Overexpression of RsRBA1 encoding a TIR-X isolated from Raphanus sativus exhibits temperature-insensitive growth inhibition in transgenic Arabidopsis thaliana

Abstract

Plant Toll/interleukin-1 receptor X (TX) proteins, defined by an N-terminal Toll/interleukin-1 receptor (TIR) domain and an uncharacterized C-terminal region, function in plant immunity by producing secondary signaling molecules that activate hypersensitive responses through nicotinamide adenine dinucleotide (NAD+) hydrolase (NADase) activity. In previous work, we characterized Arabidopsis thaliana TX12 (AtTX12) and demonstrated that its growth-inhibitory activity is insensitive to temperature and dependent on conserved amino acid residues likely required for NADase function and putative multimerization interfaces. To further investigate TX-mediated growth inhibition, we cloned and overexpressed two AtTX12-like genes from Brassicaceae species, Raphanus sativus RBA1 (RsRBA1) and Brassica oleracea TX12 (BoTX12), in transgenic Arabidopsis seedlings. Reverse transcription-polymerase chain reaction analysis showed that RsRBA1 is weakly expressed in flowers and inflorescences, whereas BoTX12 exhibits weak expression in leaves. Overexpression of RsRBA1 caused moderate growth inhibition accompanied by increased expression of defense-related genes, while BoTX12 did not induce growth inhibition or defense gene activation. This apparent loss of function in BoTX12 is likely due to the absence of three amino acid residues within the second α-helix (αB) adjacent to the BB-loop, positioned between the second β-sheet and α B. Similar to AtTX12, RsRBA1 retained activity at elevated temperatures. Together, these findings expand the known repertoire of plant defense-associated genes and highlight a potential strategy for modulating TIR activity to balance plant growth and immune responses.