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MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction

Nature volume 542, pages 105–109 (2017)Cite this article

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Abstract

Sexual reproduction in flowering plants involves double fertilization, the union of two sperm from pollen with two sex cells in the female embryo sac. Modern plant breeders increasingly seek to circumvent this process to produce doubled haploid individuals, which derive from the chromosome-doubled cells of the haploid gametophyte. Doubled haploid production fixes recombinant haploid genomes in inbred lines, shaving years off the breeding process1. Costly, genotype-dependent tissue culture methods are used in many crops2, while seed-based in vivo doubled haploid systems are rare in nature3 and difficult to manage in breeding programmes4. The multi-billion-dollar maize hybrid seed business, however, is supported by industrial doubled haploid pipelines using intraspecific crosses to in vivo haploid inducer males derived from Stock 6, first reported in 1959 (ref. 5), followed by colchicine treatment. Despite decades of use, the mode of action remains controversial6,7,8,9,10. Here we establish, through fine mapping, genome sequencing, genetic complementation, and gene editing, that haploid induction in maize (Zea mays) is triggered by a frame-shift mutation in MATRILINEAL (MTL), a pollen-specific phospholipase, and that novel edits in MTL lead to a 6.7% haploid induction rate (the percentage of haploid progeny versus total progeny). Wild-type MTL protein localizes exclusively to sperm cytoplasm, and pollen RNA-sequence profiling identifies a suite of pollen-specific genes overexpressed during haploid induction, some of which may mediate the formation of haploid seed11,12,13,14,15. These findings highlight the importance of male gamete cytoplasmic components to reproductive success and male genome transmittance. Given the conservation of MTL in the cereals, this discovery may enable development of in vivo haploid induction systems to accelerate breeding in crop plants.

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Figure 1: A mutation in MATRILINEAL is responsible for haploid induction in maize.
Figure 2: MTL is specifically found in the cytoplasm of the male gametes in pollen grains.
Figure 3: The mtl allele is responsible for pleiotropic phenotypes associated with haploid induction.

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Acknowledgements

We thank Data2Bio for the RNA-sequencing and data processing efforts. Thanks to M. Evans of the Carnegie Institute of Plant Biology for embryo sac dissection advice. We thank M. McLaughlin and T. Strebe for plant care, C. Fan for Taqman design, and K. Cox for sampling. We thank S. Dong, S. Nalapalli, H. Zhong, and A. Prairie for construct design, transformation and event production. We thank D. Skibbe and I. Jepson for advice and suggestions, and R. Sessler for proteomics efforts. We thank B. Dietrich for sequence assembly support, Q. Que and L. Shi for laboratory space and personnel, and R. Riley and E. Dunder for project support. Thanks to C. Leming, M. Dunn, S. Miles, D. Skalla, and B. Houghteling for licensing and guidance on intellectual property.

Author information

Author notes

  1. Barry Martin

    Present address: †Present address: CiBO Technologies, 155 2nd Street, Cambridge, Massachusetts 02141, USA.,

Authors and Affiliations

  1. Seeds Research, Syngenta Crop Protection, 9 Davis Drive, Research Triangle Park, North Carolina, 27709, USA

    Timothy Kelliher, Dakota Starr, Lee Richbourg, Michael L. Nuccio, Julie Green, Zhongying Chen, Jamie McCuiston, Wenling Wang, Tara Liebler & Barry Martin

  2. Syngenta Seeds, 2369 330th Street, Slater, 50244, Iowa, USA

    Satya Chintamanani & Paul Bullock

  3. Syngenta Seeds, 4133 East County Road O, Janesville, 53546, Wisconsin, USA

    Brent Delzer

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Contributions

T.K.: experimental design, management, crossing, embryo extraction, imaging, analysis, writing. D.S.: plant sampling, PCR, crossing, embryo extraction, manuscript editing. L.R.: embryo extraction for HIR determination. S.C.: experimental design, broad and fine mapping, manuscript preparation and editing. B.D.: experimental design, line acquisition, broad and fine mapping. M.N.: project initiation, experimental design, candidate gene analysis, phospholipase assays. J.G.: RNA-seq data analysis, manuscript preparation and editing. Z.C.: TALEN technology, construct design, experimental design, manuscript editing. J.M.: Taqman trial design and marker analysis for HIR determination. W.W.: Taqman trial design and marker analysis for HIR determination. T.L.: trial planning and management, plant care and pollinations. P.B.: experimental design, line acquisition, mapping, guidance and theory. B.M.: experimental design, project sponsorship, manuscript editing.

Corresponding author

Correspondence to Timothy Kelliher.

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Competing interests

A provisional patent covering the information in this manuscript was submitted on 18 November 2015.

