Diterpenoid Phytoalexin Biosynthesis Research Articles

Enhancing Crop Resilience: Insights from Labdane-Related Diterpenoid Phytoalexin Research in Rice (Oryza sativa L.).

Rice (Oryza sativa L.), as one of the most significant food crops worldwide, holds paramount importance for global food security. Throughout its extensive evolutionary journey, rice has evolved a diverse array of defense mechanisms to fend off pest and disease infestations. Notably, labdane-related diterpenoid phytoalexins play a crucial role in aiding rice in its response to both biotic and abiotic stresses. This article provides a comprehensive review of the research advancements pertaining to the chemical structures, biological activities, and biosynthetic pathways, as well as the molecular regulatory mechanisms, underlying labdane-related diterpenoid phytoalexins discovered in rice. This insight into the molecular regulation of labdane-related diterpenoid phytoalexin biosynthesis offers valuable perspectives for future research aimed at improving crop resilience and productivity.

Rice transcription factor DPF regulates stress-induced biosynthesis of diterpenoid phytoalexins.

Diterpenoid Phytoalexin Factor (DPF) is a key transcription factor involved in diterpenoid phytoalexin (DP) biosynthesis under non-stressed conditions in rice (Oryza sativa L.). Using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9, DPF knockout rice lines were generated. Treatments with abiotic stresses (copper chloride, ultraviolet light, and jasmonic acid) and biotic stress (blast fungus infection) to the knockout lines revealed that the DPF positively regulates stress-induced DP biosynthesis.

Transcriptome analysis of the molecular basis of 11‐eicosenoic acid‐mediated salt stress tolerance in rice

AbstractSalt stress is one of the major abiotic factors severely hampering rice production. Fatty acids play a crucial role in plants response to such stress. Previous studies have demonstrated the potential of unsaturated fatty acid (11‐eicosenoic acid [EA]) to enhance salt tolerance in rice, but the molecular mechanism remains unclear. Our study indicates that the exogenous application of EA could alleviate salt stress‐induced damage as well as improve the survival rate of rice seedlings. Transcriptome analysis was performed following treatment of rice seedlings with EA, NaCl, and NaCl + EA. The results showed that there were 4970, 11, 304, and 4735 differentially expressed genes (DEGs) in NaCl versus CK, EA versus CK, NaCl + EA versus NaCl, and NaCl + EA versus CK, respectively. Notably, pathways such as unsaturated fatty acid metabolism, diterpenoid phytoalexin biosynthesis, and phytohormone signal transduction were significantly enriched in NaCl + EA versus NaCl. Specifically, genes related to ethylene, abscisic acid, jasmonic acid, salicylic acid, gibberellin signaling, and fatty acid metabolism were implicated in the mitigation effect of EA on salt stress in rice. Furthermore, DEGs involved in rice diterpenoid biosynthesis were analyzed in detail, revealing that EA induced the biosynthesis of multiple diterpenoids. Metabolomic analysis showed that the levels of various fatty acids, phytohormones, and terpenes increased. The presented data may serve as a useful guide for further exploration of the various biological functions of EA in enhancing plant resilience to salt stress. Additionally, it offers a valuable list of genes for the development of crop plants with improved salt resistance.

The Mediator Subunit OsMED16 Interacts with the WRKY Transcription Factor OsWRKY45 to Enhance Rice Resistance Against Magnaporthe oryzae.

Rice blast, caused by Magnaporthe oryzae (M. oryzae), is one of the most common and damaging diseases of rice that limits rice yield and quality. The mediator complex plays a vital role in promoting transcription by bridging specific transcription factors and RNA polymerase II. Here, we show that the rice mediator subunit OsMED16 is essential for full induction of the diterpenoid phytoalexin biosynthesis genes and resistance to the ascomycetous fungus M. oryzae. Mutants of Osmed16 show reduced expression of the DP biosynthesis genes and are markedly more susceptible to M. oryzae, while transgenicplants overexpressing OsMED16 increased the expression of the DP biosynthesis genes and significantly enhanced resistance to M. oryzae. Interestingly, OsMED16 is physically associated with the WRKY family transcription factor OsWRKY45, which interacts with the phytoalexin synthesis key regulator transcription factor OsWRKY62. Further, OsMED16-OsWRKY45-OsWRKY62 complex could bind to the promoter regions of phytoalexin synthesis-related genes and activate their gene expression. Our results show that OsMED16 may enhance rice tolerance to M. oryzae via directly manipulating phytoalexin de novo biosynthesis.

