Using DIR1 to investigate long-distance signal movement during Systemic Acquired Resistance

During Systemic Acquired Resistance (SAR), a SAR-inducing infection in one leaf initiates movement of phloem-mobile signals to uninfected distant leaves to prime plants to respond in a resistant manner to subsequent infections. Our early work with the dir1-1 (defective in induced resistance) mutant in Arabidopsis demonstrated that the DIR1 protein is required for SAR and led to the hypothesis that DIR1, a lipid transfer protein (LTP), moves to distant leaves to activate SAR. To prove this hypothesis, we monitored DIR1-GFP accumulation in phloem exudates using an estrogen-SAR assay. In this assay, estrogen treatment induces DIR1-GFP expression in one leaf of dir1-1, followed by SAR-induction in the same leaf. DIR1-GFP was detected in exudates collected from local and distant leaves of SAR-induced plants using both DIR1 and GFP antibodies. This provides compelling evidence that DIR1 moves via the phloem to distant leaves to initiate priming. Our work fills a major gap in research on SAR as no other putative SAR mobile signal has been shown to move in planta to distant leaves. To discover how DIR1 enters the phloem, we took advantage of plant lines with compromised cell-to-cell movement caused by overexpression of Plasmodesmata-Located Proteins. These lines were defective for SAR, and DIR1 was not observed in distant leaf phloem exudates, supporting the idea that cell-to-cell movement of DIR1 through plasmodesmata is important for SAR signal movement. To discover new phloem proteins that play a role during SAR, we compared phloem exudate proteomes collected from mock- and SAR-induced leaves using quantitative LC-MS/MS. Numerous proteins were enriched in SAR-induced versus mock-induced phloem exudates and T-DNA knock-out lines in some of these genes were SAR-defective, indicating they contribute to SAR. Identification of SAR-specific phloem proteins may provide clues as to the protein complement of a high molecular weight DIR1-containing complex found in phloem exudates only after SAR induction. We will take advantage of DIR1's proteinaceous nature to identify proteins in the high molecular weight mobile signal complex, proteins associated with phloem loading of SAR signals and proteins involved in DIR1 perception in distant leaves.