Rapid analysis of strigolactone receptor activity in aNicotiana benthamiana dwarf14mutant

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 SLs, as well as its enantiomer GR24ent-5DS. In Nbd14a,b leaves, AtSMXL7 abundance was unaffected by GR24. Transient coexpression of AtD14 with the AtSMXL7 reporter in Nbd14a,b restored the degradation response to GR24, but required an active catalytic triad. With this platform, we evaluated the ability of several AtD14 mutants that had not been characterized in plants to target AtSMXL7 for degradation.

KARRIKIN UP-REGULATED F-BOX 1 (KUF1) imposes negative feedback regulation of karrikin and KAI2 ligand metabolism in Arabidopsis thaliana

“…S6 A ). To determine whether the remaining rac -GR24 response was mediated by D14 or KAI2, we also performed the experiment in an Nbd14a,b double mutant that has loss-of-function mutations in the two D14 homoeologs in N. benthamiana ( 61 ). We found that the SMAX1 reporter was not degraded in response to KAR 1 , KAR 2 , or rac -GR24 in Nbd14a,b leaves when KUF1 was overexpressed ( SI Appendix , Fig.…”

An N-terminal domain specifies developmental control by the SMAX1-LIKE family of transcriptional co-repressors inArabidopsis thaliana

“…These proteins are specifically targeted by D14 and not by KAI2 (Wang et al 2015;Khosla et al 2020a;White et al 2022). Finally, SMXL3-and aSMXL4-clade proteins, represented by SMXL3, SMXL4, and SMXL5 in Arabidopsis, regulate phloem development and anthocyanin abundance (Cho et al 2018;Wallner et al 2017Wallner et al , 2023Wu et al 2017;Li et al 2024).…”