An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs

Abstract

Plants contain more DICER-LIKE (DCL) enzymes and double-stranded RNA binding (DRB) proteins than other eukaryotes, resulting in increased small RNA network complexities. Analyses of single, double, triple and quadruple dcl mutants exposed DCL1 as a sophisticated enzyme capable of producing both microRNAs (miRNAs) and siRNAs, unlike the three other DCLs, which only produce siRNAs. Depletion of siRNA-specific DCLs results in unbalanced small RNA levels, indicating a redeployment of DCL/DRB complexes. In particular, DCL2 antagonizes the production of miRNAs and siRNAs by DCL1 in certain circumstances and affects development deleteriously in dcl1 dcl4 and dcl1 dcl3 dcl4 mutant plants, whereas dcl1 dcl2 dcl3 dcl4 quadruple mutant plants are viable. We also show that viral siRNAs are produced by DCL4, and that DCL2 can substitute for DCL4 when this latter activity is reduced or inhibited by viruses, pointing to the competitiveness of DCL2. Given the complexity of the small RNA repertoire in plants, the implication of each DCL, in particular DCL2, in the production of small RNAs that have no known function will constitute one of the next challenges.

Figure 1

DCL1 is the only DCL that produces miRNAs. RNA gel blots analysis of 10 μg total RNA prepared from rosette leaves of wild-type Col and dcl1 (A), and inflorescences of wild-type Col, dcl1, dcl2 dcl3 dcl4 triple mutants and dcl1 dcl2 dcl3 dcl4 quadruple mutant (B). The blots were successively probed with DNAs complementary to miRNAs and U6, which served as a loading control.

Figure 2

Analysis of RDR6-dependent endogenous ta-siRNAs content in dcl mutants. RNA gel blots analysis of 10 μg total RNA prepared from inflorescence of wild-type Col, and dcl1, dcl3 dcl4, dcl2 dcl3 dcl4, dcl1 dcl2 dcl3 dcl4 mutants. The blots were probed with DNAs complementary to ta-siRNAs, miRNAs and U6, which served as a loading control.

Figure 3

Rosette leaf morphology of single, double and triple dcl mutants involving dcl4. Pictures of 21-day-old dcl4, dcl2 dcl4, dcl3 dcl4, dcl2 dcl3 dcl4 mutants compared to the sgs3-1 allele and wild-type Col plants. The leaf phenotype was quantified by measuring the average length and width of the fifth rosette leaves of 21-day-old plants, which is indicative of the juvenile-to-adult transition. Numbers under the photographs represent the average length divided by width and the corresponding s.d.'s. Measurements were taken at the widest and longest point of each leaf. Six individual plants were measured for each class.

Figure 4

Analysis of RDR2-dependent endogenous siRNAs content in dcl mutants. RNA gel blots analysis of 10 μg total RNA prepared from inflorescence of wild-type Col, and dcl1, dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl4, dcl3 dcl4, dcl2 dcl3 dcl4, dcl1 dcl2 dcl3 dcl4. The blots were probed with DNAs complementary to siRNAs and U6, which served as a loading control.

Figure 5

Production of miRNAs in single dcl mutants. RNA gel blot analysis of 10 μg total RNA prepared from rosette leaves (A) or inflorescences (B) of wild-type Col, and dcl1, dcl2, dcl3 and dcl4 single mutants. The blots were successively probed with DNAs complementary to miRNAs and U6, which served as a loading control.

Figure 6

Phenotype of all combinations of dcl mutations involving dcl1. 65-day-old wild-type dcl1, dcl1 dcl2, dcl1 dcl3, dcl1 dcl4, dcl1 dcl2 dcl3, dcl1 dcl2 dcl4, dcl1 dcl3 dcl4, dcl1 dcl2 dcl3 dcl4 mutants were sowed in vitro, grown for 20 days under standard conditions, and transferred to soil. Photographs were taken 45 days later.