Additional information

Reviewer Information Nature thanks S. Scholten and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 The Stock 6 derivative line RWK was obtained from the University of Hohenheim in 2006 and subsequently crossed to inbreds NP2460 and NP2391 and backcrossed to RWK to generate mapping populations.

HIR testing and marker analysis were performed on both sets of mapping populations. HIRs in both populations co-segregated with the marker SM020SDQ on the long arm of chromosome 1. Several rounds of fine mapping in RWK-GP6664 BC3F3–BC3F5 populations narrowed the quantitative trait locus to a ~0.57 kb interval between SM2589 and SM2608.

Extended Data Figure 2 Quantification of Mtl RNA in haploid inducer and non-inducer pollen.

a, Splice-specific qRT–PCR results for Mtl transcripts. Three biological replicates of R1-staged anthers were tested in technical triplicate, and the average Ct and s.d. was calculated for each reaction. The relative quantity of each transcript type was compared with the endogenous control using a log2 regression of ΔCt. Two sets of primers were used to assess the relative abundance of each of the two annotated splice variants compared with a primer set that was agnostic with respect to the splice variants. The shorter transcript variant had relatively low abundance compared with the long transcript in both NP2222 (wild-type) and NP2222-HI (haploid inducer) genotypes. Expression of the mutant copies of the gene in NP2222-HI was significantly higher for all three primer pairs tested. b, Five biological replicates of fresh pollen from NP2222 and MTLTAL-FS lines were tested in technical triplicate on the generic primer, and the average Ct and s.d. were calculated for each reaction. The relative quantity of each transcript type was compared with the endogenous control using a log2 regression of ΔCt. MTLTAL-FS pollen has lower transcript abundance than NP2222 (wild-type) pollen.

Extended Data Figure 3 MTL protein sequence and activity.

a, An amino-acid alignment of the B73 predicted protein sequence of the long splice variant of the GRMZM2G471240 gene in B73 and RWK-NIL, with the predicted sequence of the mtl allele found in RWK and Stock 6 (S6). Amino acids that differ are in red; amino acids that match are indicated in normal grey text; stop codons are indicated with a full stop. Two point mutations result in amino-acid substitutions: a histidine (H) to a tyrosine (Y), and a lysine (K) to an arginine (N). These changes are not conservative; it is possible that one or both of these modifies the haploid induction phenotype—suggesting that an allelic series could be uncovered with further investigation of variants. b, Wild-type MTL and mutant (truncated) MTL encoded by the mtl allele have in vitro phospholipase activity. PLA2 phospholipase activity as measured by fluorescent liposome assay on recombinant, purified protein produced using the MTL and mtl cDNAs. Error bars, s.e.m. based on the average of four replicates.

Extended Data Figure 4 Amino-acid alignment of the publicly available MTL orthologues in eight grasses, two non-grass monocots, and thale cress.

This alignment includes maize (Z. mays), sorghum (Sorghum bicolor, 92% sequence identity to MTL), foxtail millet (Setaria italica, 85% identity), barley (Hordeum vulgare, 78% identity), Brachypodium distachyon (78% identity), Indica and Japonica variety rice (Oryza sativa var. indica and japonica, Os3g27610, 78 and 79% identity, respectively), bread wheat (Triticum aestivum, 55% identity), banana (Musa acuminata, 57% identity), oil palm (Elaeis guineesnsis, 56% identity), and Arabidopsis thaliana (52% identity). It is clear that this gene is highly conserved in the cereals, but less conserved in more distant monocots and dicots. The N terminus is missing in wheat but the meaning of this is not clear. Expression data also indicate that the rice orthologue, called PLAIIβ, is specifically expressed in pollen30, while the closest Arabidopsis homologue by amino-acid conservation, also known as PLP2, is expressed in leaves and thus is not functionally conserved. It is worth noting that the lysine (K) residue that is changed to an arginine (N) in haploid inducer lines is a conserved amino acid in the grasses.

