 gene transcripts in rice leaves by wounding and OS treatments further suggests a possible involvement of the OsPep3 peptide in rice-herbivore interactions. Correspondingly, we found that simultaneous application of OsPep3 and Mythimna loreyi OS significantly amplifies an array of defense responses in rice cells, including mitogen-activated protein kinase activation, and generation of defense-related hormones and metabolites. The induction of OsPROPEP3/4 by OsPep3 points to a positive auto-feedback loop in OsPep signaling which may contribute to additional enhancement of defense signal(s). Finally, the overexpression of the OsPep receptor OsPEPR1 increases the sensitivity of rice plants not only to the cognate OsPeps but also to OS signals. Our findings collectively suggest that HAMP-DAMP signal integration provides a critical step in the amplification of defense signaling in plants., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Pseudomonas syringae pv. tabaci causes wildfire disease by the action of tabtoxinine-β-lactam (TβL), a non-specific bacterial toxin. To better understand the molecular mechanisms of wildfire disease and its development, we focused on the phosphoinositide 3-kinase in Nicotiana benthamiana (NbPI3K) and its potential role in the disease outbreak, using l-methionine sulfoximine (MSX) as an easily accessible mimic of the TβL action. The NbPI3K-silenced plants showed accelerated induction of cell death and necrotic lesion formation by MSX, and the expression of hin1, marker gene for the programmed cell death, was strongly induced in the plants. However, the accumulation of ammonium ions, caused by MSX inhibition of glutamine sythetase activity, was not affected by the NbPI3K-silencing. Interestingly, the expression of PR-1a, a marker gene for salicylic acid (SA) innate immunity signaling, and accumulation of SA were both enhanced in the NbPI3K-silenced plants. Accordingly, the acceleration of MSX-induced cell death by NbPI3K-silencing was reduced in NahG plants, and by double silencing of NbPI3K together with the NbICS1 encoding a SA-biosynthetic enzyme. As silencing of NbPI3K accelerated the TβL-induced necrotic lesions, and lesions of wildfire disease caused by P. syringae pv. tabaci, these results suggest that the NbPI3K-related pathway might act as a negative regulator of cell death during development of wildfire disease that involves SA-dependent signaling pathway downstream of TβL action in N. benthamiana., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Plants synthesize variable mixtures of herbivore-induced plant volatiles (HIPVs) as part of their evolutionary conserved defense. To elucidate the impact of chewing herbivores with different level of adaptation on HIPV profiles in rice, we measured HIPVs released from rice seedlings challenged by either the generalist herbivore Mythimna loreyi (MYL) or the specialist Parnara guttata (PAG). Both herbivores markedly elicited the emission of HIPVs, mainly on the second and third days after attack compared to control plants. In addition, side-by-side HIPV comparisons using MYL and PAG caterpillars revealed that generalist feeding induced comparably more HIPVs relative to specialist, particularly on day two as highlighted by multivariate analysis (PLS-DA) of emitted HIPVs, and further confirmed in mimicked herbivory experiments. Here, mechanically wounded plants treated with water (WW) released more VOCs than untreated controls, and on top of this, oral secretions (OS) from both herbivores showed differential effects on volatile emissions from the wounded plants. Similar to actual herbivory, MYL OS promoted higher amounts of HIPVs relative to PAG OS, thus supporting disparate induction of rice indirect defenses in response to generalist and specialist herbivores, which could be due to the differential composition of their OS. (196 words).

Two phenolamides (PAs), p-coumaroylputrescine and feruloylputrescine strongly accumulate in rice (Oryza sativa cv. Nipponbare) leaves subjected to attack of chewing and sucking herbivores. Here we identified and characterized in vitro three novel rice genes that mediated coumaroyl-CoA/feruloyl-CoA conjugation to polyamines, putrescine and agmatine. Interestingly, two genes were highly specific for their polyamine substrates, encoding putrescine N-hydroxycinnamoyltransferase and agmatine N-hydroxycinnamoyltransferase, while the third enzyme could use both polyamines and it was therefore annotated as putrescine/agmatine N-hydroxycinnamoyltransferase. All genes were preferentially expressed in rice roots and developing flowers, and in addition, the putrescine/agmatine N-hydroxycinnamoyltransferase transcripts were strongly induced by wounding in the young rice leaves. Because the wound response of this gene was only partially suppressed in the jasmonoyl-L-isoleucine deficient plants (Osjar1), it suggests that its upregulation (as well as inducible PAs in rice) may be largely independent of jasmonoyl-L-isoleucine signaling pathway. The finding of three closely related genes with a similar and/or overlapping activity in PA biosynthesis provides another striking example of rapid diversification of plant metabolism in response to environmental stresses in nature., (© 2016 Institute of Botany, Chinese Academy of Sciences.)