BSR1, a Rice Receptor-like Cytoplasmic Kinase, Positively Regulates Defense Responses to Herbivory.

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.

OsWRKY10 extensively activates multiple rice diterpenoid phytoalexin biosynthesis genes to enhance rice blast resistance.

Phytoalexin is the main chemical weapon against pathogens in plants. Rice (Oryza sativa L.) produces a number of phytoalexins to defend against pathogens, most of which belong to the class of diterpenoid phytoalexins. Three biosynthetic gene clusters (BGCs) and a few non-BGC genes are responsible for rice diterpenoid phytoalexin biosynthesis. The corresponding regulatory mechanism of these phytoalexins in response to pathogen challenges still remains unclear. Here we identified a transcription factor, OsWRKY10, which positively regulates rice diterpenoid phytoalexin biosynthesis. Knockout mutants of OsWRKY10 obtained by CRISPR/Cas9 technology are more susceptible to Magnaporthe oryzae infection, while overexpression of OsWRKY10 enhances resistance to rice blast. Further analysis revealed that overexpression of OsWRKY10 increases accumulation of multiple rice diterpenoid phytoalexins and expression of genes in three BGCs and non-BGC genes in response to M. oryzae infection. Knockout of OsWRKY10 impairs upregulation of rice diterpenoid phytoalexin biosynthesis gene expression by blast pathogen and CuCl2 treatment. OsWRKY10 directly binds to the W-boxes or W-box-like elements (WLEs) of rice diterpenoid phytoalexin biosynthesis gene promoters to regulate gene expression. This study identified an extensive regulator (OsWRKY10) with broad transcriptional regulatory effects on rice diterpenoid phytoalexin biosynthesis genes, providing insight into the regulation of chemical defense to improve disease resistance in rice.

Identification of a small set of genes commonly regulated in rice roots in response to beneficial rhizobacteria.

Rhizosphere bacteria, whether phytopathogenic or phytobeneficial, are thought to be perceived by the plant as a threat. Plant Growth-Promoting Rhizobacteria (PGPR), such as many strains of the Azospirillum genus known as the main phytostimulator of cereals, cooperate with host plants and favorably affect their growth and health. An earlier study of rice root transcriptome, undertaken with two rice cultivars and two Azospirillum strains, revealed a strain-dependent response during the rice-Azospirillum association and showed that only a few genes, including some implicated in plant defense, were commonly regulated in all tested conditions. Here, a set of genes was selected from previous studies and their expression was monitored by qRT-PCR in rice roots inoculated with ten PGPR strains isolated from various plants and belonging to various genera (Azospirillum, Herbaspirillum, Paraburkholderia). A common expression pattern was highlighted for four genes that are proposed to be markers of the rice-PGPR interaction: two genes involved in diterpenoid phytoalexin biosynthesis (OsDXS3 and OsDTC2) and one coding for an uncharacterized protein (Os02g0582900) were significantly induced by PGPR whereas one defense-related gene encoding a pathogenesis-related protein (PR1b, Os01g0382000) was significantly repressed. Interestingly, exposure to a rice bacterial pathogen also triggered the expression of OsDXS3 while the expression of Os02g0582900 and PR1b was down-regulated, suggesting that these genes might play a key role in rice-bacteria interactions. Integration of these results with previous data led us to propose that the jasmonic acid signaling pathway might be triggered in rice roots upon inoculation with PGPR.

Jasmonate-Induced Defense Mechanisms in the Belowground Antagonistic Interaction Between Pythium arrhenomanes and Meloidogyne graminicola in Rice.

