title: The β-subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor Adi3.
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.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
authors.forename: Julian
authors.initials: J
authors.lastname: Avila
authors.forename: Oliver G
authors.initials: OG
authors.lastname: Gregory
authors.forename: Dongyin
authors.initials: D
authors.lastname: Su
authors.forename: Taunya A
authors.initials: TA
authors.lastname: Deeter
authors.forename: Sixue
authors.initials: S
authors.lastname: Chen
authors.forename: Cecilia
authors.initials: C
authors.lastname: Silva-Sanchez
authors.forename: Shouling
authors.initials: S
authors.lastname: Xu
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Martin
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Devarenne
mesh_terms: D016923:Cell Death; D005690:Galactose / Q000378:metabolism; D018551:Solanum lycopersicum / Q000166:cytology* / Q000201:enzymology*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D059828:Plant Cells / Q000201:enzymology*; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D021122:Protein Subunits; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D005690:Galactose