Bioorganic chemistry of natural products that control plant pathogens

Abstract

Developing new agrochemicals is essential for sustainable agriculture and global food security. Our group focused on natural products that control plant pathogens, conducting synthetic research across three key areas of interest: antimicrobial compounds, phytoalexins, and microbial signaling molecules. We established new methods for producing chiral allylic alcohols as useful synthetic intermediates for natural product synthesis via the enantioselective synthesis of antimicrobial agents such as peniciaculins. In the phytoalexin research, the synthesis of biosynthetic intermediates enabled the elucidation of enzyme functions in terms of their biosynthesis and the confirmation of absolute configurations, deepening our understanding of plant defense systems. Furthermore, the total synthesis and biosynthetic studies of Phytophthora mating hormones revealed a unique chemical relay system regulating sexual reproduction. These findings emphasize the importance of synthetic chemistry in advancing natural product research and offer new strategies for crop protection. Our interdisciplinary approach paves the way for future innovations in combating agricultural pests and diseases.

Introduction

Agricultural productivity is essential for ensuring global food security and economic stability. However, crops are constantly threatened by pests, diseases, and weeds, leading to substantial yield losses. The development of effective pesticides has played a crucial role in modern agriculture by mitigating these threats and increasing crop protection. Pesticides help farmers maintain high yields, reduce postharvest losses, and ensure a stable food supply for the growing global population. The importance of pesticide development extends beyond merely protecting crops. With the continuous evolution of pests and the increasing demand for sustainable agriculture, it is essential to explore new concepts in pesticide innovation.¹⁾ Advances in research have led to the development of highly specific and efficient compounds that improve pest control as well as overall agricultural practices. Integrating cutting-edge technologies, such as precision agriculture and biotechnology, further contributes to optimizing pesticide use and effectiveness. However, the emergence of pesticide resistance among pests necessitates continuous innovation in pesticide chemistry and application techniques. The pursuit of novel approaches, including the use of bioactive natural products, is crucial in addressing these challenges. The future of pesticide development lies in creating highly efficient and adaptable solutions tailored to modern agricultural needs, leveraging scientific advancements and interdisciplinary research. From this perspective, our research group has focused on natural products that control plant pathogens, developing research based on three main approaches: compounds with antimicrobial activity, phytoalexins, and microbial signaling molecules. This paper outlines the representative results for each topic.

Antimicrobial compounds

The development of effective fungicides has several challenges, including the rapid emergence of resistant fungal strains, which reduces the long-term efficacy of existing treatments. Additionally, ensuring high specificity for target pathogens while minimizing harm to beneficial microorganisms remains a substantial hurdle. High costs and lengthy approval processes further complicate innovation in this field. To address these challenges, research into natural products with antibacterial and antifungal properties offers a promising solution. Moreover, analyzing the mechanisms of action of these compounds can potentially provide insights into the development of new pesticides. Synthetic studies of natural products with antimicrobial activity are extremely important, both biologically and synthetically, and we have developed new synthetic methods based on synthetic studies of several compounds.^2–9) The representative compounds among them are shown in Fig. 1.

Fig. 1. Structures of compounds with antimicrobial activity that our group focused on.

Peniciaculins A (1) and B (2) (Fig. 1) are sesquiterpene derivatives that were isolated from the culture broth of Penicillium aculeatum SD-321, a fungus obtained from deep-sea sediment collected in South China.¹⁰⁾ Peniciaculins have been reported to exhibit inhibitory activity against some bacteria, such as phytopathogenic bacteria (Alternaria brassicae). In our synthetic studies of peniciaculins, we used the Sharpless asymmetric dihydroxylation (AD) reaction to construct the chiral tertiary alcohol moiety. Sharpless AD has the advantages of high stereoselectivity, broad substrate applicability, and simple operation using commercially available reagents. Scheme 1 depicts our peniciaculin A synthesis procedure.⁸⁾

Scheme 1. Synthesis of peniciaculin A.

