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Multiple genes recruited from hormone pathways partition maize diterpenoid defences

Nature Plants volume 5, pages 1043–1056 (2019)Cite this article

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Abstract

Duplication and divergence of primary pathway genes underlie the evolution of plant specialized metabolism; however, mechanisms partitioning parallel hormone and defence pathways are often speculative. For example, the primary pathway intermediate ent-kaurene is essential for gibberellin biosynthesis and is also a proposed precursor for maize antibiotics. By integrating transcriptional coregulation patterns, genome-wide association studies, combinatorial enzyme assays, proteomics and targeted mutant analyses, we show that maize kauralexin biosynthesis proceeds via the positional isomer ent-isokaurene formed by a diterpene synthase pair recruited from gibberellin metabolism. The oxygenation and subsequent desaturation of ent-isokaurene by three promiscuous cytochrome P450s and a new steroid 5α reductase indirectly yields predominant ent-kaurene-associated antibiotics required for Fusarium stalk rot resistance. The divergence and differential expression of pathway branches derived from multiple duplicated hormone-metabolic genes minimizes dysregulation of primary metabolism via the circuitous biosynthesis of ent-kaurene-related antibiotics without the production of growth hormone precursors during defence.

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Fig. 1: ZmKSL2 is an ent-isokaurene synthase required for kauralexin production.
Fig. 2: Two cytochrome P450s, ZmCYP71Z16 and ZmCYP71Z18, catalyse the production of kauralexins from ent-isokaurene and ent-kaurene.
Fig. 3: Kaurene oxidase-like 2 (ZmKO2) catalyses the synthesis of C-19 oxygenated kauralexins.
Fig. 4: Kauralexin reductase 2 (ZmKR2) is a steroid 5α-reductase family enzyme required for the indirect production of ent-kaurene associated defences.
Fig. 5: Coregulation, positional isomer specificity, enzyme promiscuity and a 5α-steroid reductase family enzyme partition growth and defence-related maize diterpenoid pathways.
Fig. 6: Kauralexins are required to suppress F. graminearum stalk rot.

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Data availability

Raw read sequences have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE120135. All other data that support the findings of this are available from the corresponding author upon request.

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Acknowledgements

We thank A. Steinbrenner, P. Weckwerth, K. Dressano, J. Chan, K. O’Leary, M. Broemmer, H. Riggleman, S. Reyes, S. Delgado and J. Tran for help in planting, treatments and sampling (UCSD). Thanks also to L. Smith (UCSD) for shared UCSD Biology Field Station management. This work was supported by a grant from the National Science Foundation Plant-Biotic Interactions Program (grant no. 1758976 to E.S. and P.Z.), by a Department of Energy Joint Genome Institute Community Science Program grant (no. CSP2568 to P.Z., E.S. and A.H.) and by a fellowship provided by the National Science Foundation Graduate Research Fellowship Program (to K.M.M.).

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Authors and Affiliations

  1. Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA

    Yezhang Ding, Elly Poretsky, Evan Saldivar, Mengxi Wu, Zhouxin Shen, Steven P. Briggs, Andrew Sher, Alisa Huffaker & Eric A. Schmelz

  2. Department of Plant Biology, University of California Davis, Davis, CA, USA

    Katherine M. Murphy, Sibongile Mafu & Philipp Zerbe

  3. Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA

    Bing Yang & Si Nian Char

  4. Chemistry Research Unit, Center for Medical, Agricultural, and Veterinary Entomology, US Department of Agriculture—Agricultural Research Service, Gainesville, FL, USA

    Shawn A. Christensen

  5. Institute of Ecological Agriculture, Sichuan Agricultural University, Chengdu, China

    Qiang Wang

  6. Institute of Bioinformatics, University of Georgia, Athens, GA, USA

    Lexiang Ji

  7. Department of Genetics, University of Georgia, Athens, GA, USA

    Robert J. Schmitz

  8. Department of Plant Breeding and Genetics, Cornell University, Ithaca, NY, USA

    Karl A. Kremling & Edward S. Buckler

  9. Robert W. Holley Center for Agriculture and Health, US Department of Agriculture—Agricultural Research Service, Ithaca, NY, USA

    Edward S. Buckler

  10. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

    Jörg Bohlmann

  11. Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA

    Gabriel Castro-Falcon & Chambers C. Hughes

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  1. Yezhang Ding
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Contributions

Y.D., K.M.M., A.H., P.Z. and E.A.S. designed the experiments and analysed the data. Y.D., E.P., S.A.C., L.J., R.J.S., J.B., P.Z., K.A.K. and E.S.B. designed, performed and analysed the transcriptome data. Y.D., S.M., K.M.M., P.Z. and E.A.S. performed MS experiments and MS-related data analysis. S.M., Y.D., K.M.M., Q.W., E.S. and M.W. performed and analysed the enzyme co-expression data. B.Y., S.N.C. and Y.D. designed gRNA constructs and generated the Zmksl2 maize mutants. A.S., G.C.-F. and C.C.H. performed and analysed the NMR data. Y.D., P.Z. and E.A.S. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Eric A. Schmelz.

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The authors declare no competing interests.

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Peer Review Information: Nature Plants thanks Hugues Renault and other, anonymous, reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–22 and Supplementary Table 6.

Reporting Summary

Supplementary Table 1

Southern leaf blight (SLB; Cochliobolous heterostrophus) induced gene expression in maize leaves.

Supplementary Table 2

Summary of SLB-induced expression levels of diterpene synthases and related pathway genes in maize leaves.

Supplementary Table 3

Summary of SLB-induced expression levels of the maize cytochrome P450 gene family.

Supplementary Table 4

Co-expression analyses of ZmKSL2 with maize P450 gene family and all maize genes.

Supplementary Table 5

GWAS mapping interval identified using the ratio of highly oxidized kauralexins to total kauralexins.

Supplementary Table 7

Using the ratio of kauralexin A-/B-series metabolites as a trait, GWAS identifies an interval containing steriod 5a reductase candidates.

Supplementary Table 8

Protein fold changes of DiTPS pathway enzymes in the stem tissues (Zea mays var. W22) elicited with heat-killed Fusarium venenatum.

Supplementary Table 9

Maize mapping lines used for replicated genome wide association studies (GWAS) in the Goodman diversity panel and quantitative trait loci mapping in the nested association mapping subpopulation B73 × M162W.

Supplementary Table 10

Primers used for qrtPCR analysis.

Supplementary Table 11

Primers used for mutant identification.

Supplementary Table 12

Primers used for gene cloning into the pLIFE33 expression vector from cDNA.

Supplementary Table 13

Abbreviations and accession identification numbers for diterpene synthases, kaurene oxidases, steriod 5α-reductases and CYP71 family proteins used for phylogenetic analysis in this study.

Supplementary Table 14

Gene sequences used in enzyme co-expression studies including native sequences, synthetic sequences, and codon-optimized sequences for expression in E. coli.

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Ding, Y., Murphy, K.M., Poretsky, E. et al. Multiple genes recruited from hormone pathways partition maize diterpenoid defences. Nat. Plants 5, 1043–1056 (2019). https://doi.org/10.1038/s41477-019-0509-6

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