Ectopic DICER-LIKE1 expression in P1/HC-Pro Arabidopsis rescues phenotypic anomalies but not defects in microRNA and silencing pathways

Abstract

Expression of the viral silencing suppressor P1/HC-Pro in plants causes severe developmental anomalies accompanied by defects in both short interfering RNA (siRNA) and microRNA (miRNA) pathways. P1/HC-Pro transgenic lines fail to accumulate the siRNAs that mediate RNA silencing and are impaired in both miRNA processing and function, accumulating abnormally high levels of miRNA/miRNA* processing intermediates as well as miRNA target messages. Both miRNA and RNA silencing pathways require participation of DICER-LIKE (DCL) ribonuclease III-like enzymes. Here, we investigate the effects of overexpressing DCL1, one of four Dicers in Arabidopsis thaliana, on P1/HC-Pro-induced defects in development and small RNA metabolism. Expression of a DCL1 cDNA transgene (35S:DCL1) produced a mild gain-of-function phenotype and largely rescued dcl1 mutant phenotypes. The 35S:DCL1 plants were competent for virus-induced RNA silencing but were impaired in transgene-induced RNA silencing and in the accumulation of some miRNAs. Ectopic DCL1 largely alleviated developmental anomalies in P1/HC-Pro plants but did not correct the P1/HC-Pro-associated defects in small RNA pathways. The ability of P1/HC-Pro plants to suppress RNA silencing and the levels of miRNAs, miRNA*s, and miRNA target messages in these plants were essentially unaffected by ectopic DCL1. These data suggest that P1/HC-Pro defects in development do not result from general impairments in small RNA pathways and raise the possibility that DCL1 participates in processes in addition to miRNA biogenesis.

Figure 1.

The Severity of P1/HC-Pro Phenotype in Primary Transformants Correlates with P1/HC-Pro mRNA Accumulation. (A) The mild phenotype exhibited by a subset of P1/HC-Pro primary transformants in Arabidopsis. (B) The severe phenotype exhibited by the majority of P1/HC-Pro primary transformants in Arabidopsis. (C) RNA gel blot analysis showing the levels of P1/HC-Pro mRNA in total RNA from a pool of individual primary transformants. The level of P1/HC-Pro mRNA is higher in the pool of plants displaying a severe phenotype as shown in (B) (lane 3) than that in the pool of plants displaying a mild phenotype as shown in (A) (lane 2). Lane 1 shows the absence of P1/HC-Pro mRNA in vector only–transformed Arabidopsis plants. (D) Severely phenotypic P1/HC-Pro Arabidopsis plants have narrow cotyledons as compared with those of nontransformed (nt) seedlings.

Figure 2.

Developmental Phenotypes in P1/HC-Pro Transgenic Arabidopsis Plants. (A) and (B) Branching abnormalities associated with the P1/HC-Pro transgene. The typical monopodial branching pattern of wild-type inflorescences (A), unlike the primitive branching pattern in P1/HC-Pro inflorescences (B). (C) to (F) Vascular patterning abnormalities in P1/HC-Pro transgenic Arabidopsis flowers. A sepal from a nontransgenic flower (C), as compared with a sepal from a P1/HC-Pro transgenic flower (D), showing irregular edges and reduced interconnections in the vascular system. A petal from a nontransgenic flower (E), as contrasted with a petal from a P1/HC-Pro transgenic flower (F), showing reduced interconnections in the vascular system. (G) to (J) Reduced fertility in P1/HC-Pro transgenic Arabidopsis is associated with defects in both male and female floral organs. The microsporangia (in yellow), the sites of pollen grain generation and maturation in the anther, of a nontransgenic stamen (G). The microsporangia in the anther of a P1/HC-Pro transgenic stamen (H), which are generally smaller and bound by more connective tissue in P1/HC-Pro transgenic plants than in nontransgenic plants. A pistil of a P1/HC-Pro transgenic flower (right), showing the reduced number of ovules compared with a nontransgenic pistil (left) (I). The papillae on the pistil of a P1/HC-Pro transgenic flower (right) are longer and less dense than those of nontransgenic plants (left) (J).

Figure 3.

