DNA sequencing and marker development

DNA was extracted from Yellow Pear (YP) and Gold Ball Livingston (GBL) leaf samples using the Qiagen kit (Germantown, MD) and following manufacturer’s instructions. DNA libraries were constructed and sequenced on the Illumina HiSeq2000 platform with the 101-bp paired-end mode at the Genome Technology Access Center (GTAC) at Washington University, St Louis MO (SRA SRP127270). After removing adapters and low-quality sequences, the sequences represented more than 20× coverage of the genome (aligned bases divided by 950 Mbp length of the tomato genome). High quality cleaned reads were aligned to the tomato genome SL2.4025 using NovoAlign (http://www.novocraft.com/). SNPs were identified in the genomic regions of interest using SAMtools26. High quality (>100) homozygous SNPs with coverage higher than 15× were kept for marker development. Based on the identified SNPs, we developed 26 additional dCAPS markers located between the existing markers, 12EP153 and 12EP5 (Supplementary Data 4) using dCAPS Finder (http://helix.wustl.edu/dcaps/dcaps.html) and Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/). The sov1 deletion in the promoter of SlOFP20 was identified by aligning the reads of the YP and GBL to the reference genome (Supplementary Fig. 2). After validation by Southern blotting, the structural rearrangement was further validated using primers 13EP549, 13EP550 and 13EP551 in a single PCR reaction. The wild-type band runs at 1220 bp whereas the fragment that amplifies YP DNA runs at 900 bp. The Heinz 1706 BAC clone sequence, LE_HBa0040P16 containing SlOFP20 (Genbank AC244530) allowed us to estimate the size of the sov1 deletion to 31 kb and its position in relation to the coding region of SlOFP20. The estimation of putative transcription start site of SlOFP20 is based on the tomato ESTs and cDNAs database (https://solgenomics.net/) as well as our RNA-seq data (SRA SRP090032; SRP089970).

Potato StOFP20-trait association

The M6 potato genome scaffold 814 harbored the ortholog of SlOFP2027. Primers were designed to amplify the ortholog in M6 and a linked sequence in DM1–3 (Supplementary Data 4). The marker was genotyped using 190 individuals of a segregating F 2 population derived from DM1–3 and M622. ANOVA analyses were applied to determine the significance of the association of the marker allele with the phenotype.

Alignment of the potato DM1–3 reads to the M6 scaffold 814

Reads from the 16 paired-end genomic DNA libraries of potato DM1–3 used in the de novo assembly of the reference genome (SRA029323) were aligned to M6 scaffold 814 using BWA-MEM v0.7.1328 in paired-end mode with default parameters. Alignments with MAPQ score higher than or equal to 30 were kept and visualized with Integrative Genomics Viewer (IGV v2.3.68).

Overexpression of SlOFP20

Full length SlOFP20 was amplified from DNA using primers 13EP617/618 (Supplementary Data 4) and Phusion® high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA). The fragment was cloned into the XhoI/SacI site of the pKYLX71 vector29 and expressed under the 35S promoter. The construct was sequenced to confirm that no errors were introduced during the amplification and then transformed in YP. Five independent transgenic lines were obtained and fruit and leaf shape analysis was done using 8 mature fruits or primary leaflets of each plant.

Downregulation of SlOFP20

Two artificial microRNAs (amiRNAs) targeting the coding region of SlOFP20 were designed using the WMD3 Web MicroRNA Designer (http://wmd3.weigelworld.org/) and put into the Arabidopsis MIR319a precursor backbone. The sequences of amiRNAs designed to knock-down the expression of SlOFP20 were provided in Supplementary Data 4. The engineered amiRNA precursors were synthesized at GenScript Biotech Corporation (Piscataway, NJ) and then cloned into the SacI/XbaI site of the pKYLX71 vector29. The two SlOFP20 amiRNA constructs were transformed in LA1589 resulting in 12 primary transgenic lines. The T 0 lines were evaluated for downregulation of SlOFP20 using semi-quantitative PCR. Two of the most down regulated lines per construct were backcrossed to SA29 carrying the ovate mutation. The resulting seedlings were selected for the presence of the transgene using primers EP553/554 (Supplementary Data 4) and selfed to produce families 15S91, 15S92, 15S93, and 15S94 (BC 1 F 2 ).