Next to their essential roles in plant growth and development, phytohormones play a central role in plant immunity against pathogens. In this study we studied the previously reported antagonism between the plant-pathogenic oomycete Pythium arrhenomanes and the root-knot nematode Meloidogyne graminicola, two root pathogens that co-occur in aerobic rice fields. In this manuscript, we investigated if the antagonism is related to imbalances in plant hormone levels, which could be involved in activation of plant defense. Hormone measurements and gene expression analyses showed that the jasmonate (JA) pathway is induced early upon P. arrhenomanes infection. Exogenous application of methyl-jasmonate (MeJA) on the plant confirmed that JA is needed for basal defense against both P. arrhenomanes and M. graminicola in rice. Whereas M. graminicola suppresses root JA levels to increase host susceptibility, Pythium inoculation boosts JA in a manner that prohibits JA repression by the nematode in double-inoculated plants. Exogenous MeJA supply phenocopied the defense-inducing capacity of Pythium against the root-knot nematode, whereas the antagonism was weakened in JA-insensitive mutants. Transcriptome analysis confirmed upregulation of JA biosynthesis and signaling genes upon P. arrhenomanes infection, and additionally revealed induction of genes involved in biosynthesis of diterpenoid phytoalexins, consistent with strong activation of the gene encoding the JA-inducible transcriptional regulator DITERPENOID PHYTOALEXIN FACTOR. Altogether, the here-reported data indicate an important role for JA-induced defense mechanisms in this antagonistic interaction. Next to that, our results provide evidence for induced expression of genes encoding ERF83, and related PR proteins, as well as auxin depletion in P. arrhenomanes infected rice roots, which potentially further contribute to the reduced nematode susceptibility seen in double-infected plants.

Studies on regulation mechanism of diterpenoid phytoalexin biosynthesis and its evolutional aspects

Studies on regulation mechanism of diterpenoid phytoalexin biosynthesis and its evolutional aspects

OsTGAP1 is responsible for JA-inducible diterpenoid phytoalexin biosynthesis in rice roots with biological impacts on allelopathic interaction.

Phytocassanes and momilactones are known as major diterpenoid phytoalexins (DPs), characterized by abundant production and antimicrobial activity, and their biosynthetic genes are clustered in rice genomes. The basic leucine zipper transcription factor OsTGAP1 is known to act as a regulator of the coordinated production of DPs in cultured rice cells, but in planta functions of OsTGAP1 remain largely unknown. Here, we present evidence on the biological function of OsTGAP1 in planta. In wild-type plants, OsTGAP1 is abundantly expressed in roots compared with that in shoots. Moreover, the inductive expression of OsTGAP1 under jasmonic acid (JA) treatment was only observed in a root-specific manner consistent with the JA-inducible expressions of DP biosynthetic genes in roots. In reverse genetic approaches on OsTGAP1-overexpressing and OsTGAP1-knockdown plants, expressions of the biosynthetic genes relevant for DP accumulation were found to be remarkably increased and decreased, respectively. Reporter analysis in planta revealed that OsTGAP1 activated the promoters of OsDXS3 and momilactone biosynthetic gene OsKSL4, presumably through binding to the TGACGT motif. Furthermore, cocultivation experiments with barnyard grass suggested that the allelopathic effect of knockdown and overexpression of OsTGAP1 was significantly changed compared with the controls. These results demonstrate that OsTGAP1 positively regulates DP accumulation via the transcriptional regulation of DP biosynthetic genes in rice roots, and this is indispensable for maintaining allelopathic interactions with paddy weeds by regulating the production of specialized metabolites like momilactones.

Diterpenoid phytoalexin factor, a bHLH transcription factor, plays a central role in the biosynthesis of diterpenoid phytoalexins in rice.

Rice (Oryza sativa) produces diterpenoid phytoalexins (DPs), momilactones and phytocassanes as major phytoalexins. Accumulation of DPs is induced in rice by blast fungus infection, copper chloride or UV light. Here, we describe a rice transcription factor named diterpenoid phytoalexin factor (DPF), which is a basic helix-loop-helix (bHLH) transcription factor. The gene encoding DPF is expressed mainly in roots and panicles, and is inducible in leaves by blast infection, copper chloride or UV. Expression of all DP biosynthetic genes and accumulation of momilactones and phytocassanes were remarkably increased and decreased in DPF over-expressing and DPF knockdown rice, respectively. These results clearly demonstrated that DPF positively regulates DP accumulation via transcriptional regulation of DP biosynthetic genes, and plays a central role in the biosynthesis of DPs in rice. Furthermore, DPF activated the promoters of COPALYL DIPHOSPHATE SYNTHASE2 (CPS2) and CYTOCHROME P450 MONOOXYGENASE 99A2 (CYP99A2), whose products are implicated in the biosynthesis of phytocassanes and momilactones, respectively. Mutations in the N-boxes in the CPS2 upstream region, to which several animal bHLH transcription factors bind, decreased CPS2 transcription, indicating that DPF positively regulates CPS2 transcription through the N-boxes. In addition, DPF partly regulates CYP99A2 through the N-box. This study demonstrates that DPF acts as a master transcription factor in DP biosynthesis.

