Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

Abstract

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 D14 genes in the Nicotiana benthamiana genome. The Nbd14a,b 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 Arabidopsis thaliana (AtSMXL7) after transient expression in N. benthamiana and treatment with the strigolactone analog GR24. AtSMXL7 was degraded after treatment with GR245DS, which has the stereochemical configuration of natural SLs, as well as its enantiomer GR24ent‐5DS. In Nbd14a,b leaves, AtSMXL7 abundance was unaffected by rac‐GR24 or either GR24 stereoisomer. Transient coexpression of AtD14 with the AtSMXL7 reporter in Nbd14a,b restored the degradation response to rac‐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.

Keywords

• CRISPR‐Cas9
• phytohormones
• proteolysis
• signaling
• transient expression