SlTRM CRISPR/Cas9 mutants

A CRISPR/Cas9 construct was designed to create mutations in both SlTRM3/4 and/or SlTRM5. The construct was assembled using the Golden Gate cloning method30. Two sgRNAs specifically targeting each of the two SlTRMs were amplified using the pICH86966::AtU6p::sgRNA_PDS construct (Addgene plasmid #46966, www.addgene.org) as a template with the reverse primer: 13EP639 and forward primers: 14EP292 and 14EP294 for SlTRM3/4 and SlTRM5, respectively (Supplementary Data 4). The level 1 constructs pICH47751 (Addgene #48002) and pICH47761 (Addgene #48003) were assembled using the level 0 construct pICSL01009::AtU6p (Addgene #46968) and the sgRNA PCR products to place each sgRNA under the Arabidopsis U6 promotor. Level 1 constructs, pICH47732::NOSp::NPTII (Addgene #51144), pICH47742::35S::Cas9 (Addgene #49771), pICH47751::AtU6p::sgRNA-SlTRM3/4, pICH47761::AtU6p::sgRNA-SlTRM5 and the linker pICH41780 (Addgene #48019) were then assembled into the level 2 vector pAGM4723 (Addgene #48015). All the vectors for building the CRISPR-Cas9 construct were provided by Vladimir Nekrasov and Sophien Kamoun, The Sainsbury Laboratory, Norwich Research Park, Norwich, UK.

Plant transformation and genotyping

Constructs were transformed into tomato at the Plant Transformation Facility at University of California (Davis, CA 95616) (amiRNA and overexpressor constructs), and by Dr. Joyce Van Eck, Cornell University (CRISPR-Cas9 constructs). The lines overexpressing or under-expressing SlOFP20 were genotyped for the presence of the construct with primers designed to amplify the NPTII gene conferring kanamycin resistance (EP553/554; Supplementary Data 4). For the CRISPR-Cas9 constructs, we received 16 independent T 0 lines. By sequence analyses, the majority were found to carry mutations in both SlTRM3/4 and SlTRM5 and occasionally mutations were in both alleles of one or both genes. Most lines were sterile (male and female) as no fruits with seeds were generated by selfing or with wild type pollen onto mutant styles or mutant pollen unto wild type styles. The primary transformants that were fertile (four T 0 lines) carried an in frame deletion (multiples of 3) and wild type allele for SlTRM3/4 and an in frame or frameshift mutations for SlTRM5. These fertile lines were crossed to LA1589 in order to remove the Cas9 transgene and stabilize the lines. Once stabilized, for SlTRM5 the 1 bp deletion and 1 bp insertion alleles (causing a frameshift mutation and likely a null) were backcrossed into the sov1/ovate background, which is described above for the development of the NILs.

Fruit shape analysis

Full size maturing fruits were cut longitudinally, scanned at 300 dpi, and analyzed using Tomato Analyzer v3.031,32. The following attributes were measured in most samples: fruit shape index, proximal end angle, proximal eccentricity and obovoid, which measures the pear-shapedness. Fruit Shape Index is the ratio of the maximum height length to maximum width of a fruit. Proximal end angle is the angle between best-fit lines drawn through the fruit perimeter on either side of the proximal end point at 10% (NILs) or 20% (tomato varieties). Proximal eccentricity is the ratio of the height of the internal ellipse (defined by the section of the fruit where the seeds are located) to the distance between the bottom of the ellipse and the top of the fruit. Obovoid is calculated as the maximum width (W), the height at which the maximum width occurs (y), the average width above that height (w1), and the average width below that height (w2) are calculated and a scaling function scale_ob is used for calculation: Obovoid = 1/2 × scale_ob (y) × (1 – w1/W + w2/W), if obovoid > 0, subtract 0.4; otherwise, obovoid is 0. For the potato tuber shape analyses, length and width were measured in Image J and shape index was calculated by taking the ratio of these two measurements. The length of the curved potatoes was measured by tracing the curve. Melon Fruit Shape Index was calculated with Tomato Analyzer 3.0 from scanned images. For the cucumber shape analyses, length and width were measured with calipers and the shape index was calculated by taking the ratio of the two.

