Loss-of-function alleles of ZmPLD3 cause haploid induction in maize

Abstract

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

Fig. 1. Expression characteristics of ZmPLD3.

a, Schematic diagram of conserved domains in the ZmPLD3 protein, predicted by Pfam and SMART. The C2 box refers to protein kinase C-conserved region 2; the HKD (HxKxxxxD) boxes refer to conserved catalytic regions. b, Phylogenic analysis of PLD in maize, rice and A. thaliana. The C2-PLD, PXPH-PLD and SP-PLD subfamilies are indicated by blue, green and yellow backgrounds, respectively. c, Relative expression analysis of ZmPLD3 of anther and mature pollen at different developmental stages from the wild type was determined by qRT–PCR. The values are the means ± s.d. of three biologically independent samples (each sample involved three technical repetitions).

Fig. 2. Haploid induction phenotypes of ZmPLD3 mutants.

a, ZmPLD3 structure with the CRISPR-Cas9 target sites shown. b, The insertion and deletion sites of two allelic mutations (zmpld3-1 and zmpld3-2) are shown in the alignment comparison with the wild-type (WT) sequence. c, Phenotype of the transgenic receptor LH244 (WT) and two allelic mutations (zmpld3-1 and zmpld3-2) of ears produced via self-crossing. The arrows indicate aborted kernels. Scale bars, 1 cm. d, The rates of endosperm aborted kernels (EnAR) were significantly different between the knockout lines and WT in both the self-pollinated and crossed ears. I-1, transgenic receptor line (WT); I-2, zmpld3-1; I-3, zmpld3-2; II-1, ZD958 × transgenic receptor line (WT); II-2, ZD958 × zmpld3-1; II-3, ZD958 × zmpld3-2. Value n indicates the number of ears used for evaluating the HIR and the EnAR and the seed setting rate of each genotype. The plot values are the means ± s.d.; **P < 0.01, ***P < 0.001 (two-sided Mann–Whitney test). e, PCR products of haploid and diploid plants with polymorphic markers between the transgenic receptor line and tester. A DNA marker is shown in the far right lane. H, haploid; D, diploid; LH244, transgenic receptor; ZD958, hybrid tester. f–i, Flow cytometry results (f), overall phenotypes (g), 12th leaves (h) and anthers (i) of representative haploid (left) and diploid (right) plants among the progeny of ZD958 pollinated by ZmPLD3 knockout plants (as males). Scale bars, 10 cm (f), 2 cm (g) and 1 mm (h). In e–i, experiments were repeated 206 times and similar results were obtained. Source data

Fig. 3. The synergistic effects of zmpld3 and mtl/zmpla1/nld on haploid induction phenotypes.

a, Phenotypes of ZD958 ears pollinated by LH244 (wild type), zmpld3-1, mtl/zmpla1/nld, zmdmp, zmpld3-mtl, mtl-zmdmp, zmpld3-zmdmp or zmpld3(+/–)-mtl-zmdmp. The arrows indicate aborted kernels. Scale bars, 2 cm. b, HIR of ZD958 ears pollinated by zmpld3-1, mtl/zmpla1/nld, zmdmp, zmpld3-mtl, mtl-zmdmp, zmpld3-zmdmp or zmpld3(+/–)-mtl-zmdmp. Value n indicates the number of ears used for evaluating the HIR and the EnAR and the seed setting rate of each genotype. c, The EnAR of ZD958 ears pollinated by zmpld3-1, mtl/zmpla1/nld, zmdmp, zmpld3-mtl, mtl-zmdmp, zmpld3-zmdmp or zmpld3(+/–)-mtl-zmdmp. d, The seed setting rate of ZD958 ears pollinated by LH244, zmpld3-1, mtl/zmpla1/nld, zmdmp, zmpld3-mtl, mtl-zmdmp, zmpld3-zmdmp or zmpld3(+/–)-mtl-zmdmp. The values are the means ± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001 (two-sided Mann–Whitney test) (b–d).

Fig. 4. The subcellular localization of ZmPLD3.

a–d, Transient co-expression of 35S::ZmPLD3-eGFP (at the top) or 35S::eGFP (at the bottom) with mCherry-labelled markers of the endoplasmic reticulum (ER) (a), plastids (b), Golgi apparatus (c) or cytosol (d) in maize protoplast cells, as determined by confocal laser-scanning microscopy. The experiments were repeated three times and similar results were obtained. Scale bars, 1 μm.

Fig. 5. Transcriptional profiling of multiple pathways involved in haploid induction regulated by zmpld3 and mtl/zmpla1/nld.

a, Venn diagram illustrates the overlap of DEGs shared among zmpld3, mtl/zmpla1/nld and zmpld3-mtl. The data are derived from RNA-seq of zmpld3 and mtl/zmpla1/nld and zmpld3-mtl pollen samples, each comprising two biologically independent replications. b, Venn diagram illustrates 66 pollen-specific DEGs shared among zmpld3, mtl/zmpla1/nld and zmpld3-mtl. c, GO analyses using a hypergeometric distribution of the top ten significantly enriched GO terms (FDR < 0.05) among the overlapping DEG sets was performed; those shared between zmpld3 and zmpld3-mtl, between mtl and zmpld3-mtl and between zmpld3 and mtl are shown. Colour bar, FDR.

