DIR1 is a lipid transfer protein (LTP) postulated to complex with and/or chaperone a signal(s) to distant leaves during Systemic Acquired Resistance (SAR) in Arabidopsis. DIR1 was detected in phloem sap-enriched petiole exudates collected from wild-type leaves induced for SAR, suggesting that DIR1 gains access to the phloem for movement from the induced leaf. Occasionally the defective in induced resistance1 (dir1-1) mutant displayed a partially SAR-competent phenotype and a DIR1-sized band in protein gel blots was detected in dir1-1 exudates suggesting that a highly similar protein, DIR1-like (At5g48490), may contribute to SAR. Recombinant protein studies demonstrated that DIR1 polyclonal antibodies recognize DIR1 and DIR1-like. Homology modeling of DIR1-like using the DIR1-phospholipid crystal structure as template, provides clues as to why the dir1-1 mutant is rarely SAR-competent. The contribution of DIR1 and DIR1-like during SAR was examined using an Agrobacterium-mediated transient expression-SAR assay and an estrogen-inducible DIR1-EGFP/dir1-1 line. We provide evidence that upon SAR induction, DIR1 moves down the leaf petiole to distant leaves. Our data also suggests that DIR1-like displays a reduced capacity to move to distant leaves during SAR and this may explain why dir1-1 is occasionally SAR-competent.

Systemic acquired resistance (SAR) is a plant defence response that provides immunity to distant uninfected leaves after an initial localised infection. The lipid transfer protein (LTP) Defective in Induced Resistance1 (DIR1) is an essential component of SAR that moves from induced to distant leaves following a SAR-inducing local infection. To understand how DIR1 is transported to distant leaves during SAR, we analysed DIR1 movement in transgenic Arabidopsis lines with reduced cell-to-cell movement caused by the overexpression of Plasmodesmata-Located Proteins PDLP1 and PDLP5. These PDLP-overexpressing lines were defective for SAR, and DIR1 antibody signals were not observed in phloem sap-enriched petiole exudates collected from distant leaves. Our data support the idea that cell-to-cell movement of DIR1 through plasmodesmata is important during long-distance SAR signalling in Arabidopsis.

Age-related resistance (ARR) is a plant defense response characterized by enhanced resistance to certain pathogens in mature plants relative to young plants. In Arabidopsis thaliana the transition to flowering is associated with ARR competence, suggesting that this developmental event is the switch that initiates ARR competence in mature plants (Rusterucci et al. in Physiol Mol Plant Pathol 66:222–231, 2005). The association of ARR and the floral transition was examined using flowering-time mutants and photoperiod-induced flowering to separate flowering from other developmental events that occur as plants age. Under short-day conditions, late-flowering plant lines ld-1 (luminidependens-1), soc1-2 (suppressor of overexpression of co 1-2), and FRI+ (FRIGIDA) displayed ARR before the transition to flowering occurred. Early-flowering svp-31, svp-32 (short vegetative phase), and Ws-2 were ARR-defective, whereas early-flowering tfl1-14 (terminal flower 1-14) displayed ARR at the same time as Col-0. While svp-31, svp-32 and Ws-2 produced few rosette leaves, tfl1-14 produced a rosette leaf number similar to Col-0, suggesting that the development of a minimum number of rosette leaves is necessary to initiate ARR competence under short-day conditions. Photoperiod-induced transient expression of FT (FLOWERING LOCUS T) caused precocious flowering in short-day-grown Col-0 but this was not associated with ARR competence. Under long-day conditions co-9 (constans-9) mutants did not flower but displayed an ARR response at the same time as Col-0. This study suggests that SVP is required for the ARR response and that the floral transition is not the developmental event that regulates ARR competence.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-013-0083-7) contains supplementary material, which is available to authorized users.

AtDIR1 (Defective in Induced Resistance1) is an acidic lipid transfer protein essential for systemic acquired resistance (SAR) in Arabidopsis thaliana. Upon SAR induction, DIR1 moves from locally infected to distant uninfected leaves to activate defense priming; however, a molecular function for DIR1 has not been elucidated. Bioinformatic analysis and in silico homology modeling identified putative AtDIR1 orthologs in crop species, revealing conserved protein motifs within and outside of DIR1’s central hydrophobic cavity. In vitro assays to compare the capacity of recombinant AtDIR1 and targeted AtDIR1-variant proteins to bind the lipophilic probe TNS (6,P-toluidinylnaphthalene-2-sulfonate) provided evidence that conserved leucine 43 and aspartic acid 39 contribute to the size of the DIR1 hydrophobic cavity and possibly hydrophobic ligand binding. An Arabidopsis–cucumber SAR model was developed to investigate the conservation of DIR1 function in cucumber (Cucumis sativus), and we demonstrated that phloem exudates from SAR-induced cucumber rescued the SAR defect in the Arabidopsis dir1-1 mutant. Additionally, an AtDIR1 antibody detected a protein of the same size as AtDIR1 in SAR-induced cucumber phloem exudates, providing evidence that DIR1 function during SAR is conserved in Arabidopsis and cucumber. In vitro TNS displacement assays demonstrated that recombinant AtDIR1 did not bind the SAR signals azelaic acid (AzA), glycerol-3-phosphate or pipecolic acid. However, recombinant CsDIR1 and CsDIR2 interacted weakly with AzA and pipecolic acid. Bioinformatic and functional analyses using the Arabidopsis–cucumber SAR model provide evidence that DIR1 orthologs exist in tobacco, tomato, cucumber, and soybean, and that DIR1-mediated SAR signaling is conserved in Arabidopsis and cucumber.

A whole-genome sequencing technique developed to identify fast neutron-induced deletion mutations revealed that iap1-1 is a new allele of EDS5 (eds5-5). RPS2-AvrRpt2-initiated effector-triggered immunity (ETI) was compromised in iap1-1/eds5-5 with respect to in planta bacterial levels and the hypersensitive response, while intra- and intercellular free salicylic acid (SA) accumulation was greatly reduced, suggesting that SA contributes as both an intracellular signaling molecule and an antimicrobial agent in the intercellular space during ETI. During the compatible interaction between wild-type Col-0 and virulent Pseudomonas syringae pv. tomato (Pst), little intercellular free SA accumulated, which led to the hypothesis that Pst suppresses intercellular SA accumulation. When Col-0 was inoculated with a coronatine-deficient strain of Pst, high levels of intercellular SA accumulation were observed, suggesting that Pst suppresses intercellular SA accumulation using its phytotoxin coronatine. This work suggests that accumulation of SA in the intercellular space is an important component of basal/PAMP-triggered immunity as well as ETI to pathogens that colonize the intercellular space.