Figure S2.
Genotype and phenotype of csld6KO, csld2KO, and csld2/6KO. (A) PCR-based genotyping. CSLD6KO-npt and CSLD2KO-hph vectors integrated to delete CSLD6 and CSLD2, respectively, with primers used for amplification of the 5′ and-3′ integration sites (arrows). For csld6KO-4, -11, -12, -20 and -32 (top row), 5′ integration tested with primer pair D6KOFlankF/VectorR-npt produced the expected 1,581 bp fragment, 3′ integration tested with primer pair VectorF-npt/D6KOFlankR produced the expected 1,476 bp fragment, and target deletion was verified by the absence of a product from primers D6TargetF/D6TargetR, which anneals within the CSLD6 coding sequence and amplified an 828 bp fragment in the wild type. For csld2KO (middle two rows), 5′ integration tested with primer pair D2KOFlankF/VectorR-hph produced the expected 1,557 bp fragment in 10 lines, 3′ integration tested with primer pair VectorF-hph/D2KOFlankR produced the expected 1,611 bp fragment in 7 of those lines and target deletion was verified in lines csld2KO-1, -4, -9, -10, -16, and -17 by the absence of a product from primers D2TargetF/D2TargetR, which anneal within the CSLD2 coding sequence and amplify a 217 bp fragment in the wild type. For csld2/6KO (bottom two rows), 5′ and 3′ integration of the CSLD2KO-hph vector in csld2KO-32 was tested with the same primer pairs. Target deletion was verified in lines csld2/6KO-12, -13, -38, and -77. cre-mediated deletion of the selection cassette was verified for csld2/6KO-9 and 16 by amplification across the deletion site with primers D2KOFlankF/D2KOFlankR (2,724 bp). (B) Tube structures on csld2/6KO phyllids develop through altered cell expansion. Cells surrounding a cell separation (*) elongate radially forming an abaxial bulge with separation at the apex. Cell division and expansion enlarges the bulge forming a tube that protrudes from the abaxial surface. Scale bars = 50 μm. (C) CSLD2 or CSLD6 rescues phyllid development defects. Wild-type leaf morphology was restored when csld2/6KO plants were transformed with either a CSLD2 or CSLD6 expression vector, but not an empty control vector (EV). Ratios indicate the number of transformed lines with normal gametophores over the total number of transformed lines with gametophores. csld2/6KO image is a partial duplication of Fig. 2 G. (D)CSLD2 and CSLD6 are not required for protonemal development. Quantification of chlorophyll autofluorescence images of 7-d old wild type and csld2/6KO plants regenerated from protoplasts as a proxy for total plant area. A binary image of the median plant from each line is shown above (scale bar = 250 µm). For each of two experiments, 25 plants were measured from each of six replicate plates for each genetic line. Area was normalized to the wild-type parent line. Significant differences determined by a one-way ANOVA analysis with a Tukey post hoc test (alpha = 0.05) are indicated by different letters. Source data are available for this figure: SourceData FS2.

Genotype and phenotype of csld6KO, csld2KO, and csld2/6KO. (A) PCR-based genotyping. CSLD6KO-npt and CSLD2KO-hph vectors integrated to delete CSLD6 and CSLD2, respectively, with primers used for amplification of the 5′ and-3′ integration sites (arrows). For csld6KO-4, -11, -12, -20 and -32 (top row), 5′ integration tested with primer pair D6KOFlankF/VectorR-npt produced the expected 1,581 bp fragment, 3′ integration tested with primer pair VectorF-npt/D6KOFlankR produced the expected 1,476 bp fragment, and target deletion was verified by the absence of a product from primers D6TargetF/D6TargetR, which anneals within the CSLD6 coding sequence and amplified an 828 bp fragment in the wild type. For csld2KO (middle two rows), 5′ integration tested with primer pair D2KOFlankF/VectorR-hph produced the expected 1,557 bp fragment in 10 lines, 3′ integration tested with primer pair VectorF-hph/D2KOFlankR produced the expected 1,611 bp fragment in 7 of those lines and target deletion was verified in lines csld2KO-1, -4, -9, -10, -16, and -17 by the absence of a product from primers D2TargetF/D2TargetR, which anneal within the CSLD2 coding sequence and amplify a 217 bp fragment in the wild type. For csld2/6KO (bottom two rows), 5′ and 3′ integration of the CSLD2KO-hph vector in csld2KO-32 was tested with the same primer pairs. Target deletion was verified in lines csld2/6KO-12, -13, -38, and -77. cre-mediated deletion of the selection cassette was verified for csld2/6KO-9 and 16 by amplification across the deletion site with primers D2KOFlankF/D2KOFlankR (2,724 bp). (B) Tube structures on csld2/6KO phyllids develop through altered cell expansion. Cells surrounding a cell separation (*) elongate radially forming an abaxial bulge with separation at the apex. Cell division and expansion enlarges the bulge forming a tube that protrudes from the abaxial surface. Scale bars = 50 μm. (C) CSLD2 or CSLD6 rescues phyllid development defects. Wild-type leaf morphology was restored when csld2/6KO plants were transformed with either a CSLD2 or CSLD6 expression vector, but not an empty control vector (EV). Ratios indicate the number of transformed lines with normal gametophores over the total number of transformed lines with gametophores. csld2/6KO image is a partial duplication of Fig. 2 G. (D)CSLD2 and CSLD6 are not required for protonemal development. Quantification of chlorophyll autofluorescence images of 7-d old wild type and csld2/6KO plants regenerated from protoplasts as a proxy for total plant area. A binary image of the median plant from each line is shown above (scale bar = 250 µm). For each of two experiments, 25 plants were measured from each of six replicate plates for each genetic line. Area was normalized to the wild-type parent line. Significant differences determined by a one-way ANOVA analysis with a Tukey post hoc test (alpha = 0.05) are indicated by different letters. Source data are available for this figure: SourceData FS2.

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