Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea.

Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate's near ß-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured ß-axial OH was closest to Asp51, whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)-DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (-)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain.

Pathogen-induced biosynthetic pathways encode defense-related molecules in bread wheat.

Wheat is a widely grown food crop that suffers major yield losses due to attack by pests and pathogens. A better understanding of biotic stress responses in wheat is thus of major importance. The recently assembled bread wheat genome coupled with extensive transcriptomic resources provides unprecedented new opportunities to investigate responses to pathogen challenge. Here, we analyze gene coexpression networks to identify modules showing consistent induction in response to pathogen exposure. Within the top pathogen-induced modules, we identify multiple clusters of physically adjacent genes that correspond to six pathogen-induced biosynthetic pathways that share a common regulatory network. Functional analysis reveals that these pathways, all of which are encoded by biosynthetic gene clusters, produce various different classes of compounds­namely, flavonoids, diterpenes, and triterpenes, including the defense-related compound ellarinacin. Through comparative genomics, we also identify associations with the known rice phytoalexins momilactones, as well as with a defense-related gene cluster in the grass model plant Brachypodium distachyon. Our results significantly advance the understanding of chemical defenses in wheat and open up avenues for enhancing disease resistance in this agriculturally important crop. They also exemplify the power of transcriptional networks to discover the biosynthesis of chemical defenses in plants with large, complex genomes.

Understanding the chemical language mediating maize immunity and environmental adaptation.

Diverse networks of specialized metabolites promote plant fitness by mediating beneficial and antagonistic environmental interactions. In maize (Zea mays), constitutive and dynamically formed cocktails of terpenoids, benzoxazinoids, oxylipins, and phenylpropanoids contribute to plant defense and ecological adaptation. Recent research has highlighted the multifunctional nature of many specialized metabolites, serving not only as elaborate chemical defenses that safeguard against biotic and abiotic stress but also as regulators in adaptive developmental processes and microbiome interactions. Great strides have also been made in identifying the modular pathway networks that drive maize chemical diversity. Translating this knowledge into strategies for enhancing stress resilience traits has the potential to address climate-driven yield losses in one of the world's major food, feed, and bioenergy crops.

Strigolactone deficiency induces jasmonate, sugar and flavonoid phytoalexin accumulation enhancing rice defense against the blast fungus Pyricularia oryzae.

Strigolactones (SLs) are carotenoid-derived phytohormones that regulate plant growth and development. While root-secreted SLs are well-known to facilitate plant symbiosis with beneficial microbes, the role of SLs in plant interactions with pathogenic microbes remains largely unexplored. Using genetic and biochemical approaches, we demonstrate a negative role of SLs in rice (Oryza sativa) defense against the blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae). We found that SL biosynthesis and perception mutants, and wild-type (WT) plants after chemical inhibition of SLs, were less susceptible to P. oryzae. Strigolactone deficiency also resulted in a higher accumulation of jasmonates, soluble sugars and flavonoid phytoalexins in rice leaves. Likewise, in response to P. oryzae infection, SL signaling was downregulated, while jasmonate and sugar content increased markedly. The jar1 mutant unable to synthesize jasmonoyl-l-isoleucine, and the coi1-18 RNAi line perturbed in jasmonate signaling, both accumulated lower levels of sugars. However, when WT seedlings were sprayed with glucose or sucrose, jasmonate accumulation increased, suggesting a reciprocal positive interplay between jasmonates and sugars. Finally, we showed that functional jasmonate signaling is necessary for SL deficiency to induce rice defense against P. oryzae. We conclude that a reduction in rice SL content reduces P. oryzae susceptibility by activating jasmonate and sugar signaling pathways, and flavonoid phytoalexin accumulation.

Singlet oxygen signalling and its potential roles in plant biotic interactions.

Singlet oxygen (SO) is among the most potent reactive oxygen species, and readily oxidizes proteins, lipids and DNA. It can be generated at the plant surface by phototoxins in the epidermis, acting as a direct defense against pathogens and herbivores (including humans). SO can also accumulate within mitochondria, peroxisomes, cytosol and the nucleus through multiple enzymatic and nonenzymatic processes. However, the majority of research on intracellular SO generation in plants has focused on transfer of light energy to triplet oxygen by photopigments from the chloroplast. SO accumulates in response to diverse stresses that perturb chloroplast metabolism, and while its high reactivity limits diffusion distances, it participates in retrograde signalling through the EXECUTER1 sensor, generation of carotenoid metabolites and possibly other unknown pathways. SO thereby reprogrammes nuclear gene expression and modulates hormone signalling and programmed cell death. While SO signalling has long been known to regulate plant responses to high-light stress, recent literature also suggests a role in plant interactions with insects, bacteria and fungi. The goals of this review are to provide a brief overview of SO, summarize evidence for its involvement in biotic stress responses and discuss future directions for the study of SO in defense signalling.

