Selection of a subspecies-specific diterpene gene cluster implicated in rice disease resistance

ABSTRACT Diterpenoids are the major group of antimicrobial phytoalexins in rice1,2. Here, we report the discovery of a rice diterpenoid gene cluster on chromosome 7 (_DGC7_) encoding the

entire biosynthetic pathway to 5,10-diketo-casbene, a member of the monocyclic casbene-derived diterpenoids. We revealed that _DGC7_ is regulated directly by JMJ705 through methyl

jasmonate-mediated epigenetic control3. Functional characterization of pathway genes revealed _OsCYP71Z21_ to encode a casbene C10 oxidase, sought after for the biosynthesis of an array of

medicinally important diterpenoids. We further show that _DGC7_ arose relatively recently in the _Oryza_ genus, and that it was partly formed in _Oryza_ _rufipogon_ and positively selected

for in _japonica_ during domestication. Casbene-synthesizing enzymes that are functionally equivalent to OsTPS28 are present in several species of Euphorbiaceae but gene tree analysis shows

that these and other casbene-modifying enzymes have evolved independently. As such, combining casbene-modifying enzymes from these different families of plants may prove effective in

producing a diverse array of bioactive diterpenoid natural products. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution

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PATTERN AND STRIGA SUSCEPTIBILITY Article Open access 12 August 2024 GENOME-WIDE CHARACTERIZATION OF THE SORGHUM _JAZ_ GENE FAMILY AND THEIR RESPONSES TO PHYTOHORMONE TREATMENTS AND APHID

INFESTATION Article Open access 25 February 2022 GENOME-WIDE INVESTIGATION OF CYTOKININ OXIDASE/DEHYDROGENASE (CKX) FAMILY GENES IN _BRASSICA JUNCEA_ WITH AN EMPHASIS ON YIELD-INFLUENCING

_CKX_ Article Open access 29 May 2025 DATA AVAILABILITY The sequences data of 424 O_. sativa_ accessions are available in the National Center for Biotechnology Information (NCBI) under the

BioProject PRJNA171289 (ref. 17). The single nucleotide polymorphisms information of 424 _O. sativa_ accessions is available in RiceVarMap (http://ricevarmap.ncpgr.cn/v1). The pangenome data

were acquired from the pangenome dataset (https://figshare.com/collections/Novel_sequences_structural_variations_and_gene_presence_variations_of_Asian_cultivated_rice/3876022/1 and

http://cgm.sjtu.edu.cn/3kricedb/)18,19,24. Thirteen accessions of _O. rufipogon_ were selected from 446 diverse _O. rufipogon_ accessions from Asia and Oceania, and represented all the major

genetically distinct clusters in _O. rufipogon_; the other ten wild rice are from EnsemblPlants (http://plants.ensembl.org/index.html) and National Genomics Data Center

(https://bigd.big.ac.cn/search?dbId=gwh&q=Oryza), including _O. barthii_ (AA), _O. glumipatula_ (AA), _O. glaberrima_ (AA), _O. meridionalis_ (AA), _O. longistaminata_ (AA), _O. nivara_

(AA), _O. brachyantha_ (FF), _O. punctata_ (BB) and _O. brachyantha_ (GG)24. Genes reported in the study are deposited in the NCBI. The genes can be found in GenBank or Rice Genome

Annotation Project database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml) under the following accession numbers: OsTPS28, MN833254; OsCYP71Z21, LOC_Os07g11870; OsCYP71Z2,

LOC_Os07g11739; OsCYP71Z22, LOC_Os07g11970; OsCYP71Z30, LOC_Os07g11890. Source data are provided with this paper. CHANGE HISTORY * _ 16 DECEMBER 2020 A Correction to this paper has been

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responsive pathways in the UV-B adaptation of qingke. _Mol. Plant_ 13, 112–127 (2020). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank J. D. Keasling, G. P.

Lomonossoff and Z. Zhao for their advice and their gift of the expression vectors and strains. We also thank D. R. Nelson, University of Tennessee, for help in naming the OsCYP71Z30. This

work was supported by the National Science Fund for Distinguished Young Scholars (grant no. 31625021), the State Key Programme of National Natural Science Foundation of China (grant no.

