Arabidopsis Tubby domain‐containing F‐box proteins positively regulate immunity by modulating PI4Kβ protein levels

Abstract

Summary The Tubby domain, named after the TUBBY protein in mice, binds to phosphatidylinositol 4,5‐bisphosphate. Arabidopsis has 11 Tubby domain‐containing proteins referred to as Tubby‐Like Proteins (TLPs). Of the 11 TLPs, 10 possess the N‐terminal F‐box domain, which can interact with SKP‐like proteins and form SKP1‐Cullin‐F‐box E3 ligase complexes. Although mice TUBBY has been extensively studied, plant TLPs' functions are scarcely detailed. In this study, we show that the Arabidopsis Tubby‐like protein 6 (TLP6) and its redundant homologs, TLP1, TLP2, TLP5, and TLP10, positively regulate Arabidopsis immune responses. Furthermore, in an immunoprecipitation mass spectrometry analysis to search for ubiquitination substrates of the TLPs, we identified two redundant phosphoinositide biosynthesis enzymes, phosphatidylinositol 4‐kinase β proteins (PI4Kβs), PI4Kβ1 and PI4Kβ2, as TLP interactors. Importantly, TLP6 overexpression lines fully phenocopy the phenotypes of the pi4kβ1,2 mutant, while TLP6 overexpression also leads to increased PI4Kβ2 ubiquitination and reduction in its protein level in a proteasome‐dependent manner. Most significantly, TLP6 overexpression does not further enhance the autoimmunity of the pi4kβ1,2 double mutant, supporting the hypothesis that TLP6 targets the PI4Kβs for ubiquitination and degradation. Thus, our study reveals a novel mechanism where TLPs promote plant immune responses by modulating the PI4Kβs protein levels.

Deletions within intronic T‐DNA lead to reversion of T‐DNA mutant phenotypes

“…Besides its autoimmunity-related dwarfism, and enhanced resistance to oomycete and bacterial pathogens (Antignani et al, 2015;Preuss et al, 2006;Sa sek et al, 2014), pi4kb1,2 plants also have short roots ( Sa sek et al, 2014). In our previous work, we identified PI4Kb1 as a substrate for TLP-mediated ubiquitination and protein degradation (Thulasi Devendrakumar et al, 2023). However, it is unclear why pi4kb1,2 is autoimmune.…”

“…However, due to the short read length of the Illumina reads and the presence of multiple copies of the LB to RB region, it was not possible to resolve the exact architecture of the T-DNA insertions in the PI4Kbs. Ulker et al, 2008). Compared to other organisms, Arabidopsis introns are relatively small.…”