The allylsulfide derivative 11 was prepared in an E-selective manner using Suzuki–Miyaura coupling from 10. The Sharpless AD reaction of 11 gave the corresponding diol with high enantioselectivity (97 : 3), and the sulfide moiety was oxidized with m-chloroperoxybenzoic acid to afford sulfone 12. After acetylation of the secondary hydroxy group of 12, the reductive elimination reaction using samarium diiodide yielded the key synthetic intermediate 13 with a tertiary allyl alcohol moiety. The olefin metathesis reaction with an alkene, followed by catalytic hydrogenation, gave 14. This sequence of reactions enabled the enantioselective synthesis of the carbon skeleton of peniciaculin A. Finally, Williamson ether synthesis of chloride 15 with phenol 16, followed by the deprotection of SEM (2-(trimethylsilyl)ethoxymethyl) groups, afforded peniciaculin A (1). Because the specific optical rotation sign of the synthetic compound 1 was the same as that of the natural product, we confirmed the absolute configuration of the natural product. Subsequently, common intermediate 14 was used to synthesize peniciaculin B (2). The method developed in this study for synthesizing optically active allylic alcohols, which combines AD and Julia reactions, can be applied to the total synthesis of various natural organic compounds. This reaction can produce secondary and tertiary optically active alcohols with high enantiomeric purity, and the resulting alkene moiety can be used in subsequent reactions, such as the aforementioned olefin metathesis reaction, to extend the carbon chain or modify the functional group. The number of microbial control substances synthesized in our studies was limited to those obtained from nature, and there were compounds with which it was difficult to determine the stereochemistry of natural products. It is expected that a quantitative supply of samples would facilitate the advancement of research on the mechanism of action of the compounds.

Phytoalexins

Phytoalexins are a diverse group of antifungal specialized metabolites that are synthesized de novo in plants during pathogen invasion.^11,12) These compounds play crucial roles in plant innate immunity, particularly in defense against phytopathogenic fungi, and they have been extensively investigated since their initial discovery. Several crop species have emerged as important model systems for elucidating the molecular and biochemical basis of phytoalexin biosynthesis, notably rice (Oryza sativa), soybean (Glycine max), and maize (Zea mays). These species not only serve as representative systems for studying inducible defense responses but also hold substantial agricultural importance as staple crops worldwide.

In rice, phytoalexins are predominantly diterpenoids. Key compounds include momilactones A and B,^13,14) oryzalexins A–F and S,^15–19) phytocassanes A–F,^20–22) ent-10-oxodepressin,²³⁾ and abietoryzins.²⁴⁾ The biosynthetic pathways leading to these metabolites have been extensively characterized, providing a comprehensive framework for understanding diterpenoid-mediated defense. Central to these pathways are diterpene synthases that catalyze the formation of hydrocarbon scaffolds, which are subsequently modified by tailoring enzymes, including cytochrome P450 monooxygenases (CYPs).^25–30)

Advances in genomics, transcriptomics, and metabolomics, combined with functional characterization via genetic and biochemical approaches, have greatly facilitated the identification of genes involved in phytoalexin biosynthesis. These studies not only advance our understanding of plant chemical defense systems but also provide potential strategies for engineering disease resistance in crops. The initial step in these studies is to determine the structure of the compounds. However, it is often difficult to determine the structure of phytoalexin compounds that are absent from healthy plants or are present in extremely small amounts. Therefore, synthetic chemistry research is an effective method.^31–36) The structures of representative compounds targeted in our synthetic studies are shown in Fig. 2.

Fig. 2. Structures of phytoalexins that our group focused on.

One of the major classes of diterpenoid phytoalexins in rice is the phytocassanes. The genes responsible for phytocassane biosynthesis are thought to form a cluster on chromosome 2, comprising two diterpene cyclases (OsCPS2 and OsKSL7) and six cytochrome P450s (CYP71Z6, CYP71Z7, and CYP76M5 to CYP76M8). During the functional analysis of a complementary DNA encoding the diterpene cyclase OsKSL7, which is involved in phytoalexin biosynthesis in rice, a previously unidentified diterpene hydrocarbon-like compound was detected as the major product of the enzymatic reaction between ent-copalyl diphosphate (ent-CDP) and recombinant OsKSL7 expressed in Escherichia coli (Scheme 1).²⁶⁾ This unknown compound was also observed in suspension-cultured rice cells following exogenous treatment with a chitin elicitor. Given that ent-CDP is a plausible precursor of ent-cassa-12,15-diene (19), a putative biosynthetic intermediate leading to phytocassanes, 19 was proposed as a possible identity for the unknown compound. However, owing to challenges in purifying the compound, structural characterization remained limited. This issue was resolved by comparing it to a synthetic sample obtained in our synthetic studies (Scheme 2), which confirmed that 19 was indeed a phytocassane biosynthetic intermediate and elucidated the enzymatic function of OsKSL7.³³⁾

Scheme 2. Synthesis of ent-cassa-12,15-diene.

Compound 25, the starting material for the synthesis of 19, was originally a synthetic intermediate in the total synthesis of phytocassane D (18).³²⁾ Various phytocassane derivatives, such as 2-deoxyphytocassane A (17) and 1-deoxyphytocassane C, were synthesized from 25, and using these samples as reference standards, the functions of CYP71Z7 and CYP76M7/M8 were successfully elucidated.²⁸⁾ As previously stated, these studies demonstrate that synthetic chemistry is a useful approach for elucidating enzyme functions that are difficult to determine using conventional biochemical methods.

