A pollen expressed PME gene at Tcb1 locus confers maize unilateral cross‐incompatibility

Maize unilateral cross-incompatibility (UCI) is a pre-zygotic reproductive barrier to unidirectionally prevent hybridization (Zhang et al., 2018). Gametophyte factor1 (Ga1), Gametophyte factor2 (Ga2), and Teosinte crossing barrier1 (Tcb1) are three distinct maize UCI loci. Each locus contains a pair of male and female determinants that forms three different haplotypes: S-haplotype possessing both male and female determinants, M-haplotype harbouring only male determinant, and wildtype having neither (Chen et al., 2022). During fertilization, the female determinant produces a barrier to pollen which lacks the corresponding male determinant. To date, the Tcb1 female determinant, the male and female determinants of Ga1 and Ga2 have been identified (Chen et al., 2022; Lu et al., 2019; Moran Lauter et al., 2017; Zhang et al., 2018), and all of them encode the type-II/group1 pectin methylesterase (PME) proteins which are essential to maintain the state of pectin methylesterification required for pollen tube growth (Bosh and Peter, 2005). However, the Tcb1 male determinant (termed as Tcb1-m) has not been reported, which hindered the study of the specificity of each locus and eventually deciphering the molecular mechanism of maize UCI. Maize UCI restricts gene flow among maize populations and is believed to have practical application in commercial breeding programs (Zhang et al., 2018). This practice is restricted by the presence of the corresponding allele at the same locus among elite maize lines. Here, we report the identification of the Tcb1-m and the stacking of different UCI loci to strengthen its application in maize production. Since the high sequence similarity between Tcb1 and Ga1 female determinants Tcb1-f and ZmPME3, it is conceivable that their male determinants Tcb1-m and ZmGa1P may also be conserved. A Ga1-M free maize line named 401T which has the Tcb1 male function was identified from the F2 population of 401C (Ga1-MTcb1-S/Ga1-MTcb1-S) and B73 (ga1tcb1/ga1tcb1) (Figure 1a). We designed PCR primers based on the ZmGa1P coding sequence and amplified putative Tcb1-m from 401T. A PME gene exhibiting 96.59% and 94.72% sequence identity compared with ZmGa1P at the nucleotide and amino acid level, was identified as a candidate for the Tcb1-m (Figures S1 and S2). We also performed RNA-seq profiling on 401T and B73 mature pollen. The Tcb1-m candidate was the third significantly differentially expressed genes of de novo assembled transcripts between the two lines (Table S1). A quantitative PCR (qPCR) analysis revealed that the Tcb1-m candidate was specifically expressed in 401T pollen (Figure 1b). We further conformed the high expression level in pollen of Tcb1 lines, while no expression was detected in tcb1 pollen (Figure 1c). To validate Tcb1-m candidate had the male function, two types of transgenic plants carrying the Tcb1-m candidate driven by its native promoter and ZmGa1P promoter, respectively, were developed (Figure 1d,e; Figures S3 and S4). The 401C pollinated with pollen from the T1 transgenic plants exhibited a full seed set, but those pollinated with pollen from non-transgenic plants set no seeds (Figure 1f,h). qPCR analyses confirmed the expression of the Tcb1-m candidate in the pollen of transgenic plants (Figure 1g,i). Though the Tcb1-m candidate and ZmGa1P exhibited high sequence similarities, the transgenic plants were unable to fertilize SDGa25 (Ga1-S/Ga1-S) plants (Figure S5), suggesting the Tcb1-m candidate does not possess the Ga1 male function. We thus conclude that the Tcb1-m candidate is the male determinant of the Tcb1 locus. Similar to Tcb1-f (Lu et al., 2019), the closest homologs of Tcb1-m were located at the Ga1 locus in the nested associated mapping founder line genomes (maizeGDB.org), but none of these sequences matched Tcb1-m perfectly (Figure S6). The Tcb1 locus may be a null allele in conventional maize lines. Supporting this speculation, all the 946 maize lines (Zhang et al., 2018) were identified as tcb1 alleles by cross-compatibility analysis, and none of them carried either Tcb1-m or Tcb1-f (Table S2). We further genotyped the Tcb1 locus in 21 teosinte accessions and found that 12 of them carried both the Tcb1-m and Tcb1-f (Figure 1j and Table S3). Tcb1 locus is loosely linked with the Ga1 locus with a physical distance of ~20 Mb (Figure S7). Genotyping results indicated that the ZmGa1P is present in all Ga1-M and Ga1-S lines, and in the