Functional analysis of AtTX11/12 TIR-domain proteins identifies key residues for basal and temperature-insensitive growth inhibition

Abstract

Plant Toll/interleukin-1 receptor (TIR) domains function as NADases and ribosyl-transferases generating second messengers that trigger hypersensitive responses. TIR-X (TX) proteins contain a TIR domain with or without various C-terminal domains and lack the canonical nucleotide-binding site and leucine-rich repeat domain. In a previous study, we identified an Arabidopsis thaliana activation-tagging line with severe growth defects caused by the overexpression of the AtTX12 gene. Here, we investigated the domains and specific amino acid residues required for the growth inhibition activity of AtTX12 and its homolog AtTX11. C-terminal truncation analysis revealed that the AtTX12C173Δ mutant, lacking 30 C-terminal amino acids, retained partial activity, whereas the C163Δ, lacking 40 amino acids, lost activity entirely indicating that the fifth α-helix within the TIR domain is critical for activity, while the sixth α-helix in the extra domain is dispensable. The substitution mutagenesis revealed that residues essential for enzymatic activities (E79 for NADase, C76 for 2′,3′-cAMP/cGMP synthetase), self-association (H25, E43, K142/G144, K150), and undefined roles (I97) were crucial for growth inhibition activity with varying effects. Temperature sensitivity tests revealed that the AtTX12 N36D mutant, which exhibited moderately strong growth inhibition activity at normal temperatures, became inactive under high-temperature conditions in which Enhanced Disease Susceptibility 1 (EDS1) is almost non-functional. In contrast, wild-type AtTX12 retained activity under elevated temperatures, implicating N36 in maintaining temperature-insensitive functionality. Furthermore, a slightly reduced growth inhibition phenotype induced by AtTX12 overexpression in the eds1 mutant was consistently observed under both normal and high temperatures. These results suggest that AtTX12-mediated growth inhibition integrates EDS1-dependent (temperature-sensitive) and EDS1-independent (temperature-insensitive) pathways. Our findings suggest that attenuated AtTX11/12 mutants could be used to optimize the growth-defense trade-off, enhancing plant defense with minimal growth penalties.