Failure of the tomato trans-acting short interfering RNA program to regulate AUXIN RESPONSE FACTOR3 and ARF4 underlies the wiry leaf syndrome

Abstract

Interfering with small RNA production is a common strategy of plant viruses. A unique class of small RNAs that require microRNA and short interfering (siRNA) biogenesis for their production is termed trans-acting short interfering RNAs (ta-siRNAs). Tomato (Solanum lycopersicum) wiry mutants represent a class of phenotype that mimics viral infection symptoms, including shoestring leaves that lack leaf blade expansion. Here, we show that four WIRY genes are involved in siRNA biogenesis, and in their corresponding mutants, levels of ta-siRNAs that regulate AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 are reduced, while levels of their target ARFs are elevated. Reducing activity of both ARF3 and ARF4 can rescue the wiry leaf lamina, and increased activity of either can phenocopy wiry leaves. Thus, a failure to negatively regulate these ARFs underlies tomato shoestring leaves. Overexpression of these ARFs in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and potato (Solanum tuberosum) failed to produce wiry leaves, suggesting that the dramatic response in tomato is exceptional. As negative regulation of orthologs of these ARFs by ta-siRNA is common to land plants, we propose that ta-siRNA levels serve as universal sensors for interference with small RNA biogenesis, and changes in their levels direct species-specific responses.

Figure 1.

The Morphological Spectrum of the Wiry Syndrome. (A) to (D) Heteroblasty (progressing from left to right) of wild-type (WT) tomato leaves (A), a typical strong wiry, w2-1 (B), a weak wiry, w2-3 (C), and a strong w3 allele (D) before the onset of necrosis. In all mutants, the first two to three leaves have five to seven abnormal leaflets and small intercalary folioles, while later-formed leaves lack lamina at different magnitudes. (E) and (F) Toward flowering, wild-type leaves are not changing (E), while all leaves of strong wiry plants (F) lack or nearly lack leaflets and lamina. (G) and (H) Scanning electron microscopy images of wild-type leaves (G) showing distinct initiation of trichomes on the abaxial and adaxial sides, whereas initiating needle-like wiry leaves (H) lack adaxial trichomes and the initiation of their abaxial characteristics is delayed. (I) An 8-week-old w3-1 plant with necrosis throughout its leaves. (J) Flowers of wild-type and wiry plants. w-1 flower is typical for w, w2, and w4, which are different from w3 flowers. Bars = 5 cm (A) to (D) and (I) and 2 cm in (E), (F), and (J). [See online article for color version of this figure.]

Figure 2.

wiry Mutants Are Impaired in Genes of the ta-siRNA Biogenesis Pathway. (A) to (D) A scheme of the tomato orthologs of RDR6 (A), AGO7 (B), SGS3 (C), and DCL4 (D) and the lesions identified in the different alleles. aa, amino acids; dsRBM, double-stranded RNA binding motif. (E) and (F) Different miRNAs, such as miR166, miR164, and miR390, are present in both wild-type (WT) and wiry shoots (E), but the ta-siARF derived from TAS3 could not be detected in any of the four wiry mutants (F). [See online article for color version of this figure.]

Figure 3.

A Loss of ARF3 and ARF4 Negative Regulation in the wiry Mutants. (A) to (F) In situ localization of ARF4, ARF3, and PHB transcripts in wild-type (WT) ([A] to [C]) and ago7-1 ([D] to [F]) vegetative apices. ARF4 mRNA is abaxial in leaf primordia and later it is expressed in pro-vascular and vascular strands (A). Its expression is stronger and broader in ago7-1 apices (D). ARF3 mRNA is detected in both sides of leaf primordia, with weaker expression in the SAM (B), and no change is detected in its expression pattern in ago7-1 apices (E). PHB mRNA is adaxial in leaf primordial and in the SAM center (C). It is strongly reduced in ago7-1 vegetative apices but remains adaxial in leaves. Insets in (A) and (D) show sections through P1 leaf primordia. Bars = 50 μm. (G) and (H) An RNA gel blot of RNA extracted from wild-type and wiry shoots probed with ARF3 and ARF4 cDNAs. Bottom panel is the RNA loaded. Note the high levels of ARF4 mRNA in rdr6, ago7, and sgs3 shoots (G) and the high levels of ARF3 in the dcl4 shoots (H). [See online article for color version of this figure.]