Ovary shape analysis

Anthesis ovaries were cut longitudinally and digitalized using the Olympus Szx9 (SZX-ILLB2–100) dissecting scope. The maximum length and width of ovaries were measured using ImageJ software (https://imagej.nih.gov/ij/), and from this, the ratios of the maximum length to maximum width were calculated. Three to five plants of each genotype were analyzed, and the average values each taken from 8 to 10 fruits or ovaries per plant were analyzed with Tukey’s HSD test (α < 0.05).

Cellular attributes

Anthesis ovaries were cut longitudinally with a razor blade and fixed in FAA (50% Ethanol, 10% Formaldehyde, 5% glacial acetic acid) at 4 °C overnight. The samples were dehydrated on ice with ethanol-ddH 2 O series (50, 70, 85, 95, 100% x 2), and rehydrated with ethanol-ddH 2 O series (95, 85, 70, 50, 30, 15%) at one hour for each step. The samples were rinsed with ddH 2 O twice for 20 min each. Ovaries were incubated on ice in the staining buffer (0.02 mg/ml propidium iodide, (MP Biomedicals), 0.02% DMSO) for 1 h and rinsed with ddH 2 O for 20 min twice, then dehydrated with ethanol-ddH 2 O series (50, 70, 85, 95, 100%) on ice, and 100% ethanol at room temperature overnight. Finally, the samples were treated with 1:1 ethanol: methyl salicylate for 1–2 h followed by 100% methyl salicylate (Fisher Chemical) at 4 °C for 2–3 days. The sections were imaged using a Zeiss LSM 510 META Confocal Microscope, and cell size and number assessments were made using the ImageJ software package. The proximal area of the ovary (the area above the ovules closest to the stem end of the flower) was used for the histological and cellular analysis. The length and width of the entire proximal area was measured. Numbers of parenchyma cells were counted in the middle of the proximal area in both the proximo-distal and medio-lateral direction. The length and width of parenchyma cells in the proximal area were evaluated on at least 20 cells.

Yeast two-hybrid analyses

Full-length OVATE or full length SlOFP20 were cloned into the pGBKT7 vector (Clontech, Mountain View, CA) as C-terminal fusions to the GAL4 DNA binding domain (BD). Fragments of SlTRM5, SlTRM17/20a and SlTRM25 were cloned into the pP6 vector (Hybrigenics, Paris, France) as a C-terminal fusion to the GAL4 activation domain (AD). A pair of bait and prey plasmids were transformed into the Y2HGold yeast strain following the Clontech Yeast Protocol Handbook instructions (Clontech, Mountain View, CA) and plated on minimal medium lacking Trp and Leu (SD/-Trp/-Leu) with X-α-Gal (the substrate of MEL-1 gene product, α-galactosidase). Quantification of reporter α-galactosidase activity was performed using the Clontech Yeast α-Galactosidase Assay Kit. Three single colonies of each combination were grown in liquid synthetic dropout medium lacking leucine and tryptophan at 30 °C with shaking (250 cycles/min) for 18 h. After recording the OD 600 , the supernatants were collected via centrifugation at 18,800xg for 2 min. After adding the assay buffer (PNP-α-Gal + CH 3 COONa) to the supernatants and incubating at 30 °C for 60 min, the reaction was terminated by adding the stop solution (Na 2 CO 3 ). The optical density of each sample was recorded at OD 410 and α-galactosidase activity was calculated using the formula: 1,000 × Vf × OD 410 /[(e × b) × t × Vi × OD 600 ] where t = time (min) of incubation, Vf = volume of assay (200 or 992 µl), Vi = volume of culture medium supernatant added, OD 410 = A410 of the reaction mix, OD 600 = A600 of 1 ml of culture, e × b = p-nitrophenol molar absorbtivity at 410 nm × the light path (10.5 (ml/μmol) for 200-µl format = 16.9 (ml/μmol) for 1-ml format where b = 1 cm). The empty vector was used as the negative control.