Induced plant defense responses against insect herbivores are triggered by wounding and/or perception of herbivore elicitors from their oral secretions (OS) and/or saliva. In this study, we analyzed OS isolated from two rice chewing herbivores, Mythimna loreyi and Parnara guttata. Both types of crude OS had substantial elicitor activity in rice cell system that allowed rapid detection of early and late defense responses, i.e. accumulation of reactive oxygen species (ROS) and defense secondary metabolites, respectively. While the OS from M. loreyi contained large amounts of previously reported insect elicitors, fatty acid-amino acid conjugates (FACs), the elicitor-active P. guttata's OS contained no detectable FACs. Subsequently, elicitor activity associated with the high molecular mass fraction in OS of both herbivores was identified, and shown to promote ROS and metabolite accumulations in rice cells. Notably, the application of N-linolenoyl-Gln (FAC) alone had only negligible elicitor activity in rice cells; however, the activity of isolated elicitor fraction was substantially promoted by this FAC. Our results reveal that plants integrate various independent signals associated with their insect attackers to modulate their defense responses and reach maximal fitness in nature.

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Plants defend against attack from herbivores by direct and indirect defence mechanisms mediated by the accumulation of phytoalexins and release of volatile signals, respectively. While the defensive arsenals of some plants, such as tobacco and Arabidopsis are well known, most of rice's (Oryza sativa) defence metabolites and their effectiveness against herbivores remain uncharacterized. Here, we used a non-biassed metabolomics approach to identify many novel herbivory-regulated metabolic signatures in rice. Most were up-regulated by herbivore attack while only a few were suppressed. Two of the most prominent up-regulated signatures were characterized as phenolamides (PAs), p-coumaroylputrescine and feruloylputrescine. PAs accumulated in response to attack by both chewing insects, i.e. feeding of the lawn armyworm (Spodoptera mauritia) and the rice skipper (Parnara guttata) larvae, and the attack of the sucking insect, the brown planthopper (Nilaparvata lugens, BPH). In bioassays, BPH insects feeding on 15% sugar solution containing p-coumaroylputrescine or feruloylputrescine, at concentrations similar to those elicited by heavy BPH attack in rice, had a higher mortality compared to those feeding on sugar diet alone. Our results highlight PAs as a rapidly expanding new group of plant defence metabolites that are elicited by herbivore attack, and deter herbivores in rice and other plants., (© 2015 John Wiley & Sons Ltd.)

