Scanning for New BRI1 Mutations via TILLING Analysis

Abstract

The identification and characterization of a mutational spectrum for a specific protein can help to elucidate its detailed cellular functions. BRASSINOSTEROID INSENSITIVE1 (BRI1), a multidomain transmembrane receptor-like kinase, is a major receptor of brassinosteroids in Arabidopsis (). Within the last two decades, over 20 different mutant alleles have been identified, which helped to determine the significance of each domain within BRI1. To further understand the molecular mechanisms of BRI1, we tried to identify additional alleles via targeted induced local lesions in genomes. Here, we report our identification of 83 new point mutations in, including nine mutations that exhibit an allelic series of typical phenotypes, from subtle to severe morphological alterations. We carried out biochemicalanalyses to investigate possible mechanisms of these mutations in affecting brassinosteroid signaling. A number of interesting mutations have been isolated via this study. For example, , the only weak allele identified so far with a mutation in the activation loop, showed reduced autophosphorylation activity., a subtle allele with a mutation in the extracellular portion, disrupts the interaction of BRI1 with its ligand brassinolide and coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1. , with a mutation in the extracellular portion, is a subtle defective mutant. Surprisingly, root inhibition analysis indicated that it is largely insensitive to exogenous brassinolide treatment. In this study, we found that possesses kinase activity in vivo, clarifying a previous report arguing that kinase activity may not be necessary for the function of BRI1. These data provide additional insights into our understanding of the early events in the brassinosteroid signaling pathway.

A novel mutant allele uncouples brassinosteroid-dependent and independent functions of BRI1

“…In contrast, bri1 cnu1 was much less responsive to exogenous BR and did not reach growth depression with the concentrations tested here (up to 10 nM) (Fig. 2E), as reported for other bri1 hypomorphic alleles of similar strength (Sun et al , 2017). Consistent with the mild growth phenotype, transformation with constructs encoding the two BRI1 mutant versions alone or a combination of both mutations rescued hypomorphic bri1-301 and bri1-null mutants (Fig 3A, B).…”

“…These analyses revealed that BR signalling dependent BRI1 functions are only mildly affected in bri1 cnu4 , whereas we observed a strong negative effect on RLP44 function in the regulation of vascular cell fate. Interestingly, the same mutation we report here as bri1 cnu4 , G746A (G2236A on nucleic acid level) has been recently described as bri1-711 in a tilling approach to obtain new bri1 mutants (Sun et al , 2017). Consistent with our results, bri1-711 showed subtle growth defects and mild insensitivity to exogenous application of BL.…”

“…Consistent with our results, bri1-711 showed subtle growth defects and mild insensitivity to exogenous application of BL. In addition, the accumulation of non-phosphorylated BES1 as a readout of BR signalling was similar to that of the Col-0 WT in response to BL (Sun et al , 2017). In contrast to our results obtained with bri1 cnu4 , other bri1 hypomorphic mutants such as bri1-301 and bri1-5 have negligible effects on xylem cell fate in the root, despite their pronounced effect on BR signalling (Holzwart et al , 2018).…”

Brassinosteroids: Multidimensional Regulators of Plant Growth, Development, and Stress Responses

“…Thus, it was proposed that the substitution of Ile-642 (in BRL1) or Met-657 (in BRI1) to Glu-614 (in BRL2) might interfere with the BL binding by changing the hydrophobicity of this region (She et al, 2013). Several bri1 mutants with point mutations in the island domain-LRR interface have been identified (Li and Chory, 1997;Noguchi et al, 1999;Sun et al, 2017). It remains to be demonstrated if BRI1 mutants carrying these molecular lesions are deficient in BR binding, which would further confirm the importance of this region.…”

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

“…Characterization of a mutational spectrum for a specific protein can help to elucidate its detailed cellular functions. To date, over 30 bri1 point mutants with mutant phenotypes have been identified using the approach of genetics, including bri1-5 (Noguchi et al, 1999;Sun C. et al, 2017). In addition, diverse phosphorylation sites in BRI1 were also identified; site-directed mutagenesis followed by in vitro kinase assays of BRI1 kinase domain (KD) and genetic complementation of bri1-5 revealed multiple phosphorylation sites essential for BR signaling in planta (Wang et al, 2005).…”