Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis

Abstract

Chromosome dosage has substantial effects on reproductive isolation and speciation in both plants and animals, but the underlying mechanisms are largely obscure ¹ . Transposable elements in animals can regulate hybridity through maternal small RNA ² , whereas small RNAs in plants have been postulated to regulate dosage response via neighboring imprinted genes^3,4. Here we show that a highly conserved microRNA in plants, miR845, targets the tRNA^Met primer-binding site (PBS) of long terminal repeat (LTR) retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21-22-nucleotide (nt) small RNAs in a dose-dependent fashion via RNA polymerase IV. We show that these epigenetically activated small interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (the 'triploid block'), and that natural variation for miR845 may account for 'endosperm balance' allowing the formation of triploid seeds. Targeting of the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.

Figure 1. miR845 family is expressed in pollen and targets retrotransposons

(a) miR845a and miR845b are preferentially expressed in mature pollen, consistent with complete absence in leaves and low levels in inflorescence tissue. (b) miR845 targets the PBS (uppercase) of retrotransposons where tRNAs bind to initiate reverse transcription. Predicted targets include Gypsy and Copia elements. The full list of targets is presented in Supplementary Table 1. (c) miR845 targets accumulate high levels of secondary 21- and 22-nt easiRNA in pollen. RPM, reads per million. (d) GFP sensor construct includes 3′UTR with a miR845b target site and driven by the UBIQUITIN10 (UBQ10) promoter. (e) Strong GFP fluorescence was detected in floral organs of 8 independent transgenic lines, but not in wild type Col-0 pollen. The same reporter is not silenced in Ler-0 pollen, where miR845b is not expressed. Scale bars represent 30 μm. (f) The MIR845 haplotype in Ler-0 is also found in other Arabidopsis accessions such as Bay-0, Kro-0 and Tsu-0 that produce low levels of miR845b, while Col-like accessions express high levels of miR845b in pollen. Bars represent mean Ct values of miR845b levels as measured by quantitative RT-PCR (qRT-PCR) and normalized to the levels of miR156a (n=2 technical replicates). Error bars represent range.

Figure 2. Natural variation in DNA methylation levels in Col-0 and Ler-0 pollen nuclei

(a) Bisulfite sequencing of FACS-sorted Col-0 and Ler-0 pollen nuclei revealed decreased mCHH levels in Ler-0 VN, compared to Col-0 VN. (b) Differentially methylated regions (DMRs) between Col-0 and Ler-0 VN were detected for CG and CHH methylation. mCHH hypomethylated DMR in Ler-0 VN overlapped primarily with TE features, particularly the superfamilies LTR/Gypsy and DNA/MuDR. (c) Hypomethylated mCHH DMRs in Ler-0 VN overlapped with hypomethylated loci in nrpd1a (Pol IV) and cmt2 mutant VN (both in Col-0 background). (d) Small RNA in Col-0 pollen matching hypomethylated mCHH DMRs in Ler-0 VN are depleted in wild type Ler-0 pollen, and dependent on Pol IV (nrpd1a) (21/22 and 24-nt). (e) Genome browser tracks of CG, CHG and CHH methylation levels in the VN of Col-0, Ler-0, nrpd1a and cmt2 pollen, illustrating CMT2 and RdDM-targeted TEs where CHH methylation was lost in Ler-0 VN.

Figure 3. miR845b-dependent easiRNA biogenesis from transgenes and transposons

(a) GFP sensor including 3′UTR with a miR845b target site and driven by the UBIQUITIN10 (UBQ10) promoter in wild type and dcl1-5/+ heterozygous background. GFP expression was restored in dcl1 pollen, allowing FACS-purification of wild type, dcl1, dcl2/4 and dcl1/2/4 pollen grains. Scale bars represent 30 μm. (b) Loss of GFP siRNA was detected in dcl1, dcl2/4 and dcl1/2/4 pollen grains, indicating that miR845b triggers DCL2/4-dependent secondary siRNA from the GFP transgene. (c) Small RNA sequencing from wild type and mutant FACS-sorted pollen revealed that 21- and 22-nt TE siRNA were lost in the dcl2/4 mutants, while miRNAs were depleted in dcl1. (d) miR845a and miR845b were depleted in dcl1 mutant and Ler-0 pollen, but miR845b was restored in transgenic Ler-0 plants expressing Col-MIR845b (Ler:MIR845b). (e) 21- and 22-nt TE-derived siRNA levels were also depleted in wild-type Ler-0 pollen, but restored in transgenic Ler:MIR845b. RPM, reads per million.

Figure 4. miR845b-dependent easiRNA is required for the triploid block

(a) miR845b was down-regulated 2-fold in two biologically independent replicates of diploid pollen (2n) from a T-DNA insertion mutant at the MIR845b locus (mir845b-1, Col-0 background), and abolished entirely in Ler-0. Expression of Col-MIR845b in Ler-0 2n pollen (osd1-2 mutant background, Ler:MIR845b) restored miR845b levels in the pollen of two biologically independent transgenic lines. (b) TE-derived siRNAs were mapped to aligned 5′ regions (metaplots) of LTR Gypsy elements. 21/22-nt easiRNA were depleted in mir845b-1 pollen and wild-type Ler-0 pollen compared to Col-0, but restored in Ler:MIR845b. These effects were even more pronounced in osd1-2 diploid pollen (2n). (c) Wild type Col-0 seed parents were pollinated with osd1-1 diploid (2n) pollen leading to the production of triploid seeds that abort at high frequency. When osd1-1/mir845b-1 double mutant (2n) pollen was used, seed viability increased to 35%. When osd1-2 Ler-0 (2n) pollen was used, the triploid block was also partially suppressed with 30% abnormal or collapsed seeds. However, expression of Col-MIR845b in osd1-2 Ler-0 2n pollen (Ler:MIR845b) was not sufficient to restore the triploid block. The numbers on top of each bar shows the number of seeds counted for each cross.