Plants can detect infecting fungi through the perception of chitin oligosaccharides by lysin motif receptors such as CEBiP and CERK1. A major function of CERK1 seems to be as a signaling molecule in the receptor complex formed with ligand-binding molecules and to activate downstream defense signaling. Fungal pathogens, however, have developed counter strategies to escape from the chitin-mediated detection by using effectors and/or changing their cell walls. Common structural features between chitin and Nod-/Myc-factors and corresponding receptors have suggested the close relationships between the chitin-mediated immunity and rhizobial/arbuscular mycorrhizal symbiosis. The recent discovery of the dual function of OsCERK1 in both plant immunity and mycorrhizal symbiosis sheds new light on the evolutionary relationships between defense and symbiotic systems in plants., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The csnR gene, localized at the beginning of an operon, csnR-K, which organization is conserved through many actinomycete genomes, was previously shown to repress the transcription of the chitosanase gene csnA in Streptomyces lividans. However, knowledge on the function of the whole csnR-K operon in the metabolism of chitosan (an N-deacetylated derivative of chitin) remained limited. Mutants of S. coelicolor A3(2) harboring partial or total deletions of the csnR-K operon were analyzed for their capacity to uptake glucosamine oligosaccharides (GlcN)n. The csnR-K operon was autoregulated by CsnR repressor and its transcription was inducible by GlcN oligosaccharides. The operon controlled the uptake of GlcN oligosaccharides in S. coelicolor A3(2), with a minor contribution to the consumption of monomeric GlcN but not chitin-related N-acetylated derivatives. The deletion of the whole operon abolished the uptake of GlcN oligosaccharides. The CsnEFG transporter encoded by this operon is the front door for the assimilation of chitosan-derived hydrolysis products in S. coelicolor A3(2). The ATP-binding component MsiK was essential for CsnEFG transport function. Also, deletion of msiK abolished the induction of csnA transcription by GlcN oligosaccharides., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recognition of microbe-associated molecular patterns (MAMPs) initiates pattern-triggered immunity in host plants. Pattern recognition receptors (PRRs) and receptor-like cytoplasmic kinases (RLCKs) are the major components required for sensing and transduction of these molecular patterns. However, the regulation of RLCKs by PRRs and their specificity remain obscure. In this study we show that PBL27, an Arabidopsis ortholog of OsRLCK185, is an immediate downstream component of the chitin receptor CERK1 and contributes to the regulation of chitin-induced immunity in Arabidopsis. Knockout of PBL27 resulted in the suppression of several chitin-induced defense responses, including the activation of MPK3/6 and callose deposition as well as in disease resistance against fungal and bacterial infections. On the other hand, the contribution of PBL27 to flg22 signaling appears to be very limited, suggesting that PBL27 selectively regulates defense signaling downstream of specific PRR complexes. In vitro phosphorylation experiments showed that CERK1 preferentially phosphorylated PBL27 in comparison to BIK1, whereas phosphorylation of PBL27 by BAK1 was very low compared with that of BIK1. Thus, the substrate specificity of the signaling receptor-like kinases, CERK1 and BAK1, may determine the preference of downstream RLCKs., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.

Chitin is a representative microbe-associated molecular pattern (MAMP) molecule for various fungi and induces immune responses in many plant species. It has been clarified that the chitin signaling in rice requires a receptor kinase OsCERK1 and a receptor-like protein (Os)CEBiP, which specifically binds chitin oligosaccharides. On the other hand, Arabidopsis requires a receptor kinase (At)CERK1 for chitin signaling but it is not clear whether the plant also requires a CEBiP-like molecule for chitin perception/signaling. To clarify the similarity/difference of the chitin receptor in these two model plants, we first characterized CEBiP homologs in Arabidopsis. Only one of three CEBiP homologs, AtCEBiP (LYM2), showed a high-affinity binding for chitin oligosaccharides similar to rice CEBiP. AtCEBiP also represented the major chitin-binding protein in the Arabidopsis membrane. However, the single/triple knockout (KO) mutants of Arabidopsis CEBiP homologs and the overexpressor of AtCEBiP showed chitin-induced defense responses similar to wild-type Arabidopsis, indicating that AtCEBiP is biochemically functional as a chitin-binding protein but does not contribute to signaling. Studies of the chitin binding properties of the ectodomains of At/OsCERK1 and the chimeric receptors consisting of ecto/cytosolic domains of these molecules indicated that AtCERK1 is sufficient for chitin perception by itself.

Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.