Figure 4.

Small RNAs Derived from the ARF4 and ARF3 Transcripts. (A) In the wild type (WT), ARF4-derived siRNAs (i) originate mainly from the sequence flanked by the two ta-siARF recognition sites (red arrows), and ARF3-derived siRNAs (ii) are present in low numbers and originate from all parts of the mRNA. (B) More ARF4-derived siRNAs were found in dcl4 apices (i) as well as siRNAs from the gene’s 3′ end but not from ARF3 (ii). (C) The ARF4-derived siRNAs (i) are 20 to 22 nucleotides in the wild type and primarily 22 to 24 nucleotides in dcl4 but the ARF3-derived siRNAs (ii) are 20 to 21 nucleotides in the wild type and primarily 22 nucleotides in dcl4. In (A) and (B), the x axis marks the position along the gene, but in (C), it marks the small RNA size. The y axis marks the numbers of reads.

Figure 5.

Normal and DCL4-Independent Production of TAS3 siRNAs. (A) Wild-type (WT) small RNAs derived from the TAS3-1 (i), TAS3-7 (ii), and TAS3-12 (iii) genes. Most small RNAs are derived from the fragment flanked by the miR390 recognition sites (red arrows). (B) In the dcl4 library, small RNAs from TAS3-1 (i) and TAS3-7 (ii) are present, but small RNAs from TAS3-12 are not present (iii). (C) Size distribution of small RNAs derived from the TAS3 genes. In the wild type, most are 21 nucleotides, but in the dcl4, they are shifted to 22 nucleotides. In (A) and (B), the x axis marks the position along the gene, but in (C), it marks the small RNA size. The y axis marks the numbers of reads.

Figure 6.

Loss of ARF3 and ARF4 Regulation Is the Basis of the wiry Syndrome. (A) to (C) Leaf 7 of wild-type (WT) (A), 35S:ARF3 (B), and 35S:ARF4 (C) plants. (D) A scheme of the ARF genes and construction of their ta-siRNA–insensitive forms by introduction of silent mutations into the two ta-siARF recognition sites. (E) and (F) Overexpressing the ta-siARF–insensitive form (Slm) of ARF3 (E) or ARF4 (F) in the wild type resulted in a strong wiry phenotype. (G) Expressing 35S:ARF4 in the weak ago7-3 mutant resulted in a very strong wiry phenotype. (H) to (J) Rescue of ago7-3 by downregulation of both ARF3 and ARF4 mRNA using an artificial miRNA; compare ago7-3 (H), pFIL>>amiR-ARF (I), and ago7-3 pFIL>>amiR-ARF (J). Bars = 2 cm. [See online article for color version of this figure.]

Figure 7.

Species-Specific Responses to ARF3 Activities. (A) to (C) Arabidopsis plants (A) overexpressing a ta-siARF–insensitive (m) ARF3 of either Arabidopsis (B) or tomato (C) origin have bifacial flat leaves. WT, the wild type. (D) Tomato plant overexpressing Arabidopsis mARF3. (E) to (H) Comparison of wild-type (i) and 35S:Sl-mARF3 (ii) tobacco plants. Tobacco leaves (E) are mildly modified, whereas flowers (F) are smaller and the tips of the petals are narrow. N. benthamiana leaves (G) are narrow and curled down, whereas flowers (H) are smaller and the petals are narrow and separated. (I) and (J) Leaves of normal (I) and 35S:At-mARF3 (J) potato plants. Bars = 2 cm. [See online article for color version of this figure.]