Figure 7

DCL4 is the primary producer of CMV-derived siRNAs. (A) RNA gel blot analysis of 15 μg total RNA prepared from rosette leaves of wild-type Col, dcl1, dcl2, dcl3 and dcl4 single mutants, dcl1 dcl2, dcl1 dcl3, dcl1 dcl4, dcl2 dcl3, dcl2 dcl4, dcl3 dcl4 double mutants, dcl1 dcl2 dcl3, dcl1 dcl2 dcl4, dcl2 dcl3 dcl4 triple mutants and dcl1 dcl2 dcl3 dcl4 quadruple mutant infected with CMV. Plants were grown for 10 days in vitro, transferred to soil, inoculated the day after, and RNAs were extracted from pools of 2–10 plants, 21 days after inoculation. The blots were probed with antisense radiolabeled RNA probes transcripted in vitro complementary to the region coding for the coat protein of CMV. U6 served as a loading control. (B) 31-day-old wild-type dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl4, dcl3 dcl4, dcl2 dcl3 dcl4, dcl1, dcl1 dcl2 dcl3 dcl4 mutants were sowed in vitro, grown for 10 days under standard conditions, transferred to soil, and inoculated the day after with CMV. Photographs were taken 21 days later. (C) High molecular weight RNA blot analysis of 5 μg total RNA prepared from wild-type Col, dcl1, dcl2, dcl3 and dcl4 single mutants, dcl2 dcl3, dcl2 dcl4 and dcl3 dcl4 double mutants, dcl2 dcl3 dcl4 triple mutants and dcl1 dcl2 dcl3 dcl4 quadruple mutant infected with CMV. Total RNA was extracted from a pool of 2–10 CMV-infected plants 21 days after inoculation. This experiment was repeated three times and one representative experiment is shown. Blots were probed with radiolabeled DNAs complementary to the region coding for the coat protein of CMV. 25S RNA served as a loading control.

Figure 8

DCL4 and DCL2 produce TCV siRNAs. (A) RNA gel blot analysis of 15 μg total RNA prepared from rosette leaves of wild-type Col, dcl1, dcl2, dcl3 and dcl4 single mutants, dcl1 dcl2, dcl1 dcl3, dcl2 dcl3, dcl2 dcl4, dcl3 dcl4 double mutants, dcl1 dcl2 dcl3, dcl1 dcl2 dcl4, dcl2 dcl3 dcl4 triple mutants and dcl1 dcl2 dcl3 dcl4 quadruple mutant infected with TCV. Plants were grown for 10 days in vitro, transferred to soil, inoculated the day after, and RNAs were extracted from pools of 2–10 plants, 21 days after inoculation. The blots were probed with antisense radiolabeled RNAs probes transcripted in vitro complementary to the region coding for the coat protein of TCV. U6 served as a loading control. (B) 31-day-old wild-type dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl4, dcl3 dcl4, dcl2 dcl3 dcl4, dcl1, dcl1 dcl2 dcl3 dcl4 mutants were sowed in vitro, grown for 10 days under standard conditions, transferred to soil, and inoculated the day after with TCV. Photographs were taken 21 days later. (C) High molecular weight RNA blot analysis of 5 μg total RNA prepared from wild-type Col, dcl1, dcl2, dcl3 and dcl4 single mutants, dcl2 dcl3, dcl2 dcl4 and dcl3 dcl4 double mutants, dcl2 dcl3 dcl4 triple mutants and dcl1 dcl2 dcl3 dcl4 quadruple mutant infected with TCV. Total RNA was extracted from a pool of 2–10 TCV-infected plants 21 days after inoculation. This experiment was repeated three times and one representative experiment is shown. Blots were probed with radiolabeled DNAs complementary to the region coding for the coat protein of TCV. 25S RNA served as a loading control.

Figure 9

Production of ta-siRNAs in plants infected by TCV or CMV. RNA gel blot analysis of 10 μg total RNA prepared from 1-month-old rosette leaves of wild-type Col mock-inoculated or inoculated with CMV or TCV. RNAs were extracted from pools of 2–10 plants, 21 days after inoculation. The blots were probed with DNAs complementary to ta-siRNAs and miRNAs, and U6, which served as a loading control.