The chitin elicitor-binding protein (CEBiP) from rice was the first plant lysin motif (LysM) protein for which the biological and biochemical function had been established. It belongs to a plant-specific family of extracellular LysM proteins (LYMs) for which we analyzed the phylogeny. LYMs are present in vascular plants only, where an early gene duplication event might have resulted in two types which were retained in present day genomes. LYMs consist of a signal peptide, three consecutive LysMs, separated by cysteine pairs, and a C-terminal region without any known signature, whose length allows the distinction between the two types, and which may be followed by a glycosylphosphatidylinositol (GPI) anchor motif. We analyzed a representative of each type, MtLYM1 and MtLYM2, from Medicago truncatula at the biochemical level and with respect to their expression patterns and observed some similarities but also marked differences. MtLYM1 and MtLYM2 proved to be very different with regard to abundance and apparent molecular mass on SDS-PAGE. Both undergo several post-translational modifications, including N-glycosylation and the addition of a GPI anchor, which would position the proteins at the outer face of the plasma membrane. Only MtLYM2, but not MtLYM1, showed specific binding to biotinylated N-acetylchitooctaose in a manner similar to CEBiP, which belongs to the same type. We postulate that LYM2-type proteins likely function in the perception of chitin-related molecules, whereas possible functions of LYM1-type proteins remain to be elucidated., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Multicellular organisms activate immunity upon recognition of pathogen-associated molecular patterns (PAMPs). Chitin is the major component of fungal cell walls, and chitin oligosaccharides act as PAMPs in plant and mammalian cells. Microbial pathogens deliver effector proteins to suppress PAMP-triggered host immunity and to establish infection. Here, we show that the LysM domain-containing effector protein Ecp6 of the fungal plant pathogen Cladosporium fulvum mediates virulence through perturbation of chitin-triggered host immunity. During infection, Ecp6 sequesters chitin oligosaccharides that are released from the cell walls of invading hyphae to prevent elicitation of host immunity. This may represent a common strategy of host immune suppression by fungal pathogens, because LysM effectors are widely conserved in the fungal kingdom.

The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

Innate immunity is the first line of defense against invading microorganisms in vertebrates and the only line of defense in invertebrates and plants. Bacterial glyco-conjugates, such as lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria and peptidoglycan (PGN) from the cell walls of both Gram-positive and Gram-negative bacteria, and fungal and oomycete glycoconjugates such as oligosaccharides derived from the cell wall components beta-glucan, chitin and chitosan, have been found to act as elicitors of plant innate immunity. These conserved indispensable microbe-specific molecules are also referred to as microbe-associated molecular patterns (MAMPs). Other glyco-conjugates such as bacterial extracellular polysaccharides (EPS) and cyclic glucan have been shown to suppress innate immune responses, thus conversely promoting pathogenesis. MAMPs are recognized by the plant innate immune system though the action of pattern recognition receptors (PRRs). A greater insight into the mechanisms of MAMP recognition and the description of PRRs for different microbial glyco-conjugates will have considerable impact on the improvement of plant health and disease resistance. Here we review the current knowledge about the bacterial MAMPs LPS and PGN, the fungal MAMPs beta-glucan, chitin and chitosan oligosaccharides and the bacterial suppressors EPS and cyclic glucan, with particular reference to the chemical structures of these molecules, the PRRs involved in their recognition (where these have been defined), and possible mechanisms underlying suppression.

Phospholipase D (PLD) plays an important role in plants, including responses to abiotic as well as biotic stresses. A survey of the rice (Oryza sativa) genome database indicated the presence of 17 PLD genes in the genome, among which OsPLDalpha1, OsPLDalpha5, and OsPLDbeta1 were highly expressed in most tissues studied. To examine the physiological function of PLD in rice, we made knockdown plants for each PLD isoform by introducing gene-specific RNA interference constructs. One of them, OsPLDbeta1-knockdown plants, showed the accumulation of reactive oxygen species in the absence of pathogen infection. Reverse transcription-polymerase chain reaction and DNA microarray analyses revealed that the knockdown of OsPLDbeta1 resulted in the up-/down-regulation of more than 1,400 genes, including the induction of defense-related genes such as pathogenesis-related protein genes and WRKY/ERF family transcription factor genes. Hypersensitive response-like cell death and phytoalexin production were also observed at a later phase of growth in the OsPLDbeta1-knockdown plants. These results indicated that the OsPLDbeta1-knockdown plants spontaneously activated the defense responses in the absence of pathogen infection. Furthermore, the OsPLDbeta1-knockdown plants exhibited increased resistance to the infection of major pathogens of rice, Pyricularia grisea and Xanthomonas oryzae pv oryzae. These results suggested that OsPLDbeta1 functions as a negative regulator of defense responses and disease resistance in rice.

