DICER-LIKE3 activity in Physcomitrella patens DICER-LIKE4 mutants causes severe developmental dysfunction and sterility

Abstract

Trans-acting small interfering RNAs (ta-siRNAs) are plant-specific siRNAs released from TAS precursor transcripts after microRNA-dependent cleavage, conversion into double-stranded RNA, and Dicer-dependent phased processing. Like microRNAs (miRNAs), ta-siRNAs direct site-specific cleavage of target RNAs at sites of extensive complementarity. Here, we show that the DICER-LIKE 4 protein of Physcomitrella patens (PpDCL4) is essential for the biogenesis of 21 nucleotide (nt) ta-siRNAs. In ΔPpDCL4 mutants, off-sized 23 and 24-nt ta-siRNAs accumulated as the result of PpDCL3 activity. ΔPpDCL4 mutants display severe abnormalities throughout Physcomitrella development, including sterility, that were fully reversed in ΔPpDCL3/ΔPpDCL4 double-mutant plants. Therefore, PpDCL3 activity, not loss of PpDCL4 function per se, is the cause of the ΔPpDCL4 phenotypes. Additionally, we describe several new Physcomitrella trans-acting siRNA loci, three of which belong to a new family, TAS6. TAS6 loci are typified by sliced miR156 and miR529 target sites and are in close proximity to PpTAS3 loci.

Figure 1.

Phenotypic Analyses of ΔPpDCL4 Mutants. Both independent ΔPpDCL4 mutants display an identical mutant phenotype. Representative images of one line are shown. (A) Equal amounts of protonema tissue from wild-type (WT) and the ΔPpDCL4 mutant were spotted onto solid medium and grown under long-day conditions. Colonies were photographed after 4 weeks. Note the increased protonema development in the ΔPpDCL4 mutant. Scale bar: 0.5 cm. (B) Cultivation for 8 d in darkness results in caulonema formation in WT (black arrows), but not in the ΔPpDCL4 mutant. Scale bar: 0.5 cm. (C) The ΔPpDCL4 mutant develops a high number of brachycytes (arrows). Scale bar: 0.1 mm. (D) Under long-day growth conditions, the ΔPpDCL4 mutant develops dwarfed gametophores. Scale bar: 1 mm. (E) As in (A) grown under short-day conditions. Scale bar: 1 cm. (F) The gametophores of the ΔPpDCL4 mutant are dwarfed when grown under sporophyte-inducing short-day conditions. Gametophore size in the ΔPpDCL4 mutant is indicated by arrows. Scale bar: 1 mm. (G) The ΔPpDCL4 mutant develops abnormal spathulate elongated leaves. Scale bar: 0.1 mm. (H) The ΔPpDCL4 mutant fails to develop sporophytes. Scale bar: 200 μm.

Figure 2.

Analyses of miRNAs and ta-siRNAs in ΔPpDCL4 Mutants. (A) Summed, scaled, repeat-normalized sRNA abundances for all MIRNA-derived sRNAs from wild-type and ΔPpDCL4. Units are normalized reads per 10 000. (B) Expression analysis of miR390, miR166, miR156, and miR160 in protonema tissue by RNA gel blots. U6 snRNA served as control for normalization. Numbers under blots indicate normalized expression values with respect to wild-type. (C) Chart indicating the expression of TAS3a-d derived sRNAs inferred from sequencing data of wild-type and ΔPpDCL4. (D) RNA gel blots for ta-siRNAs derived from TAS3a and TAS3c precursors in wild-type and ΔPpDCL4 mutants. U6snRNA served as control for normalization. Numbers under blots indicate normalized expression values for off-sized ta-siRNAs with respect to the 21-nt wild-type ta-siRNAs. (E) Transcript levels of ta-siRNA target genes (PpARF; Phypa_203442 and PpAP2; Phypa_65352) in wild-type (WT) and ΔPpDCL4 mutants determined by qRT–PCR. Expression levels were normalized to the constitutively expressed control gene PpEF1α. (F) RLM 5' RACE products of the miRNA and ta-siRNA target PpARF from WT and ΔPpDCL4 mutants. Arrows mark products of the expected size (1: miRNA-mediated cleavage product; 2: ta-siRNA-mediated cleavage product) that were eluted, cloned, and sequenced. Numbers above miRNA:target and ta-siRNA:target alignments indicate sequenced RACE products with the corresponding 5' end.

Figure 3.

Reduced Transcript Accumulation and Increased DNA Methylation at TAS Loci in ΔPpDCL4 Mutants. (A) Expression level of the TAS precursors TAS3a and TAS3c determined by qRT–PCR. Expression levels were normalized to the constitutively expressed control gene PpEF1α. (B) Bisulfite PCR with methylation-specific (MSP) and unmethylation-specific (USP) primers for TAS3a and TAS3c.

Figure 4.

Phenotypic Analyses of ΔPpDCL3/ΔPpDCL4 Double Mutants. (A) Equal amounts of protonema tissue from wild-type (WT) and ΔPpDCL3/ΔPpDCL4 mutants were spotted onto solid medium and developed colonies were photographed after 3 weeks. ΔPpDCL3/ΔPpDCL4 mutants show accelerated gametophore development. Scale bar: 0.5 cm. (B) WT and ΔPpDCL3/ΔPpDCL4 mutants display normal caulonema development after 10 d growth in darkness. Scale bar: 0.5 cm. (C) Gametophore size is similar in WT and ΔPpDCL3/ΔPpDCL4 mutants. Scale bar: 2 mm. (D) Sporophyte formation is rescued in ΔPpDCL3/ΔPpDCL4 mutants. Scale bar: 200 μm. (E) RNA gel blots hybridized with antisense probes of miR390, miR156, and ta-siRNAs generated from the PpTAS3a precursor in WT and ΔPpDCL3/ΔPpDCL4 double mutants. An antisense probe for U6 snRNA served as control for normalization. Numbers under blots indicate normalized miRNA expression values with respect to WT.

Figure 5.

Neighboring miR156- and miR390-Sliced TAS Loci. (A) An annotated genomic snapshot of PpTAS3a and PpTAS6a. Shaded regions indicate boundaries of indicated TAS loci, with locations and strand orientations of miRNA complementary sites and known functional ta-siRNAs shown. Insets above show the small RNA size distribution in the indicated genotypes, as well as the ‘phasing’ distributions of wild-type small RNAs in 21-nt bins (numbers on periphery indicate percentage of siRNAs in each bin). Browser track small RNA data (blue) and degradome data (magenta) show the 5' end positions from wild-type samples, with positive values indicating Watson-strand mapped reads, and negative values indicating Crick-strand mapped reads. Insets below show miRNAs aligned with miRNA complementary sites. Numbers above alignments are the number of degradome-derived 5' ends mapped to the 10th nucleotide of the alignment. (B) Evidence for trans-acting slicing directed by the PpTAS6a-derived ta-siZNF. Annota ted genomic snapshot showing protein-coding transcripts (gray), polyA+ RNAseq data (not strand-specific (Zemach et al., 2010), and degradome data. Inset below-left shows ta-siZNF/target alignment, with the number of degradome reads at the 10th nucleotide of the alignment indicated. Inset below-right shows schematic of the protein (from Phytozome).