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. 2004 May 15;18(10):1187-97.
doi: 10.1101/gad.1201404. Epub 2004 May 6.

The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development

Hervé Vaucheret  1 , Franck Vazquez, Patrice Crété, David P Bartel
Affiliations

The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development

Hervé Vaucheret et al. Genes Dev. .

Abstract

MicroRNAs (miRNAs) are endogenous 21-24-nt RNAs that can down-regulate gene expression by pairing to the messages of protein-coding genes to specify mRNA cleavage or repression of productive translation. They act within the RNA-induced silencing complex (RISC), which in animals contains a member of the Argonaute family of proteins. In the present study, we show that Arabidopsis ago1 mutants have increased accumulation of mRNAs known to be targeted for cleavage by miRNAs. In hypomorphic ago1 alleles, this compromised miRNA function occurs without a substantial change in miRNA accumulation, whereas in null alleles it is accompanied by a drop in some of the miRNAs. Therefore, AGO1 acts within the Arabidopsis miRNA pathway, probably within the miRNA-programmed RISC, such that the absence of AGO1 destabilizes some of the miRNAs. We also show that targeting of AGO1 mRNA by miR168 is needed for proper plant development, illustrating the importance of feedback control by this miRNA. Transgenic plants expressing a mutant AGO1 mRNA with decreased complementarity to miR168 overaccumulate AGO1 mRNA and exhibit developmental defects partially overlapping with those of dcl1, hen1, and hyl1 mutants showing a decrease in miRNA accumulation. miRNA targets overaccumulate in miR168-resistant plants, suggesting that a large excess of AGO1 protein interferes with the function of RISC or sequesters miRNAs or other RISC components. Developmental defects induced by a miR168-resistant AGO1 mRNA can be rescued by a compensatory miRNA that is complementary to the mutant AGO1 mRNA, proving the regulatory relationship between miR168 and its target and opening the way for engineering artificial miRNAs in plants.

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Figures

Figure 1.
Figure 1.
ago1, hen1, and hyl1 mutants exhibit overlapping developmental defects. (A) Rosettes of plants grown under short-day conditions. (B) Flowers of plants grown under long-day conditions.
Figure 2.
Figure 2.
ago1, hen1, and hyl1 mutants have increased steady-state levels of miRNA targets. RNA extracted from rosettes of isogenic wild-type or mutant siblings deriving from heterozygote parents and of untransformed plants or 2m-AGO1 transformants was quantified for the indicated mRNA by real-time quantitative PCR using primers surrounding the cleavage site. GAPDH and eEF-1(A4) were used as nontarget controls. Quantifications were normalized to that of ACTIN2, then to the value of the wild-type plants or wild-type siblings, which was arbitrarily fixed to 1.
Figure 3.
Figure 3.
miRNA accumulation in ago1 mutants. miRNA accumulation was determined by RNA gel blot analysis using 30 μg (A) or 10μg (B) of the same RNA used for RT–qPCR analyses. Blots were successively hybridized to different probes complementary to miRNAs. (A) miRNA accumulation in the ago1-26 and ago1-27 hypomorphic alleles. (B) miRNA accumulation in the ago1-3-null allele.
Figure 4.
Figure 4.
Silent mutations in the miR168 complementary site of the AGO1 mRNA induce developmental defects. (A) The WT-AGO1 mRNA naturally contains three mismatches with miR168 (in blue), including a G:U wobble pair. Silent mutations in 2m-AGO1 and 4m-AGO1 constructs introduce two and four additional mismatches (in red), reducing complementarity with miR168. ΔΔG was calculated using mfold. (B) Representative sets of transformants carrying the WT-AGO1 or 2m-AGO1 construct. (C) Proportion of transformants showing a wild-type phenotype (open bar), an ago1 phenotype caused by late cosuppression (yellow bar), or an mir-AGO1 phenotype caused by AGO1 overexpression (red bar). Plants were transformed with either an empty vector (EV) or the WT-AGO1, 2m-AGO1, or 4m-AGO1 constructs. The number of transformants analyzed is indicated in parentheses. (D) AGO1 mRNA accumulation determined by real-time quantitative PCR in untransformed plants (Col) or plants transformed with the WT-AGO1 or 2m-AGO1 constructs. Quantifications were normalized to that of ACTIN2. The value in Col was arbitrarily fixed to 1. Numbers (#) correspond to the plants shown in B.
Figure 5.
Figure 5.
Developmental defects in 2m-AGO1 transformants. (A, top row) Wild-type plant (Col) and dcl1, hen1, and hyl1 mutants. (Middle row) Wild-type plant (Col) and 2m-AGO1 transformants exhibiting curled leaves resembling those of hen1 and hyl1 mutants, at 10 d. Transformants with aberrant cotyledons were occasionally observed (right). (Bottom row) Wild-type plant (Col) and 4m-AGO1 transformants exhibiting a variety of developmental defects, including asymmetric rosette leaf formation and curled or twisted leaves, at 20 d. (B, top row) Wild-type plant (Col) and dcl1 and hyl1 mutants. (Bottom row) Adult wild-type plant (Col) and a representative 2m-AGO1 transformant exhibiting spoon-shaped or twisted anthocyaned leaves resembling those of dcl1 and hyl1 mutants. (C, top row) Inflorescence of a wild-type plant (Col) and of a representative 2m-AGO1 transformant. (Bottom row) Stems and siliques (seed pots) of the same plants. The wild-type Col plant is fertile, whereas the 2m-AGO1 transformant is sterile with aborted siliques.
Figure 6.
Figure 6.
Compensatory mutations in the MIR168a gene rescue developmental defects induced by silent mutations in the miR168 complementary site of the AGO1 mRNA. (A) The MIR168a gene encodes a primary transcript that is partially paired (unpaired nucleotides are purple). The miRNA is boxed. Compensatory mutations in the 4m-MIR168a transgene (red) conserved the structure of the primary transcript and restored pairing with the 4m-AGO1 mRNA. Original mismatches (blue) were kept. ΔΔG was calculated using mfold. (B) Representative sets of transformants carrying the 4m-AGO1 construct alone or the 4m-AGO1 and 4m-MIR168a constructs together. (C) Proportion of transformants showing a wild-type phenotype (open bar), an ago1 phenotype caused by late cosuppression (yellow bar), or a mir-AGO1 phenotype caused by AGO1 overexpression (red bar). Plants were transformed with the 4m-AGO1 or 4m-MIR168a constructs or both. The number of transformants analyzed is indicated in parentheses. (D) Accumulation of the compensatory miRNA (m4-miR168) in double transformants carrying the 4m-AGO1 and 4m-MIR168a constructs. RNA gel blot analysis was performed using 20 μg of total RNA extracted from two nontransformed plants (Col) and eight independent double transformants. The blot was hybridized with a probe complementary to 4m-miR168, stripped, rehybridized with a probe complementary to miR168, stripped, and finally rehybridized with the two probes simultaneously.
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