Identification of critical cysteine sites in brassinosteroid‐insensitive 1 and novel signaling regulators using a transient expression system

Abstract

Summary The plant hormones brassinosteroids (BRs) modulate plant growth and development. Cysteine (Cys) residues located in the extracellular domain of a protein are of importance for protein structure by forming disulfide bonds. To date, the systematic study of the functional significance of Cys residues in BR‐insensitive 1 (BRI1) is still lacking. We used brassinolide‐induced exogenous bri1‐EMS‐Suppressor 1 (BES1) dephosphorylation in Arabidopsis thaliana protoplasts as a readout, took advantage of the dramatic decrease of BRI1 protein levels during protoplast isolation, and of the strong phosphorylation of BES1 by BR‐insensitive 2 (BIN2) in protoplasts, and developed a protoplast transient system to identify critical Cys sites in BRI1. Using this system, we identified a set of critical Cys sites in BRI1, as substitution of these Cys residues with alanine residues greatly compromised the function of BRI1. Moreover, we identified two negative regulators of BR signaling, pattern‐triggered immunity compromised RLCK1 (PCRK1) and PCRK2, that were previously known to positively regulate innate immunity signaling. This work not only provides insight into the functional importance of critical Cys residues in stabilizing the superhelical conformation of BRI1‐leucine‐rich‐repeat, but also reveals that PCRK1/2 can inversely modulate BR and plant immune signaling pathways.

Comprehensive Analysis of BR Receptor Expression under Hormone Treatment in the Rubber Tree (Hevea brasiliensis Muell. Arg.)

“…BR and other hormones (ABA, ETH, GA, JA and SA) promote and antagonize each other under disparate conditions [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ]. BRI1 and BAK1 can be qualified as upstream components of the BR signal [ 49 , 50 , 51 ]. The mutant of BRI1 is insensitive to BR, but treatment with other hormones has no significant differences [ 52 ].…”

Phosphorylation status of CPK28 affects its ubiquitination and protein stability

“…CPK28 , CPK28 T76A , CPK28 S318A , ATL31 , and GFP were cloned into the vector pTF101 for gene transient expression in N. benthamiana or generation of transgenic plants. Arabidopsis BIK1 , UBQ10 , BRI1 , PCRK1 , and GFP constructs were described previously (Lu et al ., 2010, 2011; Huang et al ., 2019). CPK28 D188A , CPK28 S318A , CPK28 S318D , CPK28 T76A , CPK28 T76D , and ATL31 ΔTM (amino acids 67–368) were subcloned into the modified His‐tagged or GST‐tagged fusion protein expression vector pET28a (Novagen) or pMAL‐C2 (New England Biolabs, Hitchin, UK).…”

“…Co‐immunoprecipitation (Co‐IP) assays were performed as described previously (Huang et al ., 2019; Liu et al ., 2022).…”

“…Isolation of Arabidopsis mesophyll protoplasts and gene transient expression in protoplasts were described previously New Phytologist (Huang et al, 2019). Protein expression in protoplasts was detected by immunoblotting with corresponding antibodies.…”