Subcellular localization of tobacco MAPKs

Plants effect intracellular changes in response to a variety of external stimuli. Harpin, a bacterial elicitor from the bean halo-blight pathogen Pseudomonas syringae pv phaseolicola, and megaspermin, an oocyte elicitor secreted by Phythophtora megasperma H20, are both known to elicit a hypersensitive response (HR) iii tobacco. These elicitors also induce phosphorylation of a 48 kDa protein and a 46 kDa protein that have been identified as the Salicylic A c i d Induced Protein Kinase (SIPK), and Wound Induced Protein Kinase (WIPK) mitogen activated protein kinases (MAPKs) respectively. Previous work has shown that transient over-expression of SIPK causes lesion formation in tobacco leaves, suggesting a possible role for SIPK in regulating tobacco HR signaling. Extracellular Regulated Kinase (ERK), a mammalian homologue to SIPK and WIPK, has been shown to localize to different parts of the cell in response to a variety of stresses. The objective of this study was to determine the subcellular localization of the tobacco MAPKs, SIPK and WIPK, in response to challenge by harpin and megaspermin. Using both SIPK-GFP proteins and in situ immunolabeling of SIPK and WIPK, it was determined that the tobacco MAPKs were localized predominantly in the cytoplasm, with some nuclear localization. Upon challenge with harpin and/or megaspermin the phosphorylated forms of the probed MAPKs showed strong nuclear localization. In elicitor induced tobacco cultures, immunolabeling results showed strong nucleolar localization within the nucleus. These results are consistent with the model that activation of ERK-homologues by intracellular effectors leads to translocation of the activated MAPK from the cytoplasm to the nucleus, most likely to effect transcriptional regulation of stress-related genes.Forestry, Faculty ofGraduat