31530052) and the Hainan University Startup Fund KYQD(ZR)1866 to J.L. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * National Key Laboratory of Crop Genetic Improvement and National Center of

Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China Chuansong Zhan, Long Lei, Zixin Liu, Shen Zhou, Chenkun Yang, Xitong Zhu, Hao Guo, Feng Zhang, Meng Peng, Meng

Zhang, Yufei Li, Zixin Yang, Yangyang Sun, Yuheng Shi, Kang Li, Shuangqian Shen, Xuyang Wang, Jiawen Shao, Xinyu Jing, Zixuan Wang, Lianghuan Qu, Xianqing Liu, Ling-Ling Chen & Meng Yuan

* College of Tropical Crops, Hainan University, Haikou, China Long Lei, Hao Guo, Ling Liu, Xianqing Liu & Jie Luo * Centre for Novel Agricultural Products, Department of Biology,

University of York, York, UK Yi Li, Tomasz Czechowski & Ian Graham * College of Agriculture, Ibaraki University, Ami, Japan Morifumi Hasegawa * Graduate School of Biological Sciences,

Nara Institute of Science and Technology, Ikoma, Japan Takayuki Tohge * Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany Alisdair R. Fernie Authors * Chuansong Zhan

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J.L., L.-L.C., L.Q., M.Y. and X.L. supervised this study. C.Z., L. Lei, S.Z., Z.L., F.Z., M.Z., Y. Sun, Y. Shi, K.L., T.C., M.H., I.G., Z.Y. and T.T. participated in the material

preparation. C.Z., C.Y., Yi Li, X.W. and J.S. carried out the metabolite analyses. C.Z., Z.L., S.Z., C.Y., X.Z., H.G., M.P., M.Z., Yufei Li, Z.Y., L. Liu, S.S., J.S., X.J., Yi Li, T.T. and

Z.W. performed the data analyses. C.Z., L. Lei, Z.L., S.Z. and C.Y. performed most of the experiments. J.L., C.Z., I.G. and A.R.F. wrote the manuscript. CORRESPONDING AUTHOR Correspondence

to Jie Luo. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Plants_ thanks the anonymous reviewers

for their contribution to the peer review of this work. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 THE DISTRIBUTION OF THE WORLDWIDE COLLECTION OF RICE ACCESSIONS IN THIS STUDY. The core collection of 424 cultivated rice accessions in this

study has a wide geographic distribution. Colour dots indicate different subspecies/type of cultivated rice. EXTENDED DATA FIG. 2 FUNCTIONAL ANALYSES OF OSTPS28, OSCYP71Z2 AND OSCYP71Z21. A,

Gas chromatography of the reaction products of OsTPS28 with GGDP. GGDP, geranylgeranyl diphosphate. Casbene and neocembrene reference compounds were purified from infiltrated _N.

benthamiana_ leaves by the method described previously26. Compound 1, casbene; Compound 2, neocembrene. B, Gas chromatography of _in vitro_ enzyme assays showing the 10-keto-casbene C5

oxidase activity of yeast-expressed CYP71Z2 in the present of NADPH. Microsomes prepared from yeast containing PESC-URA empty vector were used as a negative control. 10-keto-casbene

reference compound was purified from rice leaves by the method described previously13,14. Compound 3, 10-keto- casbene; Compound 4, 5,10-diketo-casbene. C, Gas chromatography of the extracts

prepared from the leaves of _N. benthamiana_ infiltrated with OsTPS28 overexpressing vector. EXTENDED DATA FIG. 3 MASS SPECTRUM AND STRUCTURE OF 5,10- DIKETO-CASBENE. A, Mass spectrum and

structure of the product in _N. benthamiana_ leaves simultaneously overexpressing _OsTPS28_, _OsCYP71Z2_ and _OsCYP71Z21_. B, Mass spectrum of 5,10-diketo-casbene reference. LC-MS, liquid

chromatography–mass spectrometry. C, 1H NMR (left) and 13C NMR (right) results of 5,10-diketo-casbene. EXTENDED DATA FIG. 4 THE EXPRESSION PROFILES OF GENES FROM _DGC7_. The genes from

_DGC7_ are indicated in bold. The transcript abundances of indicated genes in different organs at different stages were shown: expression levels of _OsTPS28_, _OsCYP17Z2_ and _OsCYP71Z21_ is

correlated at different developmental stages. The numerical values for blue-to-red gradient represent normalized expression levels from quantitative real-time PCR analysis. EXTENDED DATA

FIG. 5 THE CASBENE-TYPE DITERPENE BIOSYNTHESIS VIA DISTINCT BIOSYNTHETIC ROUTES IN RICE AND CASTOR. The casbene-type diterpene biosynthetic pathways in rice and castor. Chr.7, chromosome 7;

GGDP, geranylgeranyl diphosphate. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Methods, Figs. 1–16 and references. REPORTING SUMMARY SUPPLEMENTARY TABLES Supplementary

Tables 1–10. SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 3 Statistical source data. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE Zhan, C., Lei, L., Liu, Z. _et al._ Selection of a subspecies-specific diterpene gene cluster implicated in rice disease resistance. _Nat. Plants_ 6, 1447–1454 (2020).

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