Localization of DIR1 at the tissue, cellular and subcellular levels during Systemic Acquired Resistance in Arabidopsis using DIR1:GUS and DIR1:EGFP reporters

DIR1 promoter:DIR1-GUS/dir1-1 lines were constructed to examine DIR1 expression. DIR1 is expressed in seedlings, flowers and ubiquitously in untreated or mock-inoculated mature leaf cells, including phloem sieve elements and companion cells. Inoculation of leaves with SAR-inducing avirulent or virulent Pseudomonas syringae pv tomato (Pst) resulted in Type III Secretion System-dependent suppression of DIR1 expression in leaf cells. Transient expression of fluorescent fusion proteins in tobacco and intercellular washing fluid experiments indicated that DIR1's ER signal sequence targets it for secretion to the cell wall. However, DIR1 expressed without a signal sequence rescued the dir1-1 SAR defect, suggesting that a cytosolic pool of DIR1 is important for the SAR response.Although expression of DIR1 decreases during SAR induction, the protein localizes to all living cell types of the vasculature, including companion cells and sieve elements, and therefore DIR1 is well situated to participate in long-distance signaling during SAR.Acquired resistance, or "immunization" of plants was originally documented more than seventy years ago in a review published by Kenneth Chester in which varying degrees of immunity were observed in plants that had recovered from an initial pathogen attack [1]. The term systemic acquired resistance (SAR) was originally used by Ross to describe systemic resistance induced by necrosis-causing viruses in tobacco [2] and is more generally defined as a defense mechanism induced by a localized infection that results in broad-spectrum resistance in distant tissues to normally virulent pathogens [3,4].Research using tobacco, cucumber and, more recently, Arabidopsis models indicates that SAR occurs in distinct stages. The first, or induction, stage is initiated when a necrosis-causing pathogen infects a leaf and results in either the formation of a localized hypersensitive response (HR) and local resistance, or in disease-induced necrosis [3]. A recent