Cloning the tomato curl3 gene highlights the putative dual role of the leucine-rich repeat receptor kinase tBRI1/SR160 in plant steroid hormone and peptide hormone signaling

Abstract

Brassinosteroids (BRs) are plant steroid hormones that are essential for normal plant development. To gain better understanding of the conservation of BR signaling, the partially BR-insensitive tomato mutant altered brassinolide sensitivity1 (abs1) was identified and found to be a weak allele at the curl3 (cu3) locus. BR content is increased in both of these mutants and is associated with increased expression of DWARF: The tomato homolog of the Arabidopsis Brassinosteroid Insensitive1 Leu-rich repeat (LRR) receptor-like kinase, named tBri1, was isolated using degenerate primers. Sequence analysis of tBRI1 in the mutants cu3 and abs1 revealed that cu3 is a nonsense mutant and that abs1 is a missense mutant. A comparison of BRI1 homolog sequences highlights conserved features of BRI1 sequences, with the LRRs in close proximity to the island domain showing more conservation than N-terminal LRRs. The most homologous sequences were found in the kinase and transmembrane regions. tBRI1 (SR160) also has been isolated as the putative receptor for systemin, a plant peptide hormone. This finding suggests a possible dual role for tBRI1 in steroid hormone and peptide hormone signaling.

Figure 1.

Phenotypes of cu3 and abs Mutants in Response to BL. (A) and (B) Photographs of seedlings at 18 days after sowing on medium containing various concentrations of BL. (A) Pairs of seedlings: wild type (left) and abs1 (right). (B) Pairs of seedlings: wild type (left) and cu3 (right). (C) Phenotypes of 54-day-old plants (dx and abs1) sprayed with water control and 10⁻⁶ M BL (+BL).

Figure 2.

BR Biosynthesis Pathway and BR Contents of Tomato Mutants. BR biosynthesis pathway adapted from Bishop and Yokota (2001), indicating the relative concentrations (ng/kg fresh weight [fw]) of BRs from wild-type (wt), cu-3, and abs1 plants. nd, not detected; -, not analyzed.

Figure 3.

Transcript Analysis. (A) An ethidium bromide–stained agarose gel harboring products from reverse transcriptase–PCR of two wild-type (wt1, L. esculentum control for abs1; wt2, L. pimpinellifolium control for cu3) and two mutant (cu3 and abs1) RNAs. Primers for actin, tBRI1, and Dwarf genes were used in the same PCR. The gel shows the PCR products after 29 cycles of PCR. (B) An ethidium bromide–stained agarose gel harboring products from reverse transcriptase–PCR of wild-type (wt; L. esculentum) and mutant (abs) RNAs from plants exposed to 10⁻⁶ M BL (+BL) or control (−BL). Primers for actin and Dwarf genes were used in the same PCR. The gel shows the PCR products after 33 cycles of PCR.

Figure 4.

Alignment of BRI1 Protein Sequences from Tomato, Arabidopsis, Pea, and Rice. Output from the alignment of rice, pea (T. Nomura, G.J. Bishop, and T. Yokota, unpublished data), and tomato (tom) sequences with the Arabidopsis (ara) BRI1 sequence using the program PileUp (Genetics Computer Group). The output was formatted using GeneDoc. Black boxes indicate identical amino acid residues. Spaces were introduced to separate domain regions as described in the text, with the LRR1 region containing 15 LRRs, the LRR2 region containing 6 LRRs, and the LRR3 region containing 4 LRRs. Currently known mutations resulting from a single base change in Arabidopsis (1-101, 1-103, 1-105, 1-113, and 1-115 [Li and Chory, 1997]; 1-1, 1-102, 1-114, and 1-117 [Friedrichsen et al., 2000]; and 1-5, 1-6, 1-8, and 1-9 [Noguchi et al., 1999]), in rice (d61-1 and d61-2 [Yamamuro et al., 2000]) and in tomato (cu3 and abs) are highlighted to indicate the change of amino acid residue.

Figure 5.

DNA Gel Blot Analysis. Autoradiograph of a DNA gel blot hybridized with ∼2.9 kb of ³²P-labeled tBRI1 sequence. Genomic DNA from the wild-type equivalent to abs (wt), the cu3 heterozygote (h), the wild-type equivalent of cu3 (wt 2), the cu3 mutant (cu3), and the abs mutant (abs) was digested with TspR1 restriction endonuclease and separated on a 0.8% agarose gel. Note the polymorphism in cu3, lacking bands of ∼0.45 and ∼0.3 kb and the presence of a 0.75-kb band.

Figure 6.

Coamplified Polymorphism Analysis of the cu3 and abs1 Mutations. Ethidium bromide–stained agarose gels highlighting the polymorphisms observed between wild-type and mutant DNAs. (A) TspRI digestion of PCR-amplified DNA, using primers TBR27 and 5-1, from genomic DNA isolated from the wild type (wt), cu3 heterozygotes (h), and cu3 tomato mutants (cu3). Note that cu3 mutants lack a TspRI site yielding DNA fragments of 445 and 309 bp. (B) HphI digestion of PCR-amplified DNA using primers TBR29 and 5-1 from genomic DNA isolated from the wild type (wt), abs1 heterozygotes (h), and abs1 tomato mutants (abs1). The 75-bp band indicated with the asterisk represents a doublet.

Figure 7.

Sequence Comparison of Different Regions of BRI1 Homologs. Pairwise sequence identity comparison of different regions of BRI1 homologs using the output from the PileUp analysis of the four BRI1 homologs identified in Figure 5.