A Tandem Array of ent-Kaurene Synthases in Maize with Roles in Gibberellin and More Specialized Metabolism.

While most commonly associated with its role in gibberellin phytohormone biosynthesis, ent-kaurene also serves as an intermediate in more specialized diterpenoid metabolism, as exemplified by the more than 800 known derived natural products. Among these are the maize kauralexins. However, no ent-kaurene synthases (KSs) have been identified from maize. The maize gibberellin-deficient dwarf-5 (d5) mutant has been associated with a loss of KS activity. The relevant genetic lesion has been previously mapped, and was found here to correlate with the location of the KS-like gene ZmKSL3. Intriguingly, this forms part of a tandem array with two other terpene synthases (TPSs). Although one of these, ZmTPS1, has been previously reported to encode a sesquiterpene synthase, and both ZmTPS1 and that encoded by the third gene, ZmKSL5, have lost the N-terminal γ-domain prototypically associated with KS(L)s, all three genes fall within the KS(L) or TPS-e subfamily. Here it is reported that all three genes encode enzymes that are targeted to the plastid in planta, where diterpenoid biosynthesis is initiated, and which all readily catalyze the production of ent-kaurene. Consistent with the closer phylogenetic relationship of ZmKSL3 with previously identified KSs from cereals, only transcription of this gene is affected in d5 plants. On the other hand, the expression of all three of these genes is inducible, suggesting a role in more specialized metabolism, such as that of the kauralexins. Thus, these results clarify not only gibberellin phytohormone, but also diterpenoid phytoalexin biosynthesis in this important cereal crop plant.

WRKY45-dependent priming of diterpenoid phytoalexin biosynthesis in rice and the role of cytokinin in triggering the reaction.

Plant activators such as benzothiadiazole (BTH) protect plants against diseases by priming the salicylic acid (SA) signaling pathway. In rice, the transcription factor WRKY45 plays a central role in this process. To investigate the mechanism involved in defense-priming by BTH and the role of WRKY45 in this process, we analyzed the transcripts of biosynthetic genes for diterpenoid phytoalexins (DPs) during the rice–Magnaporthe oryzae interaction. The DP biosynthetic genes were barely upregulated in BTH-treated rice plants, but were induced rapidly after M. oryzae infection in a WRKY45-dependent manner. These results indicate that the DP biosynthetic genes were primed by BTH through WRKY45. Rapid induction of the DP biosynthetic genes was also observed after M. oryzae infection to WRKY45-overexpressing (WRKY45-ox) plants. The changes in gene transcription resulted in accumulation of DPs in WRKY45-ox and BTH-pretreated rice after M. oryzae infection. Previously, we reported that cytokinins (CKs), especially isopentenyladenines, accumulated in M. oryzae-infected rice. Here, we show that DP biosynthetic genes are regulated by the SA/CK synergism in a WRKY45-dependent manner. Together, we propose that CK plays a role in mediating the signal of M. oryzae infection to trigger the induction of DP biosynthetic genes in BTH-primed plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-014-0221-x) contains supplementary material, which is available to authorized users.

Spatial Regulation of Defense-Related Genes Revealed by Expression Analysis using Dissected Tissues of Rice Leaves Inoculated with Magnaporthe oryzae

Spatial Regulation of Defense- Related Genes Revealed by Expression Analysis using Dissected Tissues of Rice Leaves Inoculated with Magnaporthe oryzae Gene expression profiles in response to inoculation with Magnaporthe oryzae at infected and adjacent cells obtained by microarray coupled with laser microdissection (LMD) were compared with results of microarray expression profiling using RNAs from pathogen infected whole leaves (WL). Genes whose expression was up-regulated following inoculation with the fungus were classified into two categories: group A indicates those whose increased expression was detected both in LMD- and WLmicroarrays, while group B indicates genes of which the expression was detected at significantly higher levels in WL-microarray than LMD-microarray. Interestingly, genes encoding enzymes for the biosynthesis of diterpenoid phytoalexins were exclusively found in group A, while pathogenesis-related (PR) genes were found in both groups A and B.

