Abstract
We previously demonstrated that salicylic acid-binding protein 2 (SABP2) of tobacco is an integral component of systemic acquired resistance (SAR). SABP2 is a methyl salicylate (MeSA) esterase that has high affinity for SA, which feedback inhibits its esterase activity. MeSA esterase activity is required in distal, healthy tissue of pathogen-infected plants to hydrolyze MeSA, which functions as a long-distance, phloem-mobile SAR signal; this hydrolysis releases the biologically active defense hormone SA. In this study, we examined the inhibitory interaction of SA with SABP2, and identified a synthetic SA analog, 2,2,2,2'-tetra-f luoroacetophenone (tetraFA) that, like SA, competitively inhibits the activity of SABP2 and targets esterases, which utilize MeSA as a substrate. However, in contrast to SA, tetraFA does not induce downstream defense responses and, therefore, is effective in planta at blocking SAR development in tobacco mosaic virus (TMV)-infected tobacco and Pseudomonas syringae-infected Arabidopsis. These results confirm the importance of SABP2 and MeSA for SAR development in tobacco and establish similar roles for MeSA and the orthologs of SABP2 in Arabidopsis. Moreover, they demonstrate that tetraFA can be used to determine whether MeSA and its corresponding esterase(s) play a role in SAR signaling in other plant species. In planta analyses using tetraFA, in conjunction with leaf detachment assays and MeSA quantification, were used to assess the kinetics with which MeSA is generated in pathogen-infected leaves, transmitted through the phloem, and processed in the distal healthy leaves. In TMV-infected tobacco, these studies revealed that critical amounts of MeSA are generated, transmitted, and processed between 48 and 72 h post primary infection.
Highlights
A number of studies have provided important insights into the immune response occurring in infected plant cells (4 – 6)
salicylic acid (SA) was shown to be an endogenous inhibitor of the methyl salicylate (MeSA) esterase salicylic acid-binding protein 2 (SABP2); SA binds in the active-site pocket of SABP2 (Kd ϭ 90 nM), which results in the inhibition of the catalytic activity of SABP2 [15, 19]
The MeSA esterase activity of SABP2 in the infected leaves is predicted to be completely inhibited by 72 hp1°i, based on a calculation performed by integrating the kinetic parameters with the level kinetics of MeSA and SA, where vi ϭ [S]1⁄7Vmax/{((Km(1 ϩ [I]/Ki)) ϩ [S]}, v0 ϭ [S]1⁄7Vmax/(Km ϩ [S]), i% ϭ 100 (1 Ϫ (vi/v0))
Summary
A number of studies have demonstrated that SA plays a critical role in the resistance signaling pathway(s) [1, 7]. Confirmation of SA as a critical resistance signal came from analyses of transgenic tobacco and Arabidopsis expressing the bacterial nahG gene, which encodes the SA-degrading enzyme salicylate hydroxylase. These plants failed to accumulate SA after pathogen infection, displayed reduced resistance to avirulent and virulent pathogens, and did not develop SAR or express pathogenesis-related genes in their distal leaves (8 –10). To determine whether an SABP2-like esterase activity is required for SAR in general, we identified and characterized several synthetic SA analogs that inhibit the MeSA esterase activity of SABP2 This approach was chosen as it circumvented purifying the enzyme, cloning its gene, and/or silencing its expression. TetraFA was used to determine the time during which the MeSA signal moves to the distal tissue after the 1° infection
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