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Abstract DWARF14 (D14) is an ɑ/β‐hydrolase and receptor for the plant hormone strigolactone (SL) in angiosperms. Upon SL perception, D14 works with MORE AXILLARY GROWTH2 (MAX2) to trigger polyubiquitination and degradation of DWARF53(D53)‐type proteins in the SUPPRESSOR OF MAX2 1‐LIKE (SMXL) family. We used CRISPR‐Cas9 to generate knockout alleles of the two homoeologousD14genes in theNicotiana benthamianagenome. TheNbd14a,bdouble mutant had several phenotypes that are consistent with the loss of SL perception in other plants, including increased axillary bud outgrowth, reduced height, shortened petioles, and smaller leaves. A ratiometric fluorescent reporter system was used to monitor degradation of SMXL7 fromArabidopsis thaliana(AtSMXL7) after transient expression inN. benthamianaand treatment with the strigolactone analog GR24. AtSMXL7 was degraded after treatment with GR24^5DS, which has the stereochemical configuration of natural SLs, as well as its enantiomer GR24^ent‐5DS. InNbd14a,bleaves, AtSMXL7 abundance was unaffected byrac‐GR24 or either GR24 stereoisomer. Transient coexpression of AtD14 with the AtSMXL7 reporter inNbd14a,brestored the degradation response torac‐GR24, but required an active catalytic triad. We used this platform to evaluate the ability of several AtD14 mutants that had not been characterized in plants to target AtSMXL7 for degradation.