Multi-omics revealed molecular mechanism of biphenyl phytoalexin formation in response to yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells.

KEY MESSAGE: Yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells leads to the biosynthesis of various hormones, which activates specific signaling pathways that augments biphenyl phytoalexin production. Pathogen incursions pose a significant threat to crop yield and can have a pronounced effect on agricultural productivity and food security. Biphenyl phytoalexins are a specialized group of secondary metabolites that are mainly biosynthesized by Pyrinae plants as a defense mechanism against various pathogens. Despite previous research demonstrating that biphenyl phytoalexin production increased dramatically in Sorbus aucuparia suspension cells (SASCs) treated with yeast extract (YE), the underlying mechanisms remain poorly understood. To address this gap, we conducted an in-depth, multi-omics analysis of transcriptome, proteome, and metabolite (including biphenyl phytoalexins and phytohormones) dynamics in SASCs exposed to YE. Our results indicated that exposure to YE-induced oxidative stress in SASCs, leading to the biosynthesis of a range of hormones, including jasmonic acid (JA), jasmonic acid isoleucine (JA-ILE), gibberellin A4 (GA4), indole-3-carboxylic acid (ICA), and indole-3-acetic acid (IAA). These hormones activated specific signaling pathways that promoted phenylpropanoid biosynthesis and augmented biphenyl phytoalexin production. Moreover, reactive oxygen species (ROS) generated during this process also acted as signaling molecules, amplifying the phenylpropanoid biosynthesis cascade through activation of the mitogen-activated protein kinase (MAPK) pathway. Key genes involved in these signaling pathways included SaBIS1, SaBIS2, SaBIS3, SaPAL, SaB4H, SaOMT, SaUGT1, SaLOX2, SaPR1, SaCHIB1, SaCHIB2 and SaCHIB3. Collectively, this study provided intensive insights into biphenyl phytoalexin accumulation in YE-treated SASCs, which would inform the development of more efficient disease-resistance strategies in economically significant cultivars.

The impact of climate change on maize chemical defenses.

Climate change is increasingly affecting agriculture, both at the levels of crops themselves, and by altering the distribution and damage caused by insect or microbial pests. As global food security depends on the reliable production of major crops such as maize (Zea mays), it is vital that appropriate steps are taken to mitigate these negative impacts. To do this a clear understanding of what the impacts are and how they occur is needed. This review focuses on the impact of climate change on the production and effectiveness of maize chemical defenses, including volatile organic compounds, terpenoid phytoalexins, benzoxazinoids, phenolics, and flavonoids. Drought, flooding, heat stress, and elevated concentrations of atmospheric carbon dioxide, all impact the production of maize chemical defenses, in a compound and tissue-specific manner. Furthermore, changes in stomatal conductance and altered soil conditions caused by climate change can impact environmental dispersal and effectiveness certain chemicals. This can alter both defensive barrier formation and multitrophic interactions. The production of defense chemicals is controlled by stress signaling networks. The use of similar networks to co-ordinate the response to abiotic and biotic stress can lead to complex integration of these networks in response to the combinatorial stresses that are likely to occur in a changing climate. The impact of multiple stressors on maize chemical defenses can therefore be different from the sum of the responses to individual stressors and challenging to predict. Much work remains to effectively leverage these protective chemicals in climate-resilient maize.

Anticancer Potential of Indole Phytoalexins and Their Analogues.

Indole phytoalexins, found in economically significant Cruciferae family plants, are synthesized in response to pathogen attacks or stress, serving as crucial components of plant defense mechanisms against bacterial and fungal infections. Furthermore, recent research indicates that these compounds hold promise for improving human health, particularly in terms of potential anticancer effects that have been observed in various studies. Since our last comprehensive overview in 2016 focusing on the antiproliferative effects of these substances, brassinin and camalexin have been the most extensively studied. This review analyses the multifaceted pharmacological effects of brassinin and camalexin, highlighting their anticancer potential. In this article, we also provide an overview of the antiproliferative activity of new synthetic analogs of indole phytoalexins, which were synthesized and tested at our university with the aim of enhancing efficacy compared to the parent compound.