The dasABC genes encode an ATP-binding cassette (ABC) transporter, which is one of the uptake systems for N,N'-diacetylchitobiose [(GlcNAc)(2)] in Streptomyces coelicolor A3(2), although the gene encoding the ABC subunit that provides ATP hydrolysis for DasABC has not been identified. In this study, we disrupted the sequence that is highly homologous to the msiK gene, the product of which is an ABC subunit assisting several ABC permeases in other Streptomyces species. Disruption of msiK severely affected the ability of S. coelicolor A3(2) to utilize maltose, cellobiose, starch, cellulose, chitin and chitosan, but not glucose. The msiK null mutant lacked (GlcNAc)(2)-uptake activity, but GlcNAc transport activity was unaffected. The data indicated that msiK is essential for (GlcNAc)(2) uptake, which in S. coelicolor A3(2) is governed by ABC transporters including the DasABC-MsiK system, in contrast to Escherichia coli and Serratia marcescens, in which (GlcNAc)(2) uptake is mediated by the phosphotransferase system. Interestingly, the induction of chitinase production by (GlcNAc)(2) or chitin was absent in the msiK null mutant, unlike in the parent strain M145. The defect in chitinase gene induction was rescued by expressing the His-tagged MsiK protein under the control of the putative native promoter on a multicopy plasmid. The data suggest that uptake of (GlcNAc)(2) is necessary for induction of chitinase production. The msiK gene was constitutively transcribed, whereas the transcription of dasA [(GlcNAc)(2)-binding protein gene], malE (putative maltose-binding protein gene), cebE1 (putative cellobiose-binding protein gene) and bxlE1 (putative xylobiose-binding protein gene) was induced by their corresponding sugar ligands. This is believed to be the first report to indicate that (GlcNAc)(2) uptake mediated by ABC transporters is essential for chitinase production in streptomycetes, which are known to be the main degraders of chitin in soil.

Chitin is a major component of fungal cell walls and serves as a microbe-associated molecular pattern (MAMP) for the detection of various potential pathogens in innate immune systems of both plants and animals. We recently showed that chitin elicitor-binding protein (CEBiP), plasma membrane glycoprotein with LysM motifs, functions as a cell surface receptor for chitin elicitor in rice. The predicted structure of CEBiP does not contain any intracellular domains, suggesting that an additional component(s) is required for signaling through the plasma membrane into the cytoplasm. Here, we identified a receptor-like kinase, designated CERK1, which is essential for chitin elicitor signaling in Arabidopsis. The KO mutants for CERK1 completely lost the ability to respond to the chitin elicitor, including MAPK activation, reactive oxygen species generation, and gene expression. Disease resistance of the KO mutant against an incompatible fungus, Alternaria brassicicola, was partly impaired. Complementation with the WT CERK1 gene showed cerk1 mutations were responsible for the mutant phenotypes. CERK1 is a plasma membrane protein containing three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain with autophosphorylation/myelin basic protein kinase activity, suggesting that CERK1 plays a critical role in fungal MAMP perception in plants.

We previously demonstrated that a beta-1,3-, 1,6-oligoglucan (AaGlucan) from the fungus Alternaria alternata 102 shows strong elicitor activity in tobacco BY-2 cells. We have used cDNA microarray analysis to monitor global changes in gene expression in tobacco cells treated with this A. alternata fraction or with laminarin. In total, we identified 265 genes that were induced 1 h after treatment with an AaGlucan-enriched fraction or laminarin. Among them, we characterized in detail a novel tobacco R2R3 MYB-type transcription factor homolog (NtMYBGR1) and two DC1 domain-containing genes (NtDC1A and NtDC1B). Microarray data, together with overexpression and metabolic analyses, indicated that NtMYBGR1, but not the NtDC1 proteins, primarily targets the phenylpropanoid synthesis-related genes PAL and 4CL. These results suggest that NtMYBGR1 specifically regulates defense responses in BY-2 cells by enhancing phenylpropanoid metabolism in response to AaGlucan and laminarin elicitors.