Maize phytoalexins include three major classes: zealexins,³⁷⁾ kauralexins,^38,39) and dolabralexins.⁴⁰⁾ Zealexins have macrocarpane-type sesquiterpene skeletons. They were previously isolated from maize stems inoculated with plant pathogenic fungi, including Fusarium graminearum. Subsequently, the zealexin biosynthetic pathway was elucidated, and the roles of two maize terpenoid synthases, TPS6 and TPS11, were identified.⁴¹⁾ These enzymes catalyze a series of cyclization reactions from (E,E)-farnesyl diphosphate to β-macrocarpene via β-bisabolene. Comparing the enzymatically produced β-bisabolene to an authentic standard revealed that its absolute configuration was of the S-configuration. Additionally, CYP71Z18 from maize has been proposed as a biosynthetic enzyme involved in the conversion of β-macrocarpene to zealexin A1.⁴²⁾ Although the absolute configurations of the zealexins remain unconfirmed, these findings suggest that the absolute configuration of zealexin A1 should be S. We conducted synthetic studies to confirm this hypothesis, and the results are summarized in Scheme 3.³⁶⁾

Scheme 3. Synthesis of zealexin A1.

The racemic allyl alcohol (±)-31 was synthesized from compound 29 via intermediate 30 using aldol condensation, dehydration, and Luche reduction. Lipase-catalyzed kinetic resolution of (±)-31 produced optically active alcohol (S)-31 and its corresponding acetate (R)-32, both in high enantiomeric purity. A stereospecific Johnson–Claisen rearrangement of (S)-31, followed by regioselective Dieckmann condensation, afforded bicyclic compound 34. Subsequent reduction with sodium borohydride and dehydration yielded compound 35, which, upon hydrolysis of the ester group, produced (S)-zealexin A1 (22). (R)-Zealexin A1 was similarly synthesized from (R)-31. The absolute configuration of natural zealexin A1 was determined to be S using chiral gas chromatography analysis of its corresponding methyl ester 35. Overall, our synthetic studies have provided insights into the functions of the enzymes involved in zealexin biosynthesis.

Microbial signaling molecules

Microorganisms, including bacteria, fungi, and archaea, communicate with one another via small molecules that play crucial roles in intercellular signaling. Among the regulatory systems that govern life processes, the exchange of information between individual microbial cells is particularly important because it initiates key survival strategies such as sporulation and fruiting body formation. This complex communication network is primarily mediated by chemical signals, which enable coordination not only within a single species but also across species and even biological kingdoms.⁴³⁾

One major class of signaling molecules utilized by bacteria is known as autoinducers or quorum-sensing (QS) pheromones. QS is a cell density-dependent transcriptional regulatory system that orchestrates a wide range of biological processes, including pathogenicity, biofilm formation, pigment and antibiotic production, and the acquisition of genetic competence in both Gram-negative and Gram-positive bacteria. Most QS pheromones in Gram-negative bacteria are N-acylhomoserine lactones, whereas those in Gram-positive bacteria are typically oligopeptides.

Another important group of microbial signaling molecules, often referred to as microbial hormones, triggers key developmental processes such as sporulation, sexual reproduction, and fruiting body formation, particularly in fungi. Because most bacteria and fungi do not form true multicellular organisms, the term “hormone” may not be strictly appropriate; thus, these compounds are often more accurately described as hormone-like substances or pheromones. Notably, microbial hormones differ fundamentally from QS pheromones. QS pheromones function in a threshold-dependent manner, requiring a critical concentration based on cell density to induce collective behavior. In contrast, microbial hormones function in a dose-dependent manner without a defined threshold concentration. Structurally, microbial hormones are highly diverse and generally do not share common features. Reported classes include terpenoids, steroids, butenolides, cerebrosides, and nucleic acid-derived compounds. Figure 3 depicts the structures of representative compounds targeted in our synthetic studies.^44–53)

Fig. 3. Structures of microbial signaling molecules that our group focused on.

Phytophthora, which means “plant destroyer,” includes some of the most devastating plant pathogens globally. In the mid-1840s, a catastrophic outbreak of late blight caused by Phytophthora infestans decimated potato crops across Europe and the United States. During the twentieth century, fungicides were effectively used to manage these diseases. However, the emergence and global spread of virulent, fungicide-resistant strains, particularly P. infestans, has led to a resurgence of late blight worldwide.

