The tomato cell death suppressor Adi3 is restricted to the endosomal system in response to the Pseudomonas syringae effector protein AvrPto.

María J Ek-Ramos,Julian Avila,Joel W Gray,Dongyin Su,Timothy P Devarenne,Anna C Nelson Dittrich,Shao J Dai

doi:10.1371/journal.pone.0110807

Abstract

The tomato (Solanum lycopersicum) AGC protein kinase Adi3 functions as a suppressor of cell death and was first identified as an interactor with the tomato resistance protein Pto and the Pseudomonas syringae effector protein AvrPto. Models predict that loss of Adi3 cell death suppression (CDS) activity during Pto/AvrPto interaction leads to the cell death associated with the resistance response initiated from this interaction. Nuclear localization is required for Adi3 CDS. Prevention of nuclear accumulation eliminates Adi3 CDS and induces cell death by localizing Adi3 to intracellular punctate membrane structures. Here we use several markers of the endomembrane system to show that the punctate membrane structures to which non-nuclear Adi3 is localized are endosomal in nature. Wild-type Adi3 also localizes in these punctate endosomal structures. This was confirmed by the use of endosomal trafficking inhibitors, which were capable of trapping wild-type Adi3 in endosomal-like structures similar to the non-nuclear Adi3. This suggests Adi3 may traffic through the cell using the endomembrane system. Additionally, Adi3 was no longer found in the nucleus but was visualized in these punctate endosomal-like membranes during the cell death induced by the Pto/AvrPto interaction. Therefore we propose that inhibiting nuclear import and constraining Adi3 to the endosomal system in response to AvrPto is a mechanism to initiate the cell death associated with resistance.

Highlights

Programmed cell death (PCD) is part of the hypersensitive response (HR) that occurs during the resistance response of plants to pathogens [1,2]
Our previous studies have established that GFP-Adi3 cellular localization is identical in both intact leaf mesophyll cells and isolated mesophyll protoplast cells, and that protoplasts offer a better context for Adi3 cellular localization studies [8]. These studies utilized confocal microscopy and subcellular fractionation with GFP-tagged Adi3 to show that wild-type Adi3 is localized in the nucleus (Figure 1A) as well as insoluble cellular fractions, while loss of Adi3 nuclear localization by deletion of the nuclear localization signal (NLS) or the Tloop extension (Adi3DT-loop) localizes Adi3 to many punctate intracellular membranous cellular structures (Figure 1A) [8]
Since endosomal Adi3 induces cell death due to a loss of cell death suppression (CDS) [8], the Pto/AvrPto interaction leads to HR cell death [3], and Adi3 was originally isolated based on interaction with Pto and AvrPto [6], we examined if Adi3 is endosomally localized during the HR cell death induced by AvrPto

Summary

Introduction

Programmed cell death (PCD) is part of the hypersensitive response (HR) that occurs during the resistance response of plants to pathogens [1,2]. Mutation of the NLS or deletion of the Tloop extension eliminates Adi nuclear localization and confines Adi to many intracellular punctate membranous structures, which causes cell death due to a loss of Adi CDS activity [8]. It is hypothesized that during pathogen challenge the interaction of Adi with Pto/AvrPto leads to Adi inactivation, a loss of Adi CDS, and subsequently the HR-associated PCD [7,8,9]. This raises the possibility that during Pto-mediated resistance, Adi is confined to these punctate membranous structures in order to bring about the HR PCD [8]

Methods

Results

Conclusion