Abstract
Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens.
Highlights
Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system
Some of the spore suspension– derived compounds were tentatively annotated using an in-house analyte list as metabolites from the primary metabolism (sugars, phosphosugars, organic acids, amino acids, polyamines, purine derivatives and degradation products, and pipecolic acid (Table S1)). They could originate from the germinating spores, and from the medium used for P. infestans cultivation
Secretion of metabolites into the inoculum of P. infestans was first described by Böttcher et al [16], and this subsequently enabled the identification of a MATE transporter required for export of phenylpropanoid defense compounds during the nonhost resistance response of Arabidopsis against P. infestans [17]
Summary
A substrate of the ABC transporter PEN3 stimulates bacterial flagellin (flg22)-induced callose deposition in Arabidopsis thaliana. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-ylmethyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. We have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens. In addition to constitutive barriers, induced defense mechanisms contribute to nonhost resistance, which are activated in the plant upon recognition of pathogen-associated molecular patterns (PAMPs)3 [3]. Confidence level of annotation (according to Ref. 51) is as follows: 1, verified with mass, retention time, and CID spectrum of a commercial standard or a synthesized standard (*); 2, putatively annotated compounds based on CID spectrum interpretation, data base hits, and literature
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