A GH3:GUS reporter line [50 (link)] was used as the WT moss strain. Spot cultures were grown as described previously [61 (link)], and tissue for genetic analysis was prepared as in [50 (link)]. All lines were stored in the International Moss Stock Center (http://www.moss-stock-center.org ; see Supplemental Information ).
For immunolocalizations, tissue was grown for 4 weeks in continuous light, fixed in 3:1 methanol acetic acid, dehydrated, and embedded in PEG 1600. Eight-micrometer sections were interrogated with anti-maize PIN antibodies [55 (link)] at a 1/150 dilution and anti-BIP2 (Agrisera) at a 1/50 dilution. DyLight 594 and DyLight 405 were used as secondary antibodies at a 1/300 dilution.
pin disruptants were generated and screened for insertion as described inSupplemental Information .
GUS staining was carried out as elsewhere [32 (link)]. Light micrographs were compiled using a Keyence VHX-1000 series microscope with 50× and 200× objectives. Confocal imaging was undertaken as previously described [61 (link)], except for immunolocalizations; a Leica TCS 5 was used, with excitation from the Diode 405 and HeNe 594 laser lines, and emission was collected at 410–480 nm and 600–670 nm.
For immunolocalizations, tissue was grown for 4 weeks in continuous light, fixed in 3:1 methanol acetic acid, dehydrated, and embedded in PEG 1600. Eight-micrometer sections were interrogated with anti-maize PIN antibodies [55 (link)] at a 1/150 dilution and anti-BIP2 (Agrisera) at a 1/50 dilution. DyLight 594 and DyLight 405 were used as secondary antibodies at a 1/300 dilution.
pin disruptants were generated and screened for insertion as described in
GUS staining was carried out as elsewhere [32 (link)]. Light micrographs were compiled using a Keyence VHX-1000 series microscope with 50× and 200× objectives. Confocal imaging was undertaken as previously described [61 (link)], except for immunolocalizations; a Leica TCS 5 was used, with excitation from the Diode 405 and HeNe 594 laser lines, and emission was collected at 410–480 nm and 600–670 nm.
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