teosinte accessions that harbouring Tcb1 locus. The ZmPME3 was identified in all Ga1-S lines, some Ga1-M lines, and a portion of the teosinte accessions with ZmGa1P (Figure 1k; Table S3). Together with high sequence similarities of male determinants and female determinants, the Ga1 locus (or at least Ga1 male determinant) and Tcb1 locus are probably a tandem duplicate in teosinte accessions, and diversification of them appear to have occurred after maize domestication. The prevalence of M-haplotypes of Ga1 and Ga2 among elite maize lines greatly compromises the utilization of them as reproductive barriers in commercial maize breeding programs (Chen et al., 2022). Tcb1 locus is exclusively found in teosintes, endowing it with better application values. To strengthen the application of maize UCI, we stacked each two of the three UCI loci Ga1, Ga2, and Tcb1, and pyramided three of them, and developed homozygous lines of Ga1Ga2, Ga1Tcb1, Ga2Tcb1, and Ga1Ga2Tcb1. These four types of lines completely rejected the pollen mixture of Ga1, Ga2, and Tcb1. The Ga1Ga2, Ga1Tcb1, and Ga2Tcb1 lines were cross-incompatible with each other, while could be fertilized by pollen from Ga1Ga2Tcb1. The Ga1Ga2Tcb1 line only accepted pollen of the Ga1Ga2Tcb1 genotype (Figure 1l-o). Our study provides a valuable approach to overcome the deficiencies in the utilization of a single UCI locus as a reproductive barrier. In conclusion, we cloned the male determinant Tcb1-m of the Tcb1 locus, which encoded a type-II/group1 PME. Three pairs of PMEs function as male and female determinants of Ga1, Ga2, and Tcb1, respectively, revealing the PME-mediated mechanism of maize UCI. The molecular interaction of male and female determinants in self-incompatibility (SI) confers the pistil with the ability to distinguish between self-pollen and nonself-pollen (Fujii et al., 2016). The lack of a direct physical interaction of male and female determinants of maize UCI (Figure 1p) (Chen et al., 2022) indicates a distinct mechanism from that of SI. It is fascinating to determine how the male determinant of maize UCI selectively targets cognate female determinants for barrier disruption. Our study lays a solid foundation for deciphering the identity and specificity of the three UCI loci and provides valuable plant genetic resources for the application of maize UCI. This work was financially supported by the National Natural Science Foundation of China (32101725). The authors declare no conflict of interest. H.C. designed and supervised the project. Z.Z. and K.L. performed the experiments. Z.Z. and T.Z. contributed bioinformatic analysis. H.C. and Z.Z. wrote the manuscript. Appendix S1 Supplemental materials and methods. Figure S1 Comparison of genomic DNA sequences of ZmGa1P and cb1-m. Introns are indicated by underlines. Sequence variations are highlighted in green. Figure S2 Comparison of amino acid sequences of ZmGa1P and Tcb1-m. Sequence variations are highlighted in green. Figure S3 DNA sequence for construction of ProTcb1-m:Tcb1-m transgenic vector. This entire Tcb1-m genomic sequence includes the 2577 bp promoter, 1261 bp coding sequence (blue) with intron (underline), and 2244 bp terminator. Figure S4 DNA sequence for construction of ProZmGa1P:Tcb1-m transgenic vector. This sequence includes the 3142 bp ZmGa1P promoter, 1140 bp Tcb1-m coding sequence (highlight) without intron, and 2046 bp ZmGa1P terminator. Figure S5 Examination of the Ga1 male function of Tcb1-m. ProTcb1-m:Tcb1-m and ProZmGa1P:Tcb1-m transgenic plant was used to pollinate SDGa25 (Ga1-S/Ga1-S), respectively. No seed-set was obtained which suggested that Tcb1-m does not possess the Ga1 male function. Figure S6 Alignment of Tcb1-m with its closest homologs in the NAM founder genomes. (a) Position of the closest homolog of Tcb1-m in the indicated genome. (b) Sequence variations between Tcb1-m and its closest homolog in each genome. Single nucleotide polymorphism, deletion, and insertion is indicated by blue, red, and green, respectively. Figure S7 Physical positions of Ga1 and Tcb1 loci in the NAM founder genomes. Position interval was determined according to ZmGa1P (Zhang et al., 2018) and Tcb1-S (Lu et al., 2019) mapping region, respectively. Table S1 Most significant DEGs of de novo assembled transcripts from 401T and B73 mature pollen. Table S2 Genotypes of Ga1 and Tcb1 loci in standard maize lines. Table S3 Teosinte accessions used in this study. Table S4 Primers used in this study. 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