Alternate wiring of a KNOXI genetic network underlies differences in leaf development of A. thaliana and C. hirsuta

In this study, Rast-Somssich et al. investigated morphological differences between C. hirsuta, which has complex leaves with leaflets, and its relative, A. thaliana, which has simple leaves. By transferring single genes from one species into another under their endogenous regulatory elements, the authors show that leaf form can be modified in the recipient species, extending our knowledge of how paralogous genes are regulated in a complex eukaryote.


Isolation, mapping and complementation of C. hirsuta EMS mutants
The chstm-1 mutant was isolated from EMS-treated wild type seeds by visual inspection of ~1,500 EMS M2 plants (Hay and Tsiantis 2006). PCR amplification and sequencing of the complemented the phenotype in 21 out of 47 independent chstm-1 T1 lines (Fig. S2R).
The chbp-1 allele was isolated from EMS-mutagenized chas1-1;chstm-1/+ seeds by visual inspection of ~20,000 EMS M2 plants. We reasoned that screening of chas1-1;chstm-1/+ would help us distinguish KNOXI-dependent and independent ChAS1 targets potentially obtained from the screen. Genomic DNA and cDNA of wild type and chas1-1;chbp-1 was isolated, amplified and sequenced to assemble the contigs. This resulted in the identification of a G/A point mutation that created a premature stop codon in the second intron of the ChBP locus. A CAPS marker for genotyping was designed by BspHI digestion of the cBP-CAPS-F/R PCR product (5'-CCCACCTGAAGTGGTTGATAA-3' and 5'-ATACACAAAGGCCCAACTCC-3').
The chbp-1 single mutant was isolated by back crossing chas1-1;chbp-1 several times to wild type. Introduction of a ChBP::ChBP-VENUS (ChBP-V) transgene fully complemented the downward pointing silique phenotype of cbp-1 mutants in 34 out of 48 independent chbp-1 T1 lines and partially complemented the phenotype in 12 T1 lines (Fig. S2R). 12 The par mutant was isolated from EMS-treated wild type seeds by visual inspection of ~1,500 EMS M2 plants (Hay and Tsiantis 2006). A F2 mapping population was generated by out-crossing par to a polymorphic Greek strain. For map-based cloning, genomic DNA was isolated from 131 par mutants segregating in a 3:1 Mendelian ratio. A first rough mapping linked the par mutant phenotype to a 120kb region syntenic to the southern tip of chromosome 2 of A. thaliana containing 32 predicted genes. For fine mapping, new CAPS, dCAPS or length markers were designed based upon sequence differences between BAC or C.
hirstua WT sequences and the Greek ecotype. Primers used to subsequently amplify and sequence the ChMIR164A locus were designed based upon the synteny in nucleotide sequence between C. hirsuta, A. thaliana and Brassica rapa. Following the identification of a G/A point mutation in the 19 th nucleotide of the conserved 21bp miRNA164 in par, an RsaI CAPS marker for genotyping was designed (5'-TCCATTAATGCAGCCTTTGG-3' and 5'-GGAGCTCATGTTGGAGAAGG-3'). Complementation of 10 out of 12 independent T1 C. hirsuta lines with a MIR164A::MIR164A transgene (Nikovics et al. 2006) confirmed loss of ChMIR164A function in par (Fig. S4U).
The chcuc2-1 allele was isolated from EMS-treated par seeds by visually screening ~9,000 M2 EMS plants for suppression of the par leaf phenotype. The suppressed lines were genotyped for par using the RsaI CAPS marker and the ChMIR164A locus was sequenced to exclude an intragenic suppressor. The ChCUC2 locus of wild type and par;chcuc2-1 mutants was then amplified and sequenced. Following identification of a G/A point mutation in the ChCUC2 locus of par;chcuc2-1, a SpeI CAPS marker was designed (5'-CAAACAGACTTGGGCTTAGACG-3' and 5'-AACGGAGGAAGTCATTGTGG-3'). The chcuc2-1 single mutant was eventually isolated by outcrossing par;chcuc2-1 to wild type. A CUC2::CUC2-13 VENUS transgene (Heisler et al. 2005) was sufficient to complement chcuc2 in 6 out of 9 T2 par;chcuc2-1 lines, causing a reversion back to the par phenotype (Fig. S4U).
For the C.hirsuta ChBP::ChBP-VENUS construct, the ChBP ORF was amplified from genomic DNA using the primer 5'-CCGGTACCATGGAAGAATATCAGCACAACAC-3' and 5'-CGCAGCCATGGCTGGGCCAAGACGATAAGGTCC-3'. This fragment was subcloned into the BamHI/NcoI restriction sites of the intermediate pBJ97-VENUS vector. The ChBP promoter was excised from the ChBP::VENUS -pBJ97 vector described previously and inserted into a blunted BamHI restriction site. The entire cassette was subsequently moved as NotI fragment to pMLBART.
The BP::BP-CFP transgene was generated by PCR amplification of a 5825 bp genomic fragment from the BP locus using primers (5-CTCGAGTGAAAATCGCCAAAATGTCAATACA-3) and (5-GGATCCGCTGGACCGAGACGATAAGGTCCATC-3) and cloning as a translational fusion upstream of a 9 Ala linker followed two tandem copies of eCFP (Clontech Laboratories, Inc.) and OCS 3' terminator sequences. The 8.1 kb transgene was excised with NotI and cloned into the binary vector pMLBART. This fusion protein appears to function to complement the bp-1 mutant.
14 To make the BP::VENUS reporter, the BP promoter was cloned as a BamHI fragment into the intermediate pBJ97-Venus Vector. This BP::VENUS cassette was then inserted into the NotI restriction site of pMLBART.
To make MIR164A::ChBP, the MIR164A promoter (1.9 Kb) was amplified from the MIR164A::MIR164A (Nikovics et al. 2006) using the primers 5'-GCTGATGACGTCAGCGATAATTGGGGTC-3' and 5'-ACGTGAAGATTCTCACCCCGCATTTCC-3' and sub-cloned into the vector pBJ36 (Eshed et al. 2001 (Craft et al. 2005)). 16 Binary C. hirsuta ChCUC2 constructs The ChCUC2 locus was amplified in 2 PCR reactions from a BAC clone. The primers used are 5'-TCCAAGCTTTTACTCCATGGTT-3' and 5'-CAGCCATGGCGTAGTTCCAGATGCAATCAA-3' for the amplification of 750bp of the ChCUC2 coding region with NcoI restriction sites, and 5'-GGATCCTACAGATTTTAAAGAATTCG-3' and 5'-TGCAGAAGAAGAGGATCCAAA-3' for the amplification of a 5.35Kb fragment containing the ChCUC2 upstream regulatory sequences and BamHI restriction sites. The 750bp PCR product was then first cloned into the NcoI restriction sites of the pBJ97-3xVENUS intermediate vector described above, followed by a insertion of the 5.35Kb PCR fragment using the BamHI restriction sites. The entire cassette was then cloned as a NotI fragment into the binary vector pMLBART.