Extended Data Fig. 1. Expression profiles of ZmPLD3.

Transcript levels (fragments per kilobase of exon model per million mapped fragments (FPKM)) of ZmPLD3 among different tissues based on previously published RNA sequencing (RNA-seq) data. The values are the means ± s.d. of three biologically independent samples (except for the vegetative meristem samples).

Extended Data Fig. 2. HKD motifs of phospholipase D in maize, rice and Arabidopsis thaliana.

Multiple alignment of HKD motifs of PLDs in maize, rice and Arabidopsis thaliana. The shading and asterisks denote catalytic triads.

Extended Data Fig. 3. Multiple alignment of ZmPLD3 orthologs in eight monocots and three dicots.

The amino acid sequences of ZmPLD3 and its orthologs were downloaded from the website http://www.gramene.org/. Multiple alignments including Zea mays (Zm00001d037643), Sorghum bicolor (SORBI_3009G062600, 96% sequence identity to that of ZmPLD3), Setaria viridis (SEVIR_3G102400v2, 92% identity), Setaria italica (SETIT_024724mg, 90% identity), Oryza sativa Japonica Group (Os05g0171000, 86% identity), Triticum aestivum (TraesCS1A02G115300, 84% identity), Brachypodium distachyon (BRADI_2g34290v3, 83% identity), Hordeum vulgare (HORVU1Hr1G025370, 82% identity), Beta vulgaris (BVRB_9g219660, 75% identity), Brassica napus (BnaC05g37540D, 71% identity), and Arabidopsis thaliana (AT3G15730, 71% identity) were performed. The dark- and light-green backgrounds indicate increasingly conserved positions. Three conserved domains are indicated by underlines.

Extended Data Fig. 4. Predicted protein sequence of ZmPLD3 in the wild type and mutants (zmpld3-1, zmpld3-2).

An amino acid alignment of ZmPLD3 predicted the protein sequence in LH244 (WT), with the predicted sequence of the zmpld3 allele found in the mutants of zmpld3-1 and zmpld3-2. Altered amino acids are shown in red, three conserved domains are indicated by underlines, and stop codons are indicated with full stops.

Extended Data Fig. 5. CRISPR-Cas9-mediated target mutagenesis of MTL/ZmPLA1/NLD and ZmDMP.

a, MTL/ZmPLA1/NLD structure with the CRISPR-Cas9 target sites shown. mtl/zmpla1/nld had both a 2-bp deletion and a 27-bp insertion in its target region, causing 89 changed amino acids starting from the mutation site and resulting in premature translation termination. b, ZmDMP structure with the CRISPR-Cas9 target sites shown. zmdmp had a 1-bp deletion in its target region, causing 41 changed amino acids starting from the mutation site and resulting in premature translation termination. Mutation sites of the knockout lines are shown in the alignment comparison with the wild-type (WT) sequence, respectively. The target sequences are underlined, with the protospacer-adjacent motif shown in bold type. Insertions are shown in dark red and deletions are shown by dark red dashes.

Extended Data Fig. 6. The morphological phenotypes of seedlings and mature plants did not show obvious differences between the wild type and mutants (zmpld3-1, zmpld3-2).

Seedlings at seven days after sowing (a) and mature plants producing pollen (b) of LH244 and the mutants of zmpld3-1 and zmpld3-2 are shown. Scale bars, 1 cm (a) and 15 cm (b).

Extended Data Fig. 7. Identification of haploids via polymorphic molecular markers.

The lanes from left to right show DNA markers and band performance of haploid and diploid progeny as well as two parents, with 7 molecular markers. Experiments were repeated 206 times and similar results were obtained. Source data

Extended Data Fig. 8. Gating strategies of flow cytometry used for ploidy identification.

All the haploid candidates were screened by the same gating strategies of flow cytometry. Experiments were repeated 231 times and similar results were obtained (the flow cytometry results of haploids were as the top panel, whereas the flow cytometry results of diploids were as the bottom panel).

Extended Data Fig. 9. Pollen characteristics of ZmPLD3-related mutants compared to the wild type (WT).

Detection results of pollen viability (a) and pollen germination (b) of LH244 (WT), zmpld3-1, zmpld3-mtl, zmpld3-zmdmp and zmpld3 (+/-)-mtl-zmdmp. The experiments were repeated ten times, and similar results were obtained. The arrowheads indicate aborted pollen. The arrows indicate germinated pollen. Scale bars, 15 μm. Statistical analysis of the percentage of two pollen viability types (c) and the pollen germination rate (d) for LH244 (WT), zmpld3-1, zmpld3-mtl, zmpld3-zmdmp and zmpld3 (+/-)-mtl-zmdmp. The values are the means ± s.d.; n, number of pollen grains.

Extended Data Fig. 10. Co-expression of ZmPLD3 with different cellular compartment markers in maize protoplasts.

a-e, Transient co-expression of 35S::ZmPLD3-eGFP (at the top) or 35S::eGFP (at the bottom) with mCherry-labelled markers of the plasma membrane (PM) (a), nuclear (b), prevacuolar compartment (PVC) (c), peroxisome (d) or mitochondrial red fluorescent probe MitoTracker Red (e) in maize protoplast cells, as determined by confocal laser-scanning microscopy. The experiments were repeated three times, and similar results were obtained. Scale bars, 1 μm.