N,N'-Diacetylchitobiose [(GlcNAc)(2)] induces the transcription of chitinase (chi) genes in Streptomyces coelicolor A3(2). Physiological studies showed that (GlcNAc)(2) addition triggered chi expression and increased the rate of (GlcNAc)(2) concentration decline in culture supernatants of mycelia already cultivated with (GlcNAc)(2), suggesting that (GlcNAc)(2) induced the synthesis of its own uptake system. Four open reading frames (SCO0531, SCO0914, SCO2946, and SCO5232) encoding putative sugar-binding proteins of ABC transporters were found in the genome by probing the 12-bp repeat sequence required for regulation of chi transcription. SCO5232, named dasA, showed transcriptional induction by (GlcNAc)(2) and N,N',N'''-triacetylchitotriose [(GlcNAc)(3)]. Surface plasmon resonance analysis showed that recombinant DasA protein exhibited the highest affinity for (GlcNAc)(2) (equilibrium dissociation constant [K(D)] = 3.22 x 10(-8)). In the dasA-null mutant, the rate of decline of the (GlcNAc)(2) concentration in the culture supernatant was about 25% of that in strain M145. The in vitro and in vivo data clearly demonstrated that dasA is involved in (GlcNAc)(2) uptake. Upstream and downstream of dasA, the transcriptional regulator gene (dasR) and two putative integral membrane protein genes (dasBC) are located in the opposite and same orientations, respectively. The expression of dasR and dasB, which seemed independent of dasA transcription, was also induced by (GlcNAc)(2) and (GlcNAc)(3).

Tobacco BY-2 class IV chitinases (TBC-1, TBC-3) were rapidly and transiently induced by the beta-1,3-, 1,6-glucan elicitor from Alternaria alternata 102 (AaGlucan). The full-length cDNA and 5'-flanking region of a gene encoding class IV chitinases were isolated on the basis of the amino acid sequence of TBC-1. Sequence analysis indicated that NtChitIV encoded TBC-1, TBC-3, or both. Since purified TBC-1 and TBC-3 from BY-2 cells lack a chitin binding domain in the N-terminal region, these enzymes suggested to be derived from NtChitIV by post-translational proteolytic processing. The transcripts of NtChitIV accumulated rapidly within 1h after treatment with AaGlucan. Accumulation was maximal 3h after treatment. Reporter gene assays were used to analyze the promoter regions involved in the transcriptional control of NtChitIV, and these assays revealed that the 1.89-kb NtChitIV promoter was activated by AaGlucan but not by salicylic acid (SA) or methyl jasmonate (MeJA). The AaGlucan-induced transcriptional activation via 1.89-kb NtChitIV promoter was attenuated by pretreatment with SA or MeJA. These results suggest that NtChitIV expression is particularly induced by AaGlucan and that the AaGlucan-dependent signaling pathway is different from the SA- and MeJA-dependent signaling pathways.

A novel elicitor that induces chitinases in tobacco BY-2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase-inducing activity when applied at concentrations of as low as 25 ng x mL(-1). Structural determination by methylation analysis, reducing-end analysis, MALDI-TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of beta-1,3-, 1,6-oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6-linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY-2 cells indicated that the activity of this beta-1,3-, 1,6-glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense-related genes indicated that a phenylalanine ammonia-lyase gene and a coumaroyl-CoA O-methyltransferase gene were transiently expressed by this beta-1,3-, 1,6-glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY-2 cells. In the tissue of tobacco plants, this beta-1,3-, 1,6-glucan elicitor induced the expression of basic PR-3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified beta-1,3-, 1,6-glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.

Stress-induced cell-lytic activity was found in tobacco BY-2 cells treated with various stresses. Among 14 stresses, an elicitor fraction isolated from Alternaria alternata showed the highest inducing activity. Cell-lytic activity increased for 72 h even in the control sample, treated with distilled water, and several isozymes of beta-1,3-glucanases and chitinases were found to be involved in it. In contrast, cell-lytic activity in BY-2 cells treated with a fungal elicitor reached a higher level after 60 h. The principal enzymes specifically involved in this stress-induced portion are speculated to be basic beta-1,3-glucanases. A class I beta-1,3-glucanase gene (glu1) was found to be the specific gene for the stress-induced cell-lytic activity. Its expression became observable at 24 h, and the intensity reached a maximum at about 60-72 h. The glu1 was thus assigned as a late gene. Its role in the stress response is discussed in conjunction with earlier genes such as chitinases.