A PECTIN METHYLESTERASE gene at the maize Ga1 locus confers male function in unilateral cross-incompatibility

Abstract

Unilateral cross-incompatibility (UCI) is a unidirectional inter/intra-population reproductive barrier when both parents are self-compatible. Maize Gametophyte factor1 (Ga1) is an intraspecific UCI system and has been utilized in breeding. However, the mechanism underlying maize UCI specificity has remained mysterious for decades. Here, we report the cloning of ZmGa1P, a pollen-expressed PECTIN METHYLESTERASE (PME) gene at the Ga1 locus that can confer the male function in the maize UCI system. Homozygous transgenic plants expressing ZmGa1P in a ga1 background can fertilize Ga1-S plants and can be fertilized by pollen of ga1 plants. ZmGa1P protein is predominantly localized to the apex of growing pollen tubes and may interact with another pollen-specific PME protein, ZmPME10-1, to maintain the state of pectin methylesterification required for pollen tube growth in Ga1-S silks. Our study discloses a PME-mediated UCI mechanism and provides a tool to manipulate hybrid breeding.

Fig. 1

Map-based cloning of the pollen determinant. a Physical mapping, showing Bin 4.02 narrowed to a region between markers M53 and M16 contains 8 annotated genes. Recombinants are indicated below the markers. R1-R10, recombinants. C, cross-compatible; CI, cross-incompatible. b Genome-wide association analysis of the pollen determinant. The horizontal dashed line depicts the Bonferroni-adjusted significance threshold (P = 2.15 × 10⁻⁸). c The genome-wide association signals in the 8.53–10.23 Mb interval. The peak SNP is positioned by a vertical dashed line with a red diamond. The eight annotated genes are indicated by the blue bars at the bottom. d Quantile–quantile plot under a compressed mixed linear model. e Gene structure of Zm00001d048936 in B73 (ga1) and SDGa25 (Ga1-S)

Fig. 2

Validation of Zm00001d048936 as a pollen determinant. a Quantitative expression analysis of Zm0001d048936 in pollen of Ga1-S, Ga1-M, and ga1 lines. Error bars represent the mean ± SD (n = 3). b Examination of the eight candidate gene expression in pollen of SDGa25 (Ga1-S) and B73 (ga1). ZmGAPDH was used as an internal control. c, d Cross-compatibility analyses, showing ears of self-pollinations and reciprocal crosses between the SDGa25 (Ga1-S) and B104 or Z31 (ga1, control plants). The SDGa25 ears pollinated with non-transgenic controls (FQ_N and OE_N) and transgenic plants expressing ZmGa1P. c Transgenic plants made with the construct shown in Supplementary Fig 7a, d T₂ generation of transgenic plants made with the construct shown in Supplementary Fig 8a

Fig. 3

Characterization of ZmGa1P biochemical properties. a Immuno-staining the pollen tubes of SDGa25 (Ga1-S) and J66 (ga1) using the anti-ZmGa1P antibody. Scale bar = 10 μm. b Kinetic analysis of PME activity of secretory proteins in the fraction 8 of SDGa25. Error bars represent the mean ± SD of three replicates. c Split-luciferase complementation assay showing the interaction between ZmGa1P and the PME domain (CD) of ZmPME10-1 in N. benthamiana leaves infiltrated with the construct combinations shown above. Scale bar = 1 cm. d Immuno-detecting highly methylesterified pectins in the longitudinally sectioned pollen tubes of near isogenic lines with LM20 antibody. Scale bars = 10 μm. FITC, Fluorescein isothiocyanate. e Quantification of the fluorescent signal intensity of immune-examinations, to show the varied pectin DM on ga1 pollen tube walls growing in Ga1-S silks. Error bars represent the mean ± SE (n > 15 pollen tubes). a and b indicate that the means differ according to analysis of variance and Tukey’s test (P < 0.01)

Fig. 4

Field testing the isolation of ga1 pollen from adjacent plants by Ga1 locus. a KYN3^ga1(left) and its near isogenic line KYN3^Ga1-S (right) grown surrounded by a ga1 line with purple corn in Beijing, China. b KYN3^Ga1-S (white ears) was mingled with a ga1 line with yellow corn in Jilin, China