Exploring the Antiproliferative and Modulatory Effects of 1-Methoxyisobrassinin on Ovarian Cancer Cells: Insights into Cell Cycle Regulation, Apoptosis, Autophagy, and Its Interactions with NAC.

Ovarian cancer, a highly lethal malignancy among reproductive organ cancers, poses a significant challenge with its high mortality rate, particularly in advanced-stage cases resistant to platinum-based chemotherapy. This study explores the potential therapeutic efficacy of 1-methoxyisobrassinin (MB-591), a derivative of indole phytoalexins found in Cruciferae family plants, on both cisplatin-sensitive (A2780) and cisplatin-resistant ovarian cancer cells (A2780 cis). The findings reveal that MB-591 exhibits an antiproliferative effect on both cell lines, with significantly increased potency against cisplatin-sensitive cells. The substance induces alterations in the distribution of the cell cycle, particularly in the S and G2/M phases, accompanied by changes in key regulatory proteins. Moreover, MB-591 triggers apoptosis in both cell lines, involving caspase-9 cleavage, PARP cleavage induction, and DNA damage, accompanied by the generation of reactive oxygen species (ROS) and mitochondrial dysfunction. Notably, the substance selectively induces autophagy in cisplatin-resistant cells, suggesting potential targeted therapeutic applications. The study further explores the interplay between MB-591 and antioxidant N-acetylcysteine (NAC), in modulating cellular processes. NAC demonstrates a protective effect against MB-591-induced cytotoxicity, affecting cell cycle distribution and apoptosis-related proteins. Additionally, NAC exhibits inhibitory effects on autophagy initiation in cisplatin-resistant cells, suggesting its potential role in overcoming resistance mechanisms.

Liguleless narrow and narrow odd dwarf act in overlapping pathways to regulate maize development and metabolism.

Narrow odd dwarf (nod) and Liguleless narrow (Lgn) are pleiotropic maize mutants that both encode plasma membrane proteins, cause similar developmental patterning defects, and constitutively induce stress signaling pathways. To investigate how these mutants coordinate maize development and physiology, we screened for protein interactors of NOD by affinity purification. LGN was identified by this screen as a strong candidate interactor, and we confirmed the NOD-LGN molecular interaction through orthogonal experiments. We further demonstrated that LGN, a receptor-like kinase, can phosphorylate NOD in vitro, hinting that they could act in intersecting signal transduction pathways. To test this hypothesis, we generated Lgn-R;nod mutants in two backgrounds (B73 and A619), and found that these mutations enhance each other, causing more severe developmental defects than either single mutation on its own, with phenotypes including very narrow leaves, increased tillering, and failure of the main shoot. Transcriptomic and metabolomic analyses of the single and double mutants in the two genetic backgrounds revealed widespread induction of pathogen defense genes and a shift in resource allocation away from primary metabolism in favor of specialized metabolism. These effects were similar in each single mutant and heightened in the double mutant, leading us to conclude that NOD and LGN act cumulatively in overlapping signaling pathways to coordinate growth-defense tradeoffs in maize.

Can Metabolomic Approaches Become a Tool for Improving Early Plant Disease Detection and Diagnosis with Modern Remote Sensing Methods? A Review.

The various areas of ultra-sensitive remote sensing research equipment development have provided new ways for assessing crop states. However, even the most promising areas of research, such as hyperspectral remote sensing or Raman spectrometry, have not yet led to stable results. In this review, the main methods for early plant disease detection are discussed. The best proven existing techniques for data acquisition are described. It is discussed how they can be applied to new areas of knowledge. The role of metabolomic approaches in the application of modern methods for early plant disease detection and diagnosis is reviewed. A further direction for experimental methodological development is indicated. The ways to increase the efficiency of modern early plant disease detection remote sensing methods through metabolomic data usage are shown. This article provides an overview of modern sensors and technologies for assessing the biochemical state of crops as well as the ways to apply them in synergy with existing data acquisition and analysis technologies for early plant disease detection.