Phylogenetic and protein motif analyses of TRMs

To retrieve all putative SlTRM proteins in tomato, full-length sequences of 34 Arabidopsis members of this family were used for BLAST similarity search against the International Tomato Annotation Group release 2.3 predicted proteins (ITAG 2.40) (http://solgenomics.net/) and Motif Alignment and Search Tool (MAST) search33, both at a cutoff E-value of 10−5. The MEME tool34 was used to define the conserved motifs with the following parameters: “nmotifs 8, minw 10, maxw 100, minsites 30, maxsites 120”. ClustalW was used for multiple sequence alignment procedures. The phylogenetic relationships among the all the TRMs in tomato and Arabidopsis were estimated with neighbor-joining method based on the p-distance (i.e., the phylogenetic distances were obtained by dividing the number of amino acid differences with the total number of sites compared) and 1000 bootstrap validation. The phylogenetic tree was visualized by FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Protein charge plot were generated by calculating the total charge of amino acids over a sliding window of 51 residues as described previously11.

Phylogenetic analysis of the SlOFP20 subclade

We retrieved the potato ortholog and closest paralogs (four total) by conducting reciprocal best BLAST hits. This led to four OFP members in the M635 and doubled monoploid Group Phureja clone DM1–3 516 R44 (DM)36,37. The four hits in each potato assembly with the lowest E-value statistic were identified as initial candidate sequences. In order to find the best full-length tomato protein BLAST hits of each potato candidate sequence, the amino acid sequence returned for each hit was used as query in a BLASTp search against the SL3.0 tomato annotated protein sequences. Reciprocal best BLAST hit analysis was performed using these full-length tomato proteins. Each full-length protein was used as query in tBLASTn searches against the DM1–3 and M6 assemblies. The best DM1–3 and M6 hits from each of those searches was then used as query in reciprocal BLASTp and tBLASTn searches against the tomato protein sequences and genome SL3.0 assembly respectively, in order to identify and confirm each reciprocal best BLAST hit relationship. BLAST searches used the BLOSUM62 scoring matrix and a word size of 11 amino acids. Using similar methods, we obtained the five Arabidopsis OFP1 through OFP5 and the four melon OFP proteins that showed the best BLAST hit relationship to members of the SlOFP20 subclade. We also included OVATE and its best BLAST hit AtOFP7. The resulting reciprocal best BLAST hits were aligned with ClustalW. Phylogenetic relationships between them were estimated via the neighbor-joining method and p-distance, with the phylogeny rooted by the distant Physcomitrella OFP protein and validated with 1000 bootstraps, as implemented in Geneious 10.1.338.

Phylogenetic analysis of the AtTRM1–5 clade

We used the five Arabidopsis and three tomato proteins from Supplementary Fig. 4. We identified the rice and cucumber TRMs as described for the OFP1–5 clade. Phylogenetic analysis was implemented similarly as with the OFP reciprocal best BLAST hits, but with the phylogeny rooted by the Solyc02g089050 TRM protein.

In vitro mutagenesis

Site-directed mutagenesis of selected residues for SlTRMs and OVATE/SlOFP20 was carried out with the QuikChange II XL Site-Directed Mutagenesis system (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s specifications. Oligonucleotide primers between 25 and 35 bases with a melting temperature of ≥78 °C were designed using quikchange primer design software (http://www.genomics.agilent.com/primerDesignProgram.jsp) to cover the appropriate point mutations that would lead to the desired amino acids substitutions (Supplementary Data 4). The SlTRMs and OVATE/SlOFP20 mutants were subcloned into pP6 and pGBKT7 vectors respectively (Clontech, Mountain View, CA). Mutations were verified by DNA sequence analysis of the resulting clones.

