Minisymposium 4: Signaling

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Abs # M0403: Negative Regulation of Plant Cell Death by the ACG protein kinases Adi3 and PDK1

Presenter:	Devarenne, Timothy P       Contact Presenter
Authors	Devarenne, Timothy P (A)   Gregory, Oliver G (A)   Martin, Gregory B (A) (B)
Affiliations:	(A): Boyce Thompson Inst for Plant Research (B): Plant Pathology Department, Cornell University

The protein kinase PDK1 is an upstream regulator of AGC protein kinase family members which function in many basic cellular process such as transcription, translation, cell growth, and programmed cell death (PCD). The tomato AGC kinase Adi3 was identified in a yeast three-hybrid screen as interacting with the tomato resistance protein Pto and the Pseudomonas syringae effector protein AvrPto. We previously identified Adi3 as a PDK1-interacting negative regulator of cell death. Here we strengthen the PDK1/Adi3 connection and identify Adi3 interactors that may be involved in PCD regulation. Mutation of S539 in the Adi3 activation loop to Asp, a phosphorylation mimic, produces a constitutively active Adi3 with drastically increased kinase activity, indicating that phosphorylation of S539 is required for full Adi3 activity. PDK1 radiolabeled phosphorylation of Adi3 and Adi3^S539A followed by phosphopeptide mapping shows loss of one radioactive peptide in the Adi3^S539A sample. Mass spectrometry analysis confirms that S539 of Adi3 is at least one residue phosphorylated by PDK1. Additionally, inhibition of PDK1 using the PDK1 specific inhibitor OSU-03012 can induce cell death in tomato protoplasts. Overexpression of the constitutively active Adi3^S539D in tomato protoplasts is capable of limiting OSU-03012 induced cell death. Taken together this data indicates PDK1 phosphorylates and activates Adi3 which then acts to suppress cell death. Adi3 was further confirmed as a negative regulator of cell death by showing Adi3 transient overexpression in leaf tissue could inhibit cell death caused by Pto^Y207D, a constitutively active form of the Pto resistance protein that produces HR-like cell death. In order to identify Adi3 substrates that may function in PCD regulation, a yeast two-hybrid (Y2H) screen was carried out revealing the proteins Atg8 and a RING finger E3 ubiquitin ligase as Adi3 interactors. These proteins are involved in autophagy (Atg8) and ubiquitination (RING E3 ligase), two degradation processes known to function in PCD regulation in response to pathogens. Atg8 functions as a scaffold protein in the formation of autophagic vesicles and is part of a 9 member gene family (genes a - i). Adi3 interacts with Atg8h. The Arabidopsis homolog of the E3 ligase is the uncharacterized gene At3g05545 which belongs to the H2 group of RING E3 ligases. These results suggest Adi3 may regulate cell death through the control of cellular degradation mechanisms.