The β-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3

Julian Avila,Shouling Xu,Taunya A Deeter,Sixue Chen,Oliver G Gregory,Cecilia Silva-Sanchez,Dongyin Su,Timothy P Devarenne,Gregory B Martin

doi:10.1104/pp.112.198432

Abstract

The protein kinase AvrPto-dependent Pto-interacting protein3 (Adi3) is a known suppressor of cell death, and loss of its function has been correlated with cell death induction during the tomato (Solanum lycopersicum) resistance response to its pathogen Pseudomonas syringae pv tomato. However, Adi3 downstream interactors that may play a role in cell death regulation have not been identified. We used a yeast two-hybrid screen to identify the plant SnRK1 (for Sucrose non-Fermenting-1-Related Protein Kinase1) protein as an Adi3-interacting protein. SnRK1 functions as a regulator of carbon metabolism and responses to biotic and abiotic stresses. SnRK1 exists in a heterotrimeric complex with a catalytic α-subunit (SnRK1), a substrate-interacting β-subunit, and a regulatory γ-subunit. Here, we show that Adi3 interacts with, but does not phosphorylate, the SnRK1 α-subunit. The ability of Adi3 to phosphorylate the four identified tomato β-subunits was also examined, and it was found that only the Galactose Metabolism83 (Gal83) β-subunit was phosphorylated by Adi3. This phosphorylation site on Gal83 was identified as serine-26 using a mutational approach and mass spectrometry. In vivo expression of Gal83 indicates that it contains multiple phosphorylation sites, one of which is serine-26. An active SnRK1 complex containing Gal83 as the β-subunit and sucrose nonfermenting4 as the γ-subunit was constructed to examine functional aspects of the Adi3 interaction with SnRK1 and Gal83. These assays revealed that Adi3 is capable of suppressing the kinase activity of the SnRK1 complex through Gal83 phosphorylation plus the interaction with SnRK1 and suggested that this function may be related to the cell death suppression activity of Adi3.

Highlights

The protein kinase AvrPto-dependent Pto-interacting protein3 (Adi3) is a known suppressor of cell death, and loss of its function has been correlated with cell death induction during the tomato (Solanum lycopersicum) resistance response to its pathogen Pseudomonas syringae pv tomato
In an effort to identify Adi3-interacting proteins, we carried out a Y2H screen using a cDNA prey library that has been used previously to identify proteins that interact with the tomato resistance protein kinase Pto (Zhou et al, 1995)
The results show that SlGal83S26D conferred a slight yet statistically significant decrease in SlSnRK1 SAMS phosphorylation (Fig. 5B, column 6), whereas the addition of Adi3S539D to the assay drastically lowered the phosphorylation of SAMS to a level close to that of SlSnRK1 alone (Fig. 5B, column 7)

Summary

Introduction

The protein kinase AvrPto-dependent Pto-interacting protein (Adi3) is a known suppressor of cell death, and loss of its function has been correlated with cell death induction during the tomato (Solanum lycopersicum) resistance response to its pathogen Pseudomonas syringae pv tomato. An active SnRK1 complex containing Gal as the b-subunit and sucrose nonfermenting as the g-subunit was constructed to examine functional aspects of the Adi interaction with SnRK1 and Gal. One of the plant genes identified to control PCD encodes the AGC Ser/Thr protein kinase AvrPto-dependent Pto-interacting protein (Adi3) from tomato (Solanum lycopersicum), which functions as a cell death suppressor. PKB can prevent cell death when overexpressed in the presence of PCD-inducing conditions (Arico et al, 2002; Kulp et al, 2004; Zhu et al, 2004) These similarities in function, cell localization, and signaling between Adi and PKB suggest that Adi may be the functional homolog of PKB in plants (Devarenne et al, 2006; EkRamos et al, 2010)

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Results

Conclusion