Local Application of Acibenzolar-S-Methyl Treatment Induces Antiviral Responses in Distal Leaves of Arabidopsis thaliana.

Systemic acquired resistance (SAR) is a plant defense mechanism that provides protection against a broad spectrum of pathogens in distal tissues. Recent studies have revealed a concerted function of salicylic acid (SA) and N-hydroxypipecolic acid (NHP) in the establishment of SAR against bacterial pathogens, but it remains unknown whether NHP is also involved in SAR against viruses. We found that the local application of acibenzolar-S-methyl (ASM), a synthetic analog of SA, suppressed plantago asiatica mosaic virus (PlAMV) infection in the distal leaves of Arabidopsis thaliana. This suppression of infection in untreated distal leaves was observed at 1 day, but not at 3 days, after application. ASM application significantly increased the expression of SAR-related genes, including PR1, SID2, and ALD1 after 1 day of application. Viral suppression in distal leaves after local ASM application was not observed in the sid2-2 mutant, which is defective in isochorismate synthase 1 (ICS1), which is involved in salicylic acid synthesis; or in the fmo1 mutant, which is defective in the synthesis of NHP; or in the SA receptor npr1-1 mutant. Finally, we found that the local application of NHP suppressed PlAMV infection in the distal leaves. These results indicate that the local application of ASM induces antiviral SAR against PlAMV through a mechanism involving NHP.

Acibenzolar-S-methyl-mediated restriction of loading of plantago asiatica mosaic virus into vascular tissues of Nicotiana benthamiana.

Long-distance movement via vascular tissues is an essential step for systemic infection by plant viruses. We previously reported that pre-treatment of Nicotiana benthamiana with acibenzolar-S-methyl (ASM) both suppressed the accumulation of plantago asiatica mosaic virus (PlAMV) in inoculated leaves and delayed the long-distance movement to uninoculated upper leaves. These two effects occurred independently of each other. However, it remained unclear where and when the viral long-distance movement is inhibited upon ASM treatment. In this study, we found that ASM treatment restricted the loading of GFP-expressing PlAMV (PlAMV-GFP) into vascular tissues in the inoculated leaves. This led to delays in viral translocation to the petiole and the main stem, and to untreated upper leaves. We used cryohistological fluorescence imaging to show that ASM treatment affected the viral localization and reduced its accumulation in the phloem, xylem, and mesophyll tissues. A stem girdling experiment, which blocked viral movement downward through phloem tissues, demonstrated that ASM treatment could inhibit viral systemic infection to upper leaves, which occurred even with viral downward movement restricted. Taken together, our results showed that ASM treatment affects the loading of PlAMV-GFP into the vascular system in the inoculated leaf, and that this plays a key role in the ASM-mediated delay of viral long-distance movement.

Acibenzolar-S-Methyl Restricts Infection of Nicotiana benthamiana by Plantago Asiatica Mosaic Virus at Two Distinct Stages.

Plant activators, including acibenzolar-S-methyl (ASM), are chemical compounds that stimulate plant defense responses to pathogens. ASM treatment inhibits infection by a variety of plant viruses, however, the mechanisms of this broad-spectrum and strong effect remain poorly understood. We employed green fluorescent protein (GFP)-expressing viruses and Nicotiana benthamiana plants to identify the infection stages that are restricted by ASM. ASM suppressed infection by three viral species, plantago asiatica mosaic virus (PlAMV), potato virus X (PVX), and turnip mosaic virus (TuMV), in inoculated cells. Furthermore, ASM delayed the long-distance movement of PlAMV and PVX, and the cell-to-cell (short range) movement of TuMV. The ASM-mediated delay of long-distance movement of PlAMV was not due to the suppression of viral accumulation in the inoculated leaves, indicating that ASM restricts PlAMV infection in at least two independent steps. We used Arabidopsis thaliana mutants to show that the ASM-mediated restriction of PlAMV infection requires the NPR1 gene but was independent of the dicer-like genes essential for RNA silencing. Furthermore, experiments using protoplasts showed that ASM treatment inhibited PlAMV replication without cell death. Our approach, using GFP-expressing viruses, will be useful for the analysis of mechanisms underlying plant activator-mediated virus restriction.