Synthesis and Antibacterial Activity Evaluation of Biphenyl and Dibenzofuran Derivatives as Potential Antimicrobial Agents against Antibiotic-Resistant Bacteria.

The escalating prevalence of antibiotic-resistant bacteria has led to a serious global public health problem; therefore, there is an urgent need for the development of structurally innovative antibacterial agents. In our study, a series of biphenyl and dibenzofuran derivatives were designed and synthesized by Suzuki-coupling and demethylation reactions in moderate to excellent yields (51-94% yield). Eleven compounds exhibited potent antibacterial activities against the prevalent antibiotic-resistant Gram-positive and Gram-negative pathogens, among which compounds 4'-(trifluoromethyl)-[1,1'-biphenyl]-3,4,5-triol (6i) and 5-(9H-carbazol-2-yl) benzene-1,2,3-triol (6m) showed the most potent inhibitory activities against methicillin-resistant Staphylococcus aureus and multidrug-resistant Enterococcus faecalis with MIC (minimum inhibitory concentration) values as low as 3.13 and 6.25 µg/mL, respectively. Compounds 3',5'-dimethyl-[1,1'-biphenyl]-3,4,4',5-tetraol (6e), 4'-fluoro-[1,1'-biphenyl]-3,4,5-triol (6g), and 4'-(trifluoromethyl)-[1,1'-biphenyl]-3,4,5-triol (6i) showed comparable inhibitory activities with ciprofloxacin to Gram-negative bacterium carbapenems-resistant Acinetobacter baumannii. Study of the structure-activity relationship indicated that a strong electron-withdrawing group on the A ring and hydroxyl groups on the B ring of biphenyls were beneficial to their antibacterial activities, and for benzo-heterocycles, N-heterocycle exhibited optimal antibacterial activity. These results can provide novel structures of antibacterial drugs chemically different from currently known antibiotics and broaden prospects for the development of effective antibiotics against antibiotic-resistant bacteria.

Lack of antagonism between salicylic acid and jasmonate signalling pathways in poplar.

Salicylic acid (SA) and jasmonic acid (JA) often play distinct roles in plant defence against pathogens. Research from Arabidopsis thaliana has established that SA- and JA-mediated defences are more effective against biotrophs and necrotrophs, respectively. These two hormones often interact antagonistically in response to particular attackers, with the induction of one leading to suppression of the other. Here, we report a contrasting pattern in the woody perennial Populus: positive SA-JA interplay. Using genetically engineered high SA lines of black poplar and wild-type lines after exogenous hormone application, we quantified SA and JA metabolites, signalling gene transcripts, antifungal flavonoids and resistance to rust (Melampsora larici-populina). Salicylic acid and JA metabolites were induced concurrently upon rust infection in poplar genotypes with varying resistance levels. Analysis of SA-hyperaccumulating transgenic poplar lines showed increased jasmonate levels, elevated flavonoid content and enhanced rust resistance, but no discernible reduction in growth. Exogenous application of either SA or JA triggered the accumulation of the other hormone. Expression of pathogenesis-related (PR) genes, frequently used as markers for SA signalling, was not correlated with SA content, but rather activated in proportion to pathogen infection. We conclude that SA and JA pathways interact positively in poplar resulting in the accumulation of flavonoid phytoalexins.

Root-specific camalexin biosynthesis controls the plant growth-promoting effects of multiple bacterial strains.

Plants in their natural ecosystems interact with numerous microorganisms, but how they influence their microbiota is still elusive. We observed that sulfatase activity in soil, which can be used as a measure of rhizosphere microbial activity, is differently affected by Arabidopsis accessions. Following a genome-wide association analysis of the variation in sulfatase activity we identified a candidate gene encoding an uncharacterized cytochrome P450, CYP71A27 Loss of this gene resulted in 2 different and independent microbiota-specific phenotypes: A lower sulfatase activity in the rhizosphere and a loss of plant growth-promoting effect by Pseudomonas sp. CH267. On the other hand, tolerance to leaf pathogens was not affected, which agreed with prevalent expression of CYP71A27 in the root vasculature. The phenotypes of cyp71A27 mutant were similar to those of cyp71A12 and cyp71A13, known mutants in synthesis of camalexin, a sulfur-containing indolic defense compound. Indeed, the cyp71A27 mutant accumulated less camalexin in the roots upon elicitation with silver nitrate or flagellin. Importantly, addition of camalexin complemented both the sulfatase activity and the loss of plant growth promotion by Pseudomonas sp. CH267. Two alleles of CYP71A27 were identified among Arabidopsis accessions, differing by a substitution of Glu373 by Gln, which correlated with the ability to induce camalexin synthesis and to gain fresh weight in response to Pseudomonas sp. CH267. Thus, CYP71A27 is an additional component in the camalexin synthesis pathway, contributing specifically to the control of plant microbe interactions in the root.