A key biological process in Phytophthora species is sexual reproduction, which occurs in heterothallic species via the interaction of two distinct mating types: A1 and A2. Mating type A1 secretes hormone α1 (36), inducing oospore formation in A2, whereas A2 secretes hormone α2 (37), inducing oospore formation in A1. These oospores are characterized by thick double walls, enabling them to survive extreme environmental conditions such as desiccation and freezing. Controlling Phytophthora with conventional fungicides remains challenging. Sexual reproduction is believed to contribute to the rapid evolution and dissemination of fungicide-resistant strains.

Despite numerous studies, their chemical structures have remained elusive because of their low natural abundance. Over 70 years after Ashby identified the hormones,⁵⁴⁾ Ojika and colleagues successfully isolated hormone α1 from 1830 L of Phytophthora nicotianae A1 culture broth.⁵⁵⁾ Spectroscopic analysis revealed the planar structure of hormone α1, and the absolute configurations at C-3 and C-15 were determined using nuclear magnetic resonance analysis of the corresponding bis-MTPA ester. Finally, the absolute configuration of hormone α1 was elucidated based on the total synthesis conducted by our research group (Fig. 3).⁴⁷⁾ Our α1 synthesis procedure is shown in Scheme 4.

Scheme 4. Synthesis of Phytophthora mating hormone α1.

The halogen–metal exchange of (R)-citronellyl iodide (42) with tert-butyllithium, followed by nucleophilic addition to an aldehyde, afforded alcohol 43. Subsequent protection of the hydroxy group in 43 as a benzyl ether, oxidative cleavage of its alkene, and Wittig olefination of the resulting aldehyde produced compound 45. Reduction of 45 followed by bromination of the resulting alcohol provided allylic bromide 46. Coupling of 46 with a known sulfone and subsequent reductive desulfonation afforded 47, bearing the full carbon framework of hormone α1. The final key transformation in the total synthesis of hormone α1 involved the stereoselective introduction of a tertiary hydroxy group at C-11 via a Sharpless AD–deoxygenation sequence. Thus, dihydroxylation of 47 with AD-mix-α proceeded with excellent stereoselectivity to give diol 48. Monomesylation of 48, followed by base-mediated mesyloxy group elimination and regioselective reduction of the resulting epoxide with diisobutylaluminum hydride, afforded tertiary alcohol 49. Finally, removal of the benzyl group, Dess–Martin oxidation of the liberated hydroxy group, and mild deprotection of the two tert-butyldimethylsilyl groups produced optically pure (3R,7R,11R,15R)-36.

Next, we synthesized the remaining three isomers at C-7 and C-11 and conducted bioassays on all of them. The results showed that only the (7R,11R)-isomer of 36 exhibited biological activity, allowing us to determine the absolute configuration of the natural hormone α1. Furthermore, following the same procedure, we successfully determined the absolute configuration of the natural hormone α2 (37) (Fig. 3).⁴⁸⁾

As previously stated, the structures of the two mating hormones produced by Phytophthora were elucidated using synthetic chemical approaches, revealing that they are structurally very similar. Notably, mating hormone α2 is structurally similar to phytol (Fig. 4, 50), a compound found ubiquitously in plants. This observation led to the hypothesis that the Phytophthora mating hormones are biosynthesized from phytol.

Fig. 4. Biosynthesis of Phytophthora mating hormones.

To test this hypothesis, we synthesized deuterium-labeled phytol and supplemented it into the culture medium of an α2-producing P. nicotianae A2 strain. This resulted in a dramatic increase in α2 production. Furthermore, analysis of the α2 isolated from the culture medium confirmed the incorporation of deuterium, indicating that it was derived from the labeled phytol (50). In contrast, when phytol was added to the culture medium of the α1-producing A1 strain, no substantial change in α1 production was observed. Interestingly, when deuterium-labeled α2 (51) was added to the culture medium of the A1 strain, α1 production increased considerably, and the isolated α1 was found to contain deuterium (52). These findings strongly support a biosynthetic model in which phytol serves as the precursor for α2 in the A2 strain. The secreted α2 then functions as a signaling molecule, triggering oospore formation in the A1 strain. In response, the A1 strain metabolizes α2 to produce and secrete α1, inducing oospore formation in the A2 strain. This reciprocal exchange of signaling molecules—a “chemical relay” system—represents a sophisticated interstrain communication mechanism.⁴⁸⁾ Such a system may be a characteristic of the evolutionary adaptation of Phytophthora as a phytopathogen. Furthermore, we have reported the structure–activity relationships of hormones α1 and α2,⁵⁰⁾ elucidated the structures of key biosynthetic intermediates formed during the oxidation of phytol to α2,⁵²⁾ and demonstrated that α2 functions as an antagonist in the biosynthesis of α1.⁵¹⁾ The outline of the Phytophthora sexual reproduction system is shown in Fig. 5. We anticipate that these findings will provide a foundation for developing novel strategies to control Phytophthora infections.

