DUF581-9 (At2g44670; FLZ3) negatively regulates SnRK1 activity by interference with T-loop phosphorylation

Abstract

In plants, SUCROSE NON-FERMENTING RELATED KINASE 1 (SnRK1) is a key energy-sensor that orchestrates large-scale transcriptional reprogramming to maintain cellular homeostasis under energy deficit. SnRK1 activity is under tight negative control, although the exact mechanisms leading to its activation are far from being understood. We show that the Arabidopsis DOMAIN OF UNKNOWN FUNCTION (DUF581) protein DUF581-9/FLZ3 binds to the catalytic SnRK1α subunit KIN10 to inhibit its activation by GRIK-dependent T-loop phosphorylation. Overexpression of DUF581-9 in Arabidopsis dampens SnRK1 signalling and interferes with adaptation to dark-induced starvation. The presence of DUF581-9 significantly reduced SnRK1 activity in protoplasts and in vitro. This was accompanied by a reduction in T175 T-loop phosphorylation and also diminished KIN10 auto-phosphorylation. Furthermore, DUF581-9 reduced binding of the up-stream activating kinase GRIK2 to KIN10, explaining the reduced KIN10 T-loop phosphorylation. Ectopically expressed DUF581-9 protein was rapidly turned over by the proteasome when Arabidopsis plants were subjected to starvation treatment, likely releasing its inhibitory activity on the SnRK1 complex. Taken together, our results support a model in which DUF581-9 negatively regulates SnRK1 activity under energy sufficient conditions. Turnover over of the protein provides a rapid way for SnRK1 activation under energy deficit without the need of de-novo protein synthesis.

Direct and indirect responses of the Arabidopsis transcriptome to an induced increase in trehalose 6-phosphate

“…FLZ family proteins (Jamsheer et al, 2015) are negative regulators of SnRK1 and are implicated in interactions between SnRK1 and TOR (Nietsch et al, 2014; 2016; Jamsheer and Lamxi, 2015; Jamsheer et al, 2015; 2018a; 2018b; 2022; Bortlik et al, 2022). Comparison of the iTPS response with the CRF and the tSnRK1α1 response added to the evidence that expression of the FLZ family is highly regulated by C status.…”

“…As discussed above, induction of TPS8, TPS9 , TPS10 and TPS11 under C starvation may be due to falling Tre6P levels and might derepress SnRK1. The FLZ family proteins are also emerging as important modulators of SnRK1 signaling (Nietsch et al, 2014; 2016; Jamsheer and Lamxi, 2015; Jamsheer et al, 2015; 2018a; 2018b; 2022; Bortlik et al, 2022). Our analysis of their CRFs adds to the evidence that FLZ expression is strongly regulated by C status.…”

XopM, a FFAT motif containing type-III effector protein fromXanthomonas, suppresses PTI responses at the plant plasma membrane

“…Pull-down followed by LC-MS/MS analysis was basically carried out as previously described (Bortlik et al, 2023). In short, on bead trypsin digest of GFP-tagged proteins were treated previously with 100 µl of 0.1 % RapiGest SF (Waters, Eschborn) followed by reduction and alkylation.…”

“…Subsequently, desalting of peptides was carried out according to Witzel et al (2019). Protein digest were analysed using the Thermo Fisher Q Exactive high field mass spectrometer by reverse-phase HPLC-ESI-MS/MS using the Dionex UltiMate 3000 RSLC nano System coupled to the Q Exactive High Field (HF) Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific) and a Nano-electrospray Flex ion source (Thermo Fisher Scientific) as described by Bortlik et al (2023) with slight modifications. In short, samples were separated on a 25 cm EASY-Spray column (75 µm inner diameter) at 40 °C.…”