182 8 APRIL 2022¥VOL 376 ISSUE 6589 science.orgSCIENCE
Fig. 2. Effects ofTaCol-B5in T 1 ,T 2 , and T 3 trans-
genic wheat plants.(AtoC) Assessment of
TaCol-B5in T 1 transgenic wheat in the greenhouse
(GH). In addition to visual differences in adult
plant phenotype (transgenic eventTaCol-B5-OE49)
(A), significant differences were observed between
transgenic plants (+) and nontransgenic plants (–)
for SNS (B) and grain number (C), averaged
across the fourTaCol-B5overexpression families.
(DtoH) Assessment ofTaCol-B5in T 2 transgenic
wheat in the field. From single-row field plots in
Jiangsu, China (D), significant differences were
observed between T 2 transgenic plants (+) and
nontransgenic plants (–) in the number of spikelet
nodes per spike (E), spike length (F), the number of
grains per spike (G), and the number of spikes
per plant (H). A two-tailed Student’sttest was used
to determine the significance level, and actual
Pvalues are shown in the figures. Thenvalues
indicate the number of spikes [(B), (C), and
(E) to (G)] or plants (H) as characterized. (Iand
J) Assessment ofTaCol-B5in T 3 transgenic wheat
in the field. Adult transgenic plants (+) and non-
transgenic plants (–) are shown in standard field
plots, with a drainage ditch arranged between plots
(I). Same comparison as in (I), but with a black cloth
used as a background (J). Additional phenotypic
information is provided in table S3.
Fig. 3. Interaction and phosphorylation ofTaCol-B5
byTaK4.(A) Cotransformed cells were grown on
plates lacking two amino acids (−Leu/−Trp) and on
plates lacking four amino acids (−Ade/−His/−Leu/
−Trp). Four colony solutions diluted to different levels
(10^0 to 10−^3 ) were inoculated on the same plate for
each protein pair. Only yeast cells harboring theTaCol-
B5 bait andTaK4 prey combination grew. (B) Three
amino acid (a.a.) substitutions betweenTaCol-B5
andTacol-B5, and two of them that could be
differentially phosphorylated, are indicated in red.
(C) Phosphorylation ofTaCol-B5 byTaK4.Aninvitro
kinase assay was performed with purified His-tagged
TaCOL-B5 proteins. The phosphorylated (+Phos)
and nonphosphorylated (−Phos) proteins were
separated on a Phos-tag (50mM) 10% polyacrylamide
gel (top image), and the proteins from the same reaction
were run on a non-Phos-tag gel used as a control
(bottom image). The +Phos and−Phos proteins were
analyzed by Western blotting (WB) with an anti-His tag
antibody. The +PhosTaCol5 protein showed lower
electrophoretic mobility (indicated by a red arrow) than
its−Phos counterparts (indicated by a black arrow).
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