Design, Synthesis and Antiproliferative Evaluation of Bis-Indole Derivatives with a Phenyl Linker: Focus on Autophagy.

This work deals with the study of the synthesis of new bis-indole analogues with a phenyl linker derived from indole phytoalexins. Synthesis of target bis-indole thiourea linked by a phenyl linker was achieved by the reaction of [1-(tert-butoxycarbonyl)indol-3-yl]methyl isothiocyanate with p-phenylenediamine. By replacing the sulfur of the thiocarbonyl group in bis-indole thiourea with oxygen using mesityl nitrile oxide, a bis-indole homodimer with a urea group was obtained. A cyclization protocol utilizing bis-indole thiourea and methyl bromoacetate was applied to synthesize a bis-indole homodimer with a thiazolidin-4-one moiety. Bis-indole homodimers derived from 1-methoxyspirobrassinol methyl ether were prepared by bromospirocyclization methodology. Among the synthesized analogues, compound 49 was selected for further study. To evaluate the mode of the mechanism of action, we used flow cytometry, Western blot, and spectroscopic analyses. Compound 49 significantly inhibited the proliferation of lung cancer cell line A549 with minimal effects on the non-cancer cells. We also demonstrated that compound 49 induced autophagy through the upregulation of Beclin-1, LC3A/B, Atg7 and AMPK and ULK1. Furthermore, chloroquine (CQ; an autophagy inhibitor) in combination with compound 49 decreased cell proliferation and induced G1 cell cycle arrest and apoptosis. Compound 49 also caused GSH depletion and significantly potentiated the antiproliferative effect of cis-platin.

A rice root-knot nematode Meloidogyne graminicola-resistant mutant rice line shows early expression of plant-defence genes.

MAIN CONCLUSION: Resistance to rice root-knot nematode Meloidogyne graminicola in a mutant rice line is suggested to be conferred by higher expression of several genes putatively involved in damage-associated molecular pattern recognition, secondary metabolite biosynthesis including phytoalexins, and defence-related genes. Meloidogyne graminicola has emerged as the most destructive plant-parasitic nematode disease of rice (Oryza sativa L.). Genetic resistance to M. graminicola is one of the most effective methods for its management. A M. graminicola-resistant O. sativa ssp. indica mutant line-9 was previously identified through a forward genetic screen (Hatzade et al. Biologia 74:1197-1217, 2019). In the present study, we used RNA-Sequencing to investigate the molecular mechanisms conferring nematode resistance to the mutant line-9 compared to the susceptible parent JBT 36/14 at 24 h post-infection. A total of 674 transcripts were differentially expressed in line-9. Early regulation of genes putatively related to nematode damage-associated molecular pattern recognition (e.g., wall-associated receptor kinases), signalling [Nucleotide-binding, Leucine-Rich Repeat (NLRs)], pathogenesis-related (PR) genes (PR1, PR10a), defence-related genes (NB-ARC domain-containing genes), as well as a large number of genes involved in secondary metabolites including diterpenoid biosynthesis (CPS2, OsKSL4, OsKSL10, Oscyp71Z2, oryzalexin synthase, and momilactone A synthase) was observed in M. graminicola-resistant mutant line-9. It may be suggested that after the nematode juveniles penetrate the roots of line-9, early recognition of invading nematodes triggers plant immune responses mediated by phytoalexins, and other defence proteins such as PR proteins inhibit nematode growth and reproduction. Our study provides the first transcriptomic comparison of nematode-resistant and susceptible rice plants in the same genetic background and adds to the understanding of mechanisms underlying plant-nematode resistance in rice.

A (conditional) role for labdane-related diterpenoid natural products in rice stomatal closure.

