Rapid analysis of strigolactone receptor activity in a <i>Nicotiana benthamiana dwarf14</i> mutant

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 homoeologous <i>D14</i> genes in the <i>Nicotiana benthamiana</i> genome. The <i>Nbd14a,b</i> double 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 from <i>Arabidopsis thaliana</i> (AtSMXL7) after transient expression in <i>N. benthamiana</i> and 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 ^{<i>ent</i>-5DS}. In <i>Nbd14a,b</i> leaves, AtSMXL7 abundance was unaffected by <i>rac</i>-GR24 or either GR24 stereoisomer. Transient coexpression of AtD14 with the AtSMXL7 reporter in <i>Nbd14a,b</i> restored the degradation response to <i>rac</i>-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.