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

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.

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.

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Output from the alignment of rice, pea (T. Nomura, G.J. Bishop, and T. Yokota, unpublished data), and tomato (tom) sequences with the Arabidopsis (ara) BRI1 sequence using the program PileUp (Genetics Computer Group).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.

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.

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.