Oscyp71Z2 involves diterpenoid phytoalexin biosynthesis that contributes to bacterial blight resistance in rice

Bacterial blight is one of the most destructive rice diseases, which caused by Xoo, and results in yield losses, endangering worldwide food security. Diterpenoid phytoalexins, a type of antimicrobials produced in rice, are critical for resistance to fungal and bacterial pathogens. This article reports the characterization of the cytochrome P450 gene Oscyp71Z2, which belongs to the CYP71Z subfamily. Overexpression of Oscyp71Z2 in rice enhanced resistance to Xoo at the booting stage. The accumulation of phytoalexins was rapidly and strongly induced in Oscyp71Z2-overexpressing plants, and the transcript levels of genes related to the phytoalexin biosynthesis pathway were elevated. The H2O2 concentration in Oscyp71Z2-overexpressing plants was reduced in accordance with the increase in ROS-scavenging ability due to the induction of SOD as well as POD and CAT activation. We also showed that suppression of Oscyp71Z2 had no significantly effect on disease resistance to Xoo in rice. These results demonstrated that Oscyp71Z2 plays an important role in bacterial blight resistance by regulating the diterpenoid phytoalexin biosynthesis and H2O2 generation.

78. Identification of a new gene involved in the regulation of biosynthesis of diterpenoid phytoalexins in rice

78. Identification of a new gene involved in the regulation of biosynthesis of diterpenoid phytoalexins in rice

A rice fungal MAMP‐responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis

Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti-microbial secondary metabolites. Mitogen-activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor-induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4DD-induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP-triggered defense responses.

15. A gene cluster on rice chromosome 2 that is involved in biosynthesis of diterpenoid phytoalexins

15. A gene cluster on rice chromosome 2 that is involved in biosynthesis of diterpenoid phytoalexins

Effects of a bile acid elicitor, cholic acid, on the biosynthesis of diterpenoid phytoalexins in suspension-cultured rice cells

An elicitor of rice defense responses was recently isolated from human feces and was identified as cholic acid (CA). Pathogen infection in rice leaves induces phytocassanes and momilactones, both of which are major diterpenoid phytoalexins in rice, whereas CA mainly induces phytocassanes. We established a high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry protocol for the rapid and accurate quantification of phytocassanes and momilactones. Using this method, we showed that CA preferentially induced the formation of phytocassanes in suspension-cultured rice cells, while a fungal chitin oligosaccharide elicitor induced that of both phytocassanes and momilactones. We further investigated the effects of CA on the expression of diterpene cyclase genes involved in phytoalexin biosynthesis. CA induced the transcription of the genes OsCPS2 ( OsCyc2) and OsKSL7 ( OsDTC1), which are involved in phytocassane biosynthesis, to a greater extent than the genes OsCPS4 ( OsCyc1) and OsKSL4, which are involved in momilactone biosynthesis. OsCPS2 was particularly strongly induced, suggesting that it is one of the main mechanisms by which CA induces high levels of phytocassanes.

Uncovering the complex metabolic network underlying diterpenoid phytoalexin biosynthesis in rice and other cereal crop plants

Rice ( Oryza sativa) is a staple food crop and serves as a model cereal crop plant for scientific study. Phytochemical investigations of the agronomically devastating rice blast disease have identified a number of rice phytoalexins exhibiting significant direct anti-fungal activity against the causative agent, Magneporthe grisea. Current evidence strongly indicates that these phytoalexins, largely a family of labdane-related diterpenoids, are important as general antibiotics, and that similar phytoalexins are produced more broadly throughout the cereal crop family. From the extensive sequence information available for rice it has been possible to functionally identify the genes for the enzymes catalyzing the two consecutive cyclization reactions that initiate biosynthesis of these labdane-related diterpenoid phytoalexins. This has led to several insights into the underlying evolution of diterpene biosynthesis throughout the cereal crop family. The hydrocarbon olefins resulting from cyclization must be further elaborated to form bioactive natural products and, because not much is currently known, necessarily speculative biosynthetic pathways for these processes are presented. Given the significant antibiotic activity of the labdane-related diterpenoid phytoalexins from rice, and the presence of similar secondary metabolism throughout the cereal crop plant family, study of this type of biosynthesis will continue to be an area of active investigation.