Overexpression of DCL1 mRNA Largely Complements the dcl1-8 Mutation. (A) The predicted domains of the DCL1 protein from Schauer et al. (2002). NLS is the nuclear localization signal, and DUF refers to domain of unknown function 283. The location of 5′ and 3′ probes used in (B) and (C) and the sequence content of the major DCL1 transcripts are indicated. The dashed line at the beginning of the 2.5-kb RNA represents the known heterogeneity in the 5′ end of this transcript containing intron 14 sequences. (B) 35S:DCL1 seedlings accumulate high levels of DCL1 mRNA. RNA gel blot analysis of RNA isolated from 10-d seedlings of wild-type plants or plants homozygous for 35S:DCL1 transgene (line 12), as indicated, using the 5′ or 3′ hybridization probes indicated in (A). Arrows indicate the location of the 6.2-kb full-length DCL1 transcript as well as the 4.0- and 2.5-kb smaller DCL1 transcripts. Ethidium bromide staining of 25S rRNA is shown as a loading control. (C) Adult 35S:DCL1 plants accumulate high levels of DCL1 mRNA. RNA gel blot analysis of RNA from rosette leaves, stems, or flowers of adult wild-type plants, 35S:DCL1 transgenic plants, P1/HC-Pro transgenic plants, and P1/HC-Pro × 35S:DCL1 plants, as indicated, using the 5′ hybridization probe indicated in (A). The location of the 6.2-kb full-length and the 4.0-kb DCL1 transcripts is indicated. Ethidium bromide staining of 25S rRNA is shown as a loading control. (D) Rosette leaf morphology is affected by altered levels of DCL1 activity. 35S:DCL1 Arabidopsis plants sometimes show abaxialized (curled up) rosette leaves as shown in the left picture, in contrast with dcl1-8 mutant rosette leaves, which are adaxialized (curled under) as shown in the middle picture. The dcl1-8 adaxialized rosette leaf phenotype is largely complemented in plants expressing the 35S:DCL1 transgene (right picture). Arrows indicate adaxialized leaves. (E) RNA gel blot showing the levels of the indicated miRNAs in rosette leaves of young dcl1-8 rosette leaves in the absence (−) or presence (+) of the 35S:DCL1 transgene as compared with those in the equivalent tissues of 35S:DCL1 plants or wild-type plants. The location of molecular weight RNA markers of 20 and 30 nucleotides are indicated to the right of the figure. Ethidium bromide (EtBr) staining of the predominant RNA species in the low molecular weight fraction is shown as a loading control.

Figure 4.

The 35S:DCL1 Locus Largely Rescues Morphological Features of P1/HC-Pro–Expressing Arabidopsis Plants. (A) Ectopic DCL1 rescues developmental defects in P1/HC-Pro transgenic plants. The morphology of the rosette leaves and flowers is shown for wild-type (Columbia), homozygous 35S:DCL1, hemizygous P1/HC-Pro, and doubly hemizygous P1/HC-Pro × 35S:DCL1 lines. The heavily serrated leaves and sterile flowers of the P1/HC-Pro plants are almost completely suppressed in the P1/HC-Pro × 35S:DCL1 lines. (B) P1/HC-Pro mRNA is expressed in P1/HC-Pro × 35S:DCL1offspring. RNA gel blot analysis showing levels of P1/HC-Pro mRNA in the leaves, stems, and flowers of P1/HC-Pro transgenic plants and the equivalent tissues from P1/HC-Pro × 35S:DCL1 plants, as indicated. Ethidium bromide staining of 25S rRNA is shown as a loading control.

Figure 5.

The Accumulation of miRNAs in 35S:DCL1, P1/HC-Pro, and P1/HC-Pro × 35S:DCL1 Plants. The accumulation of the indicated miRNAs and miRNA*s was determined from RNA gel blot analysis of low molecular weight RNA. RNA was isolated from the indicated tissues of adult plants at a late stage of flowering. The same RNA samples were fractionated on two gels. Probes to miR162, miR164, miR164*, miR167, and miR167* were hybridized to a blot derived from one gel, whereas the probe to miR172 was hybridized to a blot derived from the other gel. Ethidium bromide (EtBr) staining of the predominant RNA species in the low molecular weight fraction is shown as a loading control.

Figure 6.

The Accumulation of miRNA Target mRNAs in 35S:DCL1, P1/HC-Pro, and P1/HC-Pro × 35S:DCL1 Plants. The accumulation of the indicated mRNA targets was determined from RNA gel blot analysis of high molecular weight RNA isolated from the indicated plants. Ethidium bromide staining of 25S rRNA is shown as a loading control.

Figure 7.

35S:DCL1 Does Not Rescue P1/HC-Pro Suppression of either Sense Transgene–Induced or Virus-Induced RNA Silencing. (A) Sense transgene–induced RNA silencing was monitored by RNA gel blot analysis of high molecular weight RNA to detect GUS mRNA (top blot) and of low molecular weight RNA to detect GUS siRNAs (bottom blot). The RNAs in lanes 1 to 5 are derived from plants hemizygous for the L1 GUS-silenced locus. The presence or absence of the P1/HC-Pro and 35S:DCL1 transgenes is indicated above the blot. The RNA in lane 2 is from a single plant hemizygous for both 35S:DCL1 and L1 transgenes and is representative of four separate individuals that were assayed. The RNAs in lanes 3 and 4 are from two different individual plants hemizygous for all three transgenes. Lanes 5 and 6 show RNA from control plants lacking both P1/HC-Pro and 35S:DCL1 transgenes and either hemizygous (lane 5) or homozygous (lane 6) for the L1 locus. (B) VIGS was assayed by RNA gel blot analysis of high molecular weight RNA to detect CH42 mRNA (top blot) and of low molecular weight RNA to detect CH42 siRNA (bottom blot). RNA was isolated from tissue pooled from 10 plants at 21 d after bombardment with the CH42 silencing vector. The presence or absence of the P1/HC-Pro and 35S:DCL1 transgenes is indicated above the blot. Ethidium bromide (EtBr) staining of 25S rRNA and the predominant RNA species in the low molecular weight fraction are shown as loading controls in (A) and (B). The molecular weight of the indicated small RNA species in (A) and (B) was inferred from the migration of DNA oligonucleotides that were subsequently standardized to RNA markers.