Extended Data Table 1 Average HIRs, origin details, and haplotype data for 19 inducer and 9 non-inducers queried for GRMZM2G471240 and GRMZM2G062320 (PGM)
Full size table
Extended Data Table 2 Reproductive phenotypes in complementation and pollen competition assays
Full size table
Extended Data Table 3 Haploid induction test crosses of the GRMZM2G471240RNAi and GRMZM2G062320RNAi lines
Full size table
Extended Data Table 4 Haploid induction and kernel abortion rate data for several ears crossed by T1 plants biallelic for frame-shift mutations in GRMZM2G471240 (MTL) but lacking the TALEN tDNA
Full size table
Extended Data Table 5 Proteins off and on in NP2222 and NP2222-HI pollen samples, including MTL, which is found in NP2222 but not NP2222-HI pollen
Full size table
Extended Data Table 6 List of genes significantly downregulated in RWK and NP2222-HI pollen compared with their near isogenic lines
Full size table

Supplementary information

Supplementary Tables 1-7

This zipped file comprises Supplementary Tables 1-7 as follows: (1) An alignment of the PLAIIβ cDNA sequence from B73, RWK-NIL (“NIL”), RWK, and Stock 6 (“ST6”), with the 4 bp insertion highlighted, and the predicted start codon in green text, and the predicted stop codons in red text; (2) Genes found to be significantly differentially up-regulated in RWK pollen versus RWK-NIL pollen; (3) Genes found to be significantly differentially down-regulated in RWK pollen versus RWK-NIL pollen; (4) Genes found to be significantly differentially up-regulated in NP2222-HI pollen versus NP2222 pollen; (5)Genes found to be significantly differentially down-regulated in NP2222-HI pollen versus NP2222 pollen; (6) Primers used in the study and (7) Example of how the putative haploid induction rate of MTLTAL-FS lines was determined using Taqman assays. (ZIP 394 kb)

Z-stack optical sections NP2222 pollen grain - Parts 1-4

Part 1 - Wild-type NP2222 pollen grain showing no fluorescent signal except the auto-fluorescence in the exine layer; Part 2 - NP2222 germinated pollen grain showing no fluorescent signal in either the grain or the tube; Part 3 - NP2222 embryo sac 21 hours after pollination by NP2222 pollen showing no fluorescent signal at the micropyle; Part 4 - NP2222 embryo sac 18 hours after pollination by NP2222 pollen showing no fluorescent signal in the GFP channel. (AVI 8572 kb)

Z-stack optical section of mtl-GFP pollen grain - Parts 1-4

Part 1 - mtl-GFP pollen grain showing no fluorescent signal; Part 2 - mtl-GFP germinated pollen grain showing no fluorescent signal in either the grain or the tube; Part 3 - NP2222 embryo sac 21 hours after pollination by mtl-GFP pollen showing no fluorescent signal at the micropyle; Part 4 - NP2222 embryo sac 18 hours after pollination by mtl-GFP pollen showing no fluorescent signal in the GFP channel. (AVI 18915 kb)

Z-stack optical section of MTL-GFP pollen grain

Z-stack optical section of MTL-GFP pollen grain showing fluorescence in the cytoplasm but not the nucleus of the two SCs. (AVI 2083 kb)

Z-stack optical section of MTL-GFP pollen grain - Parts 1-3

Z-stack optical section of MTL-GFP pollen grain showing fluorescence in the two SCs; Part 2 - Z-stack optical section of MTL-GFP pollen grain showing fluorescence in the two SCs; Part 3 - Z-stack optical section of MTL-GFP pollen grain showing fluorescence in the two SCs shortly after being applied to maize silks. (AVI 455 kb)

Z-stack optical section of MTL-GFP germinated pollen grain – Parts 1-5

Part 1 - showing fluorescence consistent with the stringy morphology of the male germ unit’s cytoplasmic compartment; Part 2 - showing fluorescence in the male germ unit; Part 3 - showing (on the right) fluorescence consistent with the male germ unit’s cytoplasm, and (on the left) MTL-GFP pollen grain that failed to germinate and has fluorescence in the two SCs trapped inside the grain; Part 4 - showing fluorescence in the pollen tube and Part 5 - showing fluorescence in the pollen tube consistent with the male germ unit. (AVI 2285 kb)

Z-stack optical section of an NP2222 embryo sac 18 hours after pollination by MTL-GFP pollen

Z-stack optical section of an NP2222 embryo sac 18 hours after pollination by MTL-GFP pollen showing fluorescent signal in two SCs in the area of the degenerated synergid cell. (AVI 990 kb)

Z-stack optical section of an NP2222 embryo sac 18 hours after pollination by MTL-GFP pollen - Parts 1 and 2

Part 1 - showing fluorescent signal in two SCs in the area of the degenerated synergid cell. This image contains propidium iodide counter staining to show where both gametophytic and sporophytic nuclei are located; Part 2 - Close-up of Part 1 showing the MTL-GFP signal in the structures that appear to be the SC cytoplasm. The GFP signal does not appear to overlap or closely border a region with dense propidium iodide staining, suggesting that the GFP is staining a non-nuclear compartment. (AVI 2276 kb)

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Kelliher, T., Starr, D., Richbourg, L. et al. MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature 542, 105–109 (2017). https://doi.org/10.1038/nature20827

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