Fig. 5. Outline of Phytophthora reproduction system.

Conclusion

We explored the potential of natural products in the development of novel agrochemicals using three major research approaches: antimicrobial compounds, phytoalexins, and microbial signaling molecules. Our synthetic studies of natural antimicrobial compounds, such as peniciaculins, have provided novel methods for constructing characteristic structures with high enantioselectivity. In our phytoalexin research, the synthesis of key biosynthetic intermediates, such as ent-cassa-12,15-diene and zealexin A1, has allowed us to elucidate enzymatic functions and absolute configurations critical to understanding plant defense mechanisms. Furthermore, the total synthesis and biosynthetic studies of Phytophthora mating hormones clarified their absolute structures and unveiled a unique chemical relay system regulating sexual reproduction, offering new perspectives for plant–pathogen control. Collectively, these results emphasize the importance of synthetic chemistry not only in the structural and functional study of natural products but also in the development of innovative strategies to address agricultural challenges. Future research will focus on integrating synthetic, biological, and ecological approaches to create sustainable solutions for crop protection.

Acknowledgments

I would like to express my deepest gratitude to the late Prof. Kenji Mori, Prof. Hirosato Takikawa, and Prof. Goro Yabuta, who introduced me to the fields of synthetic organic chemistry and pesticide science. My sincere appreciation extends to all my collaborators, and especially to my students, who worked tirelessly and with great dedication to bring my ideas and plans to fruition. I am also sincerely grateful to Prof. Hiromasa Kiyota for kindly nominating me for this award and for his ongoing encouragement and support. Finally, I would like to express my heartfelt thanks to everyone who has been part of this journey.