Rice (Oryza sativa) is the staple food for over half the world's population. Drought stress imposes major constraints on rice yields. Intriguingly, labdane-related diterpenoid (LRD) phytoalexins in maize (Zea mays) affect drought tolerance, as indicated by the increased susceptibility of an insertion mutant of the class II diterpene cyclase ZmCPS2/An2 that initiates such biosynthesis. Rice also produces LRD phytoalexins, utilizing OsCPS2 and OsCPS4 to initiate a complex metabolic network. For genetic studies of rice LRD biosynthesis the fast-growing Kitaake cultivar was selected for targeted mutagenesis via CRISPR/Cas9, with an initial focus on OsCPS2 and OsCPS4. The resulting cps2 and cps4 knockout lines were further crossed to create a cps2x4 double mutant. Both CPSs also were overexpressed. Strikingly, all of the cv Kitaake cps mutants exhibit significantly increased susceptibility to drought, which was associated with reduced stomatal closure that was evident even under well-watered conditions. However, CPS overexpression did not increase drought resistance, and cps mutants in other cultivars did not alter susceptibility to drought, although these also exhibited lesser effects on LRD production. The results suggest that LRDs may act as a regulatory switch that triggers stomatal closure in rice, which might reflect the role of these openings in microbial entry.

Rice contains a biosynthetic gene cluster associated with production of the casbane-type diterpenoid phytoalexin ent-10-oxodepressin.

Diterpenoids play important roles in rice microbial disease resistance as phytoalexins, as well as acting in allelopathy and abiotic stress responses. Recently, the casbane-type phytoalexin ent-10-oxodepressin was identified in rice, but its biosynthesis has not yet been elucidated. Here ent-10-oxodepressin biosynthesis was investigated via co-expression analysis and biochemical characterisation, with use of the CRISPR/Cas9 technology for genetic analysis. The results identified a biosynthetic gene cluster (BGC) on rice chromosome 7 (c7BGC), containing the relevant ent-casbene synthase (OsECBS), and four cytochrome P450 (CYP) genes from the CYP71Z subfamily. Three of these CYPs were shown to act on ent-casbene, with CYP71Z2 able to produce a keto group at carbon-5 (C5), while the closely related paralogues CYP71Z21 and CYP71Z22 both readily produce a keto group at C10. Together these C5 and C10 oxidases can elaborate ent-casbene to ent-10-oxodepressin (5,10-diketo-ent-casbene). OsECBS knockout lines no longer produce casbane-type diterpenoids and exhibit impaired resistance to the rice fungal blast pathogen Magnaporthe oryzae. Elucidation of ent-10-oxodepressin biosynthesis and the associated c7BGC provides not only a potential target for molecular breeding, but also, gives the intriguing parallels to the independently assembled BGCs for casbene-derived diterpenoids in the Euphorbiaceae, further insight into plant BGC evolution, as discussed here.

Antifungal Biphenyl Derivatives from Sorbus pohuashanensis Leaves Infected by Alternaria tenuissi and Their Effect against Crop Pathogens.

Eight natural biphenyl-type phytoalexins exhibiting antifungal effect were isolated from the leaves of Sorbus pohuashanensis, which invaded by Alternaria tenuissi, and their growth inhibition rate towards A. tenuissi were 50.3 %, 54.0 %, 66.4 %, 58.8 %, 48.5 %, 51.0 %, 33.3 %, and 37.0 %, respectively. In vivo activity assay verified the protective effect of these natural biphenyls on tobacco leaves. The observation of mycelial morphology revealed that these compounds possessed adverse effects on mycelial growth of A. tenuissi. Subsequently, the most potent active compounds, 3',4',5'-trimethoxy[1,1'-biphenyl]-4-ol (3) and 3,4,4',5-tetramethoxy-1,1'-biphenyl (4), were conducted to the further antifungal evaluation and showed significant activity against the other four crop pathogens, Fusarium graminearum, Helminthosporium maydis, Sclerotinia sclerotiorum, and Exserohilum turcicum. Further, the structure-activity relationships and biosynthesis of these compounds were speculated in this work.

Rice momilactone gene cluster: transcriptional response to barnyard grass (Echinochloa crus-galli).

Expression of genes involved in diterpene biosynthesis, especially momilactone and gibberellins (GAs), in rice plants (Oryza sativa L.) in response to barnyard grass (Echinochloa crus-galli) stress was examined. The three analyzed class II diterpene synthases had the highest fold change expression. Transcription patterns of genes for two homologs of momilactone synthases, OsMAS and OsMAS2, suggests their distinct roles in response to the presence of barnyard grass.