OsTDL1A binds to the LRR domain of rice receptor kinase MSP1, and is required to limit sporocyte numbers

Abstract

Hybrids lose heterotic yield advantage when multiplied sexually via meiosis. A potential alternative breeding system for hybrids is apospory, where female gametes develop without meiosis. Common among grasses, apospory begins in the nucellus, where aposporous initials (AIs) appear near the sexual megaspore mother cell (MeMC). The cellular origin of AIs is obscure, but one possibility, suggested by the mac1 and msp1 mutants of maize and rice, is that AIs are apomeiotic derivatives of the additional MeMCs that appear when genetic control over sporocyte numbers is relaxed. MULTIPLE SPOROCYTES1 (MSP1) encodes a leucine-rich-repeat receptor kinase, which is orthologous to EXS/EMS1 in Arabidopsis. Like mac1 and msp1, exs/ems1 mutants produce extra sporocytes in the anther instead of a tapetum, causing male sterility. This phenotype is copied in mutants of TAPETUM DETERMINANT1 (TPD1), which encodes a small protein hypothesized to be an extracellular ligand of EXS/EMS1. Here we show that rice contains two TPD1-like genes, OsTDL1A and OsTDL1B. Both are co-expressed with MSP1 in anthers during meiosis, but only OsTDL1A and MSP1 are co-expressed in the ovule. OsTDL1A binds to the leucine-rich-repeat domain of MSP1 in yeast two-hybrid assays and bimolecular fluorescence complementation in onion cells; OsTDL1B lacks this capacity. When driven by the maize Ubiquitin1 promoter, RNA interference against OsTDL1A phenocopies msp1 in the ovule but not in the anther. Thus, RNAi produces multiple MeMCs without causing male sterility. We conclude that OsTDL1A binds MSP1 in order to limit sporocyte numbers. OsTDL1A-RNAi lines may be suitable starting points for achieving synthetic apospory in rice.

Figure 1

Homologs of Arabidopsis protein TAPETUM DETERMINANT1 (TPD1). a: Unrooted dendrogram based on full-length protein sequences of TPD1, its closest homologs and other related proteins in Arabidopsis and rice. Rice proteins are boxed. Accession numbers for cDNAs: TPD1, AY394849; AtTDL1, BX816721; OsTDL1A, AK108523; OsTDL1B, AK121594. b: Full-length protein alignments for TPD1, AtTDL1, OsTDL1A and OsTDL1B. Sequences were aligned with the BCM Search Launcher program (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html). Residues identical to those of TPD1 are highlighted in black. Signal peptides predicted by SignalP-NN are underlined. A line has been placed above the region that is most highly conserved among the four proteins.

Figure 2

Gene expression in developing ovules and other tissues. RT-PCR for OsTDL1A, OsTDL1B and MSP1 genes with RNA from various tissues. Meiosis in the anther and the ovule is most commonly seen in 3-mm spikelets.

Figure 3

RNA in situ hybridization for MSP1, OsTDL1A and OsTDL1B transcripts in the ovule and anther of 3-mm spikelets from wild-type plants (WT) and homozygous msp1 mutants. Antisense probes were used, except where indicated. Arrows point to megaspore mother cells (MeMCs). Dotted lines indicate the margins of the nucellus. Scale bar: 20 μm.

Figure 4

Assay for physical interaction between the leucine-rich-repeat (LRR) domain of MSP1 and either OsTDL1A or OsTDL1B. (a) yeast two-hybrid analysis: i, empty DNA-binding domain with activation domain; ii, empty DNA-binding domain with activation domain of OsTDL1A or OsTDL1B; iii, empty activation domain with DNA-binding domain of MSP1 LRR region; iv, DNA-binding domain of MSP1 LRR region with activation domain of OsTDL1A or OsTDL1B. (b) Bimolecular complementation analysis: i, YN-MSP1Δ+YC; ii, YN+YC-OsTDL1A/OsTDL1B; iii, YN-MSP1Δ+YC-OsTDL1A/OsTDL1B.

Figure 5

Molecular analysis of OsTDL1A-RNAi plants. (a) Spikelets of T₀ plants: 1–7, independent transformants (4359, 4363, 4374, 4375, 4376, 4378 and 4379, respectively); C, non-transgenic control. DNA and RNA were extracted from 3-mm spikelets and analyzed by PCR for HPT and GUS, and by RT-PCR for GUS, OsTDL1A, OsTDL1B and MSP1. (b) Spikelets of T₁ progeny from T₀ transformant 4363: 1–4, T₁ progeny; C, control plant. RNA was extracted from 3-mm spikelets and analyzed by RT-PCR for GUS and OsTDL1A. Longitudinal sections of 3-mm spikelets were stained with acridine orange. Leptotene–pachytene–zygotene figures are indicated by arrows. Scale bars: 20 μm.

Figure 6

Histology of wild-type cv. Nipponbare, OsTDL1A-RNAi plant #4363 and homozygous msp1 mutant. (a–e) 3-mm spikelets. (f) 7-mm spikelets. (a, c, e, f) Fast green-safranin O staining. (b, d) Aniline blue staining. (a) Transverse section of anther. (b) Longitudinal section of anther. (c–f) Longitudinal sections of ovule. Leptotene–pachytene–zygotene figures are indicated by arrows. Scale bars: 25 μm.