References

• 1) N. Umetsu and Y. Shirai: Development of novel pesticides in the 21st century. J. Pestic. Sci. 45, 54–74 (2020).
• 2) A. Yajima, H. Takikawa and K. Mori: Synthesis of (6R*,7R*)-7-hydroxy-6,ll-cyclofarnes-3(15)-en-2-one, the racemate of the antibacterial sesquiterpene from Premna oligotricha, and Its (6R*,7S*) isomer. Liebigs Ann. 1996, 891–897 (1996).
• 3) A. Yajima and K. Mori: Synthesis of both the enantiomers of incrustoporin, an antibiotic from Incriistoporia carneola. Liebigs Ann. 1996, 1091–1093 (1996).
• 4) A. Yajima, F. Saitou, M. Sekimoto, S. Maetoko and G. Yabuta: Synthesis and absolute configuration of cordiaquinone K, antifungal and larvicidal meroterpenoid isolated from the Panamanian plant, Cordia curassavica. Tetrahedron Lett. 44, 6915–6918 (2003).
• 5) A. Yajima, F. Saitou, M. Sekimoto, S. Maetoko, T. Nukada and G. Yabuta: Synthesis of cordiaquinone J and K via B-alkyl Suzuki–Miyaura coupling as a key step and determination of the absolute configuration of natural products. Tetrahedron 61, 9164–9172 (2005).
• 6) A. Yajima, A. Yamaguchi, F. Saitou, T. Nukada and G. Yabuta: Asymmetric synthesis of abietane diterpenoids via B-alkyl Suzuki–Miyaura coupling. Formal total asymmetric synthesis of 12-deoxyroyleanone and cryptoquinone. Tetrahedron 63, 1080–1084 (2007).
• 7) A. Yajima, Y. Iizuka, T. Katsuta and T. Nukada: Synthesis and absolute configuration of formosusin A, a specific inhibitor of mammalian DNA polymerase β. Tetrahedron Lett. 57, 2012–2015 (2016).
• 8) A. Yajima, I. Shirakawa, N. Shiotani, K. Ueda, H. Murakawa, T. Saito, R. Katsuta and K. Ishigami: Practical synthesis of aromatic bisabolanes: Synthesis of 1,3,5- bisabolatrien-7-ol, peniciaculin A and B, and hydroxysydonic acid. Tetrahedron 92, 132253 (2021).
• 9) N. T. N. Anh, D. Miyaji, K. Osaki-Oka, T. Saito, A. Ishihara and A. Yajima: Synthesis and antifungal activity of the proposed structure of a volatile compound isolated from the edible mushroom Hypsizygus marmoreus. J. Pestic. Sci. 47, 17–21 (2022).
• 10) X.-D. Li, X.-M. Li, G.-M. Xu, P. Zhang and B.-G. Wang: Antimicrobial phenolic bisabolanes and related derivatives from Penicillium aculeatum SD-321, a deep sea sediment-derived fungus. J. Nat. Prod. 78, 844–849 (2015).
• 11) I. Ahuja, R. Kissen and A. M. Bones: Phytoalexins in defense against pathogens. Trends Plant Sci. 17, 73–90 (2012).
• 12) S. Ahmed and N. Kovinich: Regulation of phytoalexin biosynthesis for agriculture and human health. Phytochem. Rev. 20, 483–505 (2021).
• 13) T. Kato, C. Kabuto, N. Sasaki, M. Tsunagawa, H. Aizawa, K. Fujita, Y. Kato, Y. Kitahara and N. Takahashi: Momilactones, growth inhibitors from rice, Oryza sativa L. Tetrahedron Lett. 14, 3861–3864 (1973).
• 14) D. Cartwright, P. Langcake, R. J. Pryce, D. P. Leworthy and J. P. Ride: Chemical activation of host defence mechanisms as a basis for crop protection. Nature 267, 511–513 (1977).
• 15) T. Akatsuka, O. Kodama, H. Sekido, Y. Kono and S. Takeuchi: Novel phytoalexins (oryzalexins A, B and C) isolated from rice blast leaves infected with Pyricularia oryzae. Part I: Isolation, characterization and biological activities of oryzalexins. Agric. Biol. Chem. 49, 1689–1694 (1985).
• 16) H. Sekido, T. Endo, R. Suga, O. Kodama, T. Akatsuka, Y. Kono and S. Takeuchi: Oryzalexin D (3,7-dihydroxy-(+)-sandaracopimaradiene), a new phytoalexin isolated from blast-infected rice leaves. J. Pestic. Sci. 11, 369–372 (1986).
• 17) H. Kato, O. Kodama and T. Akatsuka: Oryzalexin E, A diterpene phytoalexin from UV-irradiated rice leaves. Phytochemistry 33, 79–81 (1993).
• 18) H. Kato, O. Kodama and T. Akatsuka: Oryzalexin F, a diterpene phytoalexin from UV-irradiated rice leaves. Phytochemistry 36, 299–301 (1994).
• 19) O. Kodama, W. X. Li, S. Tamogami and T. Akatsuka: Oryzalexin S, a novel stemarane-type Diterpene rice phytoalexin. Biosci. Biotechnol. Biochem. 56, 1002–1003 (1992).
• 20) J. Koga, M. Shimura, K. Oshima, N. Ogawa, T. Yamauchi and N. Ogasawara: Phytocassanes A, B, C and D, novel diterpene phytoalexins from rice, Oryza sativa L. Tetrahedron 51, 7907–7918 (1995).
