Arabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance

Abstract

In animals, double-stranded short interfering RNA (siRNA) and single-stranded microRNA (miRNA) regulate gene expression by targeting homologous mRNA for cleavage or by interfering with their translation, respectively. siRNAs are processed from injected or transgene-derived, long, perfect double-stranded RNA (dsRNA), while miRNAs are processed from short, imperfect dsRNA precursors transcribed from endogenous intergenic regions. In plants, both siRNAs and miRNAs activate cleavage of homologous RNA targets, but little is known about the genes controlling their production or action. The SGS2/SDE1 protein contributes to produce transgene siRNA, while DCL1 and HEN1 contribute to endogenous miRNA accumulation. Here, we show that: i) SGS2, SGS3, AGO1, and HEN1 contribute to produce transgene siRNA involved in sense posttranscriptional gene silencing (S-PTGS); ii) HEN1, but not SGS2, SGS3, or AGO1, contributes to the accumulation of the endogenous miR171 miRNA and to the cleavage of Scarecrow target mRNA by miR171; iii) SGS2, SGS3, AGO1, and HEN1 contribute to resistance against cucumber mosaic virus, but not to siRNA and IR-PTGS triggered by hairpin transgenes directly producing perfect dsRNA; and iv) the actions of HEN1 in miRNA/development and siRNA/S-PTGS can be uncoupled by single-point mutations at different positions in the protein.

Figure 1. Phenotype and Virus Sensitivity of Line L1 and the 23-2 Mutant

(A) Phenotype of line L1 and of the 23-2 mutant 2 weeks after mock infection or infection by CMV. All plants are the same age, indicating the delay in flowering of the 23-2 mutant. (B) A close-up of (A) showing the leaf phenotype of the 23-2 mutant. (C) CMV RNA accumulation in line L1 and in the 23-2 mutant, mock infected or infected by CMV. Standardization with a 25S rDNA probe is shown below.

Figure 2. GUS mRNA and siRNA Accumulation in Wild-Type and Mutant Plants

(A) mRNA extracted from leaves was hybridized with a GUS DNA probe. An ethidium bromide-stained gel is shown for standardization. The ratio between GUS and 25S signals is indicated below. (B) siRNA extracted from leaves was hybridized with antisense RNA probes corresponding to the 5′ part (position 1–558), central part (position 558–789), or 3′ part (position 789–1865) of the GUS coding sequence. Similar results were obtained with sense probes. Hybridization with 5S RNA is shown for standardization. The ratio between GUS and 5S signals is indicated below.

Figure 3. miR171 miRNA and SCL mRNA Accumulation in Wild-Type and Mutant Plants

(A) Small RNA extracted from wild-type and mutant flowers was hybridized with a probe complementary to miR171. (B) Total RNA extracted from wild-type and mutant flowers was quantified for SCL6-III mRNA relative accumulation by real-time PCR by using primers surrounding the cleavage site. Quantifications are normalized with actine2 transcript. The wild-type value is 1. AU, arbitrary unit.