Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana

Abstract

We investigated the potential of double-stranded RNA interference (RNAi) with gene activity in Arabidopsis thaliana. To construct transformation vectors that produce RNAs capable of duplex formation, gene-specific sequences in the sense and antisense orientations were linked and placed under the control of a strong viral promoter. When introduced into the genome of A. thaliana by Agrobacterium-mediated transformation, double-stranded RNA-expressing constructs corresponding to four genes, AGAMOUS (AG), CLAVATA3, APETALA1, and PERIANTHIA, caused specific and heritable genetic interference. The severity of phenotypes varied between transgenic lines. In situ hybridization revealed a correlation between a declining AG mRNA accumulation and increasingly severe phenotypes in AG (RNAi) mutants, suggesting that endogenous mRNA is the target of double-stranded RNA-mediated genetic interference. The ability to generate stably heritable RNAi and the resultant specific phenotypes allows us to selectively reduce gene function in A. thaliana.

Figure 1

Gene constructs used to analyze dsRNA effects. In p35S∷A-GUS-S, gene-specific sequences (open boxes with arrows indicating the orientation) in the antisense (A) and sense (S) orientations were linked with a 1,022-bp fragment of the GUS gene (hatched box) and controlled by the 35S promoter (solid arrow). A schematic structure of the predicted dsRNA stem with a single-stranded loop generated by p35S∷A-GUS-S constructs is shown. In p35S∷A-NOS∷S, gene-specific sequences in the antisense and sense orientations were controlled by the 35S promoter and the nopaline synthase promoter, respectively (open arrow). p35S∷A contains gene-specific sequences in the antisense orientation under control of the 35S promoter. pNOS∷S contains gene-specific sequences in the sense orientation driven by the nopaline synthase promoter. Solid box, the 3′ end of nopaline synthase.

Figure 2

Flowers of wild-type, ag-1 and AG (RNAi) plants. (A) Wild-type flowers have four sepals, four petals, six stamens, and two carpels. (B) ag-1 flowers consist of an indeterminate number of whorls of sepals and petals in the pattern (sepals, petals, petals)_n, with no staminoid or carpeloid tissue. (C–I) AG (RNAi) flowers with different severity of phenotypes. (C) Strong mutant flowers phenocopied ag-1. (D) Longitudinal section of a strong mutant flower showing a large number of sepals and petals produced by an indeterminate floral meristem (FM). (E) Weak mutant flower. The stamens fail to elongate and the anthers are slightly petaloid (arrowhead), with no pollen. (F) Intermediate mutant flower with some sepals and petals removed. Anthers are partially transformed into petaloid tissue (arrowheads). The gynoecium is bulged at the top (arrow, F), with inner organs such as carpels (arrowhead, G) and/or petals (arrowheads, H). (I) Intermediate/strong mutant flowers have the repeated pattern of sepals, petals, petals formed in outer whorls and an incomplete flower in the center (arrow). AG (RNAi) plants are in the Wassilewskija background; therefore, internode elongation between successive internal flowers are seen in intermediate/strong (I) and strong mutant flowers (C).

Figure 3

Effects of AG dsRNA on levels of AG mRNA and AG protein. (A–E) An autoradiogram of the tissue hybridized with an AG anti-mRNA probe. The tissue is from wild-type (A and F), weak (B and G), intermediate (C and H), intermediate/strong (D and I), and strong (E and J) AG (RNAi) mutant plants. (A–E) Hybridization signals declined gradually with increasingly severe phenotypes. (F–J) The bright-field/dark-field double exposures of longitudinal section through the inflorescence meristems with stage 2–5 flowers. The silver grains representing AG mRNA expression were made to appear yellow with the use of a yellow filter. The number indicated corresponds to the development stage of flowers (43). im, inflorescence meristem. (Bar = 50 μm.) (K and L) An autoradiogram of the tissue hybridized with an AG sense probe. The tissue is from wild-type (K and M) and intermediate AG (RNAi) mutant plants (L and N). (O) Western blot analysis of AG protein. The anti-AG antibody recognizes the carboxyl-terminal part of the AG protein from aa 220–285 which is absent in the AG-1 protein (27, 42); thus, ag-1 is a control of the specificity of the antibody. Whereas AG protein is weakly expressed in weak (w) and intermediate (i) AG (RNAi) mutants compared with wild type (Wt), it is not detected at levels above background in intermediate/strong (i/s) and strong (s) AG (RNAi) mutants.

Figure 4

Phenotypes of wild-type, CLV3 (RNAi), and clv3–2 plants. Wild-type and CLV3 (RNAi) mutants are in the ecotype Wassilewskija, whereas clv3–2 is in the ecotype Landsberg erecta which has reduced internode elongation. The inflorescence meristems are enlarged in CLV3 (RNAi) (B) and clv3–2 (C) compared with wild type (A). (D) Wild-type flower. (E) CLV3 (RNAi) and (F) clv3–2 flowers have additional organs. (G–I) Cross section of gynoecia showing that the wild-type gynoecium (G) consists of two carpels, and gynoecia in CLV3 (RNAi) (H) and clv3–2 (I) have four carpels.

Figure 5

Phenotypes of wild-type, ap1 and AP1 (RNAi) flowers. (A) Wild-type flower. (B) ap1–5 flower. (C) ap1–4 flower. (D) ap1–1 flower. (E–H) Flowers from weak (E), intermediate (F), intermediate/strong (G), and strong (H) AP1 (RNAi) plants. Arrowheads indicate leaf- or bract-like first whorl organs. The numbered arrows indicate the primary (1), secondary (2), and tertiary (3) flowers. The black arrows in C and G indicate leaf-like or staminoid second-whorl organs.

Figure 6

Effects of PAN dsRNA on crc-1 transgenic plants. (A and D) crc-1. (B and E) crc-1 pan-3 and (C and F) crc-1; PAN (RNAi) flowers have extra organs and unfused gynoecia.