• 21) J. Koga, N. Ogawa, T. Yamauchi, M. Kikuchi, N. Ogasawara and M. Shimura: Functional moiety for the antifungal activity of phytocassane E, a diterpene phytoalexin from rice. Phytochemistry 44, 249–253 (1997).
• 22) K. Horie, Y. Inoue, M. Sakai, Q. Yao, Y. Tanimoto, J. Koga, H. Toshima and M. Hasegawa: Identification of UV-induced diterpenes including a new diterpene phytoalexin, phytocassane F, from rice leaves by complementary GC/MS and LC/MS approaches. J. Agric. Food Chem. 63, 4050–4059 (2015).
• 23) Y. Inoue, M. Sakai, Q. Yao, Y. Tanimoto, H. Toshima and M. Hasegawa: Identification of a novel casbane-type diterpene phytoalexin, ent-10-oxodepressin, from rice leaves. Biosci. Biotechnol. Biochem. 77, 760–765 (2013).
• 24) K. Kariya, A. Fujita, M. Ueno, T. Yoshikawa, M. Teraishi, Y. Taniguchi, K. Ueno and A. Ishihara: Natural variation of diterpenoid phytoalexins in rice: Aromatic diterpenoid phytoalexins in specific cultivars. Phytochemistry 211, 113708 (2023).
• 25) E. A. Schmelz, A. Huffaker, J. W. Sims, S. A. Christensen, X. Lu, K. Okada and R. J. Peters: Biosynthesis, elicitation and roles of monocot terpenoid phytoalexins. Plant J. 79, 659–678 (2014) and references cited therein.
• 26) E. M. Cho, A. Okada, H. Kenmoku, K. Otomo, T. Toyomasu, W. Mitsuhashi, T. Sassa, A. Yajima, G. Yabuta, K. Mori, H. Oikawa, H. Toshima, N. Shibuya, H. Nojiri, T. Omori, M. Nishiyama and H. Yamane: Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor. Plant J. 37, 1–8 (2004).
• 27) Y. Wu, M. L. Hillwig, Q. Wang and R. J. Peters: Parsing a multifunctional biosynthetic gene cluster from rice: Biochemical characterization of CYP71Z6 & 7. FEBS Lett. 585, 3446–3451 (2011).
• 28) Z. Ye, K. Yamazaki, H. Minoda, K. Miyamoto, S. Miyazaki, H. Kawaide, A. Yajima, H. Nojiri, H. Yamane and K. Okada: In planta functions of cytochrome P450 monooxygenase genes in the phytocassane biosynthetic gene cluster on rice chromosome 2. Biosci. Biotechnol. Biochem. 82, 1021–1030 (2018).
• 29) C. Zhan, L. Lei, Z. Liu, S. Zhou, C. Yang, X. Zhu, H. Guo, F. Zhang, M. Peng, M. Zhang, Y. Li, Z. Yang, Y. Sun, Y. Shi, K. Li, L. Liu, S. Shen, X. Wang, J. Shao, X. Jing, Z. Wang, Y. Li, T. Czechowski, M. Hasegawa, I. Graham, T. Tohge, L. Qu, X. Liu, A. R. Fernie, L.-L. Chen, M. Yuan and J. Luo: Selection of a subspecies-specific diterpene gene cluster implicated in rice disease resistance. Nat. Plants 6, 1447–1454 (2020).
• 30) K. Kariya, H. Mori, M. Ueno, T. Yoshikawa, M. Teraishi, Y. Yabuta, K. Ueno and A. Ishihara: Identification and evolution of a diterpenoid phytoalexin oryzalactone biosynthetic gene in the genus Oryza. Plant J. 118, 358–372 (2024).
• 31) A. Yajima and K. Mori: Absolute configuration of phytocassanes as proposed on the basis of the CD spectrum of synthetic (+)-2-deoxyphytocassane A. Tetrahedron Lett. 41, 351–354 (2000).
• 32) A. Yajima and K. Mori: Synthesis and absolute configuration of (−)-phytocassane D, a diterpene phytoalexin isolated from the rice plant, Oryza sativa. Eur. J. Org. Chem. 2000, 4079–4091 (2000).
• 33) A. Yajima, K. Mori and G. Yabuta: Total synthesis of ent-cassa-12,15-diene, a putative precursor of rice phytoalexins, phytocassanes A-E. Tetrahedron Lett. 45, 167–169 (2004).
• 34) A. Yajima, A. Yamaguchi, T. Nukada and G. Yabuta: Asymmetric total synthesis of ent-sandaracopimaradiene, a biosynthetic intermediate of oryzalexins. Biosci. Biotechnol. Biochem. 71, 2822–2829 (2007).
• 35) A. Yajima, K. Toda, K. Okada, H. Yamane, M. Yamamoto, M. Hasegawa, R. Katsuta and T. Nukada: Stereocontrolled total synthesis of (±)-3β-hydroxy-9β-pimara-7,15-diene, a putative biosynthetic intermediate of momilactones. Tetrahedron Lett. 52, 3212–3215 (2011).
• 36) A. Yajima, M. Shimura, T. Saito, R. Katsuta, K. Ishigami, A. Huffaker and E. A. Schmelz: Synthesis and determination of absolute configuration of zealexin A1, a sesquiterpenoid phytoalexin from Zea mays. Eur. J. Org. Chem. 2021, 1174–1178 (2021).
• 37) A. Huffaker, F. Kaplan, M. M. Vaughan, N. J. Dafoe, X. Ni, J. R. Rocca, H. T. Alborn, P. E. A. Teal and E. A. Schmelz: Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize. Plant Physiol. 156, 2082–2097 (2011).
