Ratiometric Measurement of Protein Abundance after Transient Expression of a Transgene in Nicotiana benthamiana

Background

Karrikins (KARs) are butenolide compounds found in smoke that stimulate seed germination and enhance seedling photomorphogenesis of Arabidopsis thaliana (Flematti, 2004; Nelson et al., 2009, 2010 and 2012). KAR responses in Arabidopsis require the α/β-hydrolase KARRIKIN INSENSITIVE2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) (Sun and Ni, 2011; Waters et al., 2012). KAI2 can bind KAR₁ in vitro and is thought to function as a KAR receptor (Guo et al., 2013). KAI2 works with the F-box protein MORE AXILLARY GROWTH2 (MAX2) to mediate KAR responses, likely through polyubiquitination and degradation of SUPPRESSOR OF MAX2 1 (SMAX1) (Nelson et al., 2011; Stanga et al., 2013; Waters et al., 2017). This signaling mechanism is highly similar to that of the plant hormone strigolactone (SL). In SL signaling, DWARF14 (D14)/DECREASED APICAL DOMINANCE2 (DAD2), which is an ancient paralog of KAI2, works with MAX2 to target a subset of SMAX1-LIKE proteins for degradation (Hamiaux et al., 2012; Waters et al., 2012; Jiang et al., 2013; Zhou et al., 2013; Soundappan et al., 2015; Wang et al., 2015). Interestingly, KAI2 and D14 are both responsive to rac-GR24, a commonly used racemate of synthetic SL analogs. However, they show different preferences for the two enantiomers in this mixture, only one of which has a stereochemical configuration that mimics natural SLs (Scaffidi et al., 2014; Waters et al., 2015; Flematti et al., 2016).

In addition to mediating KAR responses, there is growing evidence that the primary role of KAI2 is the recognition of an unknown endogenous signal known as KAI2 ligand (KL) (Conn and Nelson, 2015). Identification of KL will require a highly specific assay for the activation of KAI2. Recently, we found evidence that SMAX1 degradation occurs after KAR and rac-GR24 treatment, and this response requires MAX2 and KAI2 (Khosla et al., 2020b). This observation led us to develop a bioassay for SMAX1 degradation that is appealing as a direct and specific readout of KAI2 activation.

We generated a series of ratiometric reporter vectors (pRATIO) that can be used to assess protein abundance in vivo by monitoring the relative abundance of two co-expressed fluorescent or bioluminescent reporters, one of which is fused to a target protein of interest (Khosla et al., 2020a). Rather than express a pair of target and reference genes from two promoters on a single vector or two co-transformed vectors, in the pRATIO system the target and reference genes are transcribed on the same mRNA. Importantly, pRATIO vectors can normalize for differences in transformation efficiency or transgene expression across samples by encoding a 2A ribosomal skipping peptide from foot-and-mouth disease virus (FMDV) between the target and reference genes. During translation, the nascent 2A peptide blocks the ribosomal exit channel. This disrupts formation of the bond between the C-terminal Gly and Pro residues of the 2A peptide. Translation can resume on the mRNA, producing two proteins in near stoichiometric ratios (Luke and Ryan, 2018).

Here we present a detailed protocol for using the pRATIO system. As an example, we monitor KAR- and rac-GR24- induced proteolysis of Arabidopsis SMAX1 that has been transiently expressed in Nicotiana benthamiana.