Construction of plasmids for transient transformation

For constructs used in the transient assays, full-length wild type or mutant coding sequences (CDS) of OVATE, SlOFP20, SlTRM3/4, SlTRM5 and SlTRM25 were cloned into pENTR/D-TOPO Gateway entry vector (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. The coding regions were recombined into binary destination expression vectors pH7RWG2 (Cauliflower mosaic virus (CaMV) 35S promoter-driven) for C-terminal RFP (red fluorescent protein) fusions39,40 or pSITE-2CA and pSITE-2NA (2 × 35S) for N-terminal and C-terminal GFP (green fluorescent protein) fusions, respectively41. For the BiFluorescence Complementation (BiFC) experiments, full length wild-type OVATE and SlTRM25 were cloned as C-terminal or N-terminal fusions to the C-terminal or N-terminal fragment of the Yellow Fluorescent Protein (YFP) in the BiFC vectors pYN-1 (N-terminal fusion of YFP 1–158 ), pYC-1 (N-terminal fusion of YFP 159–238 ), p2YN (C-terminal fusion of YFP 1–158 ) and p2YC (C-terminal fusion of YFP 159–238 )42 and co-expressed in all 8 combinations in N. benthamiana to determine the best usage of the BiFC vectors. Based on the overall YFP intensity and numbers of cells expressing YFP, the pYN-1 and p2YC vectors were used for SlTRMs and OVATE, respectively.

Transient expression of proteins in N. benthamiana

Agrobacterium tumefaciens strain C58C1 was used for the transient transformations. Colonies carrying the binary plasmids were grown at 28 °C on LB medium plates that contained 50 µg/ml gentamycin and 25 µg/ml rifampicin for selection of the strain, and 100 µg/ml spectinomycin for selection of the binary vectors, pH7RWG2 (carrying OVATE or SlOFP20 with a C-terminal RFP tag), pSITE-2CA (carrying SlTRM3/4 or SlTRM25 with a N-terminal GFP tag), and pSITE-2NA (carrying SlOFP20 with a C-terminal GFP tag)39,40,41. For agroinfiltration, single colonies were grown in liquid LB supplemented with gentamycin, rifampicin and spectinomycin overnight (28 °C, 220 cycles per minute). Fifty μl of the agro suspension was added to 5 ml LB with the same antibiotics for another overnight incubation under the same conditions. The agrobacteria were pelleted by centrifuging at 1610×g for 20 min or 2200×g for 15 min. The cells were resuspended in infiltration buffer containing 10 mM MgCl 2 , 10 mM MES, PH 5.7 and 150 mM acetosyringone at pH 5.6 and adjusted to an OD 600 of 0.2–0.3. The cells were incubated at room temperature for 3 h without shaking prior to infiltration. To enhance transient expression of the fusion proteins, the viral suppressor of gene silencing p19 protein was coexpressed in most of the experiments. For co-infiltration, equal volumes of cultures were mixed and infiltrated into N. benthamiana leaves through the abaxial surface using a 1-ml needleless syringe (Becton, Dickinson and Company). Plants were then kept in a growth room at 24/22 °C with a 16/8 h light/dark photoperiod for 48–72 h.

Epifluorescence and confocal microscopy

N. benthamiana leaf samples (approximately 0.25 cm2 near the infiltrated area) were collected at 70–90 h post-infiltration, mounted in water and viewed directly with a Zeiss LSM 880 confocal scanning microscope using an oil immersion objective 40× Plan-Apochromat 1.4NA (numerical aperture of 1.4). Fluorescence was excited using 488 nm and 543 nm light for GFP and RFP, respectively. GFP and RFP emission fluorescence was selectively detected at 490–540 and 550–630 nm using the Zen 2.3 SP1 software. For each experiment, 50–100 cells in two independent leaves that expressed both proteins were evaluated. For the BiFC experiments, YFP fluorescence was excited using 514 nm laser and detected at 520–550 nm using the Leica TCS SP5 confocal scanning microscope with a 25x objective.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.