• 38) E. A. Schmelza, F. Kaplan, A. Huffaker, N. J. Dafoe, M. M. Vaughan, X. Ni, J. R. Rocca, H. T. Alborn and P. E. Teal: Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize. Proc. Natl. Acad. Sci. U.S.A. 108, 5455–5460 (2011).
• 39) Y. Ding, K. M. Murphy, E. Poretsky, S. Mafu, B. Yang, S. N. Char, S. A. Christensen, E. Saldivar, M. Wu, Q. Wang, L. Ji, R. J. Schmitz, K. A. Kremling, E. S. Buckler, Z. Shen, S. P. Briggs, J. Bohlmann, A. Sher, G. Castro-Falcon, C. C. Hughes, A. Huffaker, P. Zerbe and E. A. Schmelz: Multiple genes recruited from hormone pathways partition maize diterpenoid defences. Nat. Plants 5, 1043–1056 (2019).
• 40) K. M. Murphy, T. Dowd, A. Khalil, S. N. Char, B. Yang, B. J. Endelman, P. M. Shih, C. Topp, E. A. Schmelz and P. Zerbe: A dolabralexin-deficient mutant provides insight into specialized diterpenoid metabolism in maize. Plant Physiol. 192, 1338–1358 (2023).
• 41) T. G. Köllner, C. Schnee, S. Li, A. Svatoš, B. Schneider, J. Gershenzon and J. Degenhardt: Protonation of a neutral (S)-β-bisabolene intermediate is involved in (S)-β-macrocarpene formation by the maize sesquiterpene synthases TPS6 and TPS11. J. Biol. Chem. 283, 20779–20788 (2008).
• 42) H. Mao, J. Liu, F. Ren, R. J. Peters and Q. Wang: Characterization of CYP71Z18 indicates a role in maize zealexin biosynthesis. Phytochemistry 121, 4–10 (2016).
• 43) A. Yajima: Recent progress in the chemistry and chemical biology of microbial signaling molecules: quorum-sensing pheromones and microbial hormones. Tetrahedron Lett. 55, 2773–2780 (2014) and references cited therein.
• 44) A. Yajima, A. A. N. van Brussel, J. Schripsema, T. Nukada and G. Yabuta: Synthesis and stereochemistry-activity relationship of small bacteriocin, an autoinducer of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum. Org. Lett. 10, 2047–2050 (2008).
• 45) A. Yajima, N. Imai, A. R. Poplawsky, T. Nukada and G. Yabuta: Synthesis of a proposed structure for the diffusible extracellular factor of Xanthomonas campestris pv. campestris. Tetrahedron Lett. 51, 2074–2077 (2010).
• 46) A. Yajima, N. Kawanishi, J. Qi, T. Asano, Y. Sakagami, T. Nukada and G. Yabuta: Synthesis and biological activity of a stereoisomeric mixture of the mating horomone of Phytophthora. Tetrahedron Lett. 48, 4601–4603 (2007).
• 47) A. Yajima, Y. Qin, Z. Zhou, N. Kawanishi, X. Xiao, J. Wang, D. Zhang, Y. Wu, T. Nukada, G. Yabuta, J. Qi, T. Asano and Y. Sakagami: Synthesis and absolute configuration of hormone α1. Nat. Chem. Biol. 4, 235–237 (2008).
• 48) M. Ojika, S. D. Molli, H. Kanazawa, A. Yajima, K. Toda, T. Nukada, H. Mao, R. Murata, T. Asano, J. Qi and Y. Sakagami: The second Phytophthora mating hormone defines interspecies biosynthetic crosstalk. Nat. Chem. Biol. 7, 591–593 (2011).
• 49) A. Yajima, K. Toda, S. D. Molli, M. Ojika and T. Nukada: Synthesis of the four stereoisomers of Phytophthora mating hormone α2 and a concise synthesis of mating hormone α1. Tetrahedron 67, 8887–8894 (2011).
• 50) S. D. Molli, J. Qi, A. Yajima, K. Shikai, T. Imaoka, T. Nukada, G. Yabuta and M. Ojika: Structure-activity relationship of α hormones, the mating factors of phytopathogen Phytophthora. Bioorg. Med. Chem. 20, 681–686 (2012).
• 51) L. Zhang, A. Yajima and M. Ojika: The Phytophthora mating hormone α2 is an antagonist of the counter hormone α1. Biosci. Biotechnol. Biochem. 80, 1062–1065 (2016).
• 52) S. Ariyoshi, Y. Imazu, R. Ohguri, R. Katsuta, A. Yajima, T. Shibata and M. Ojika: Identification of biosynthetic intermediates for the mating hormone α2 of the plant pathogen Phytophthora. Biosci. Biotechnol. Biochem. 85, 1802–1808 (2021).
• 53) A. Yajima, R. Katsuta, M. Shimura, A. Yoshihara, T. Saito, K. Ishigami and K. Kai: Disproof of the proposed structures of bradyoxetin, a putative Bradyrhizobium japonicum signaling molecule, and HMCP, a putative Ralstonia solanacearum quorum-sensing molecule. J. Nat. Prod. 84, 495–502 (2021).
• 54) S. F. Ashby: Strains and taxonomy of Phytophthora palmivora Butl. (P. faberi Maubl.). Trans. Br. Mycol. Soc. 14, 18–38 (1929).
• 55) J. Qi, T. Asano, M. Jinno, K. Matsui, K. Atsumi, Y. Sakagami and M. Ojika: Characterization of a Phytophthora mating hormone. Science 309, 1828 (2005).