Metabolite-based Defense Mechanisms

Lipoxygenases (LOX; linoleate:oxygen oxidoreductase EC 1.13.11.12) consist of a class of enzymes that catalyze the regio‐ and stereo specific dioxygenation of polyunsaturated fatty acids. Here we characterize two proteins that belong to the less studied class of 9‐LOXs, Solanum tuberosum StLOX1 and Arabidopsis thaliana AtLOX1. The proteins were recombinantly expressed in E. coli and the product specificity of the enzymes was tested against different fatty acid substrates. Both enzymes showed high specificity against all tested C18 fatty acids and produced (9S )‐hydroperoxides. However, incubation of the C20 fatty acid arachidonic acid with AtLOX1 gave a mixture of racemic hydroperoxides. On the other hand, with StLOX1 we observed the formation of a mixture of products among which the (5S )‐hydroperoxy eicosatetraenoic acid (5S‐ H(P)ETE) was the most abundant. Esterified fatty acids were no substrates. We used site directed mutagenesis to modify a conserved valine residue in the active site of StLOX1 and examine the importance of space within the active site, which has been shown to play a role in determining the positional specificity. The Val576Phe mutant still catalyzed the formation of (9S )‐hydroperoxides with C18 fatty acids, while it exhibited altered specificity against arachidonic acid and produced mainly (11S )‐H(P)ETE. These data confirm the model that in case of linoleate 9‐LOX binding of the substrate takes place with the carboxyl‐group first.

Infections with Phytophthora infestans, the causal agent of potato and tomato late blight disease, are difficult to control and can lead to considerable agricultural losses. Thus, the development of new effective agents against the pathogen is of great interest. In previous work, (E)-4-oxohexadec-2-enoic acid (3) was isolated from Hygrophorus eburneus, which exhibited fungicidal activity against Cladosporium cucumerinum. Here, the inhibitory effect of 3 on P. infestans spore germination and mycelium growth in vitro is demonstrated. The in vivo effect on infections of whole potato plants was investigated by spraying plants with the sodium salt of 3, sodium (2E)-4-oxohexadec-2-enoic acid (4), prior to P. infestans inoculation. Additionally, the influence of 3 on mycelium growth of Colletotrichum coccodes, the causal agent of potato black dot disease, was analyzed. In all approaches, a significant inhibition of pathogen development was achieved. Importantly, the unsaturated fatty acid exerted no toxic effect when sprayed on plants, a prerequisite for its commercial use.

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, γ-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate–treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.

To elucidate the molecular mechanisms underlying pathogen‐associated molecular pattern (PAMP)‐induced defense responses in potato (Solanum tuberosum ), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep‐13, a PAMP from Phytophthora , induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive‐like cell death. We have previously shown that SA is required for Pep‐13‐induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12‐oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F‐box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep‐13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep‐13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA‐deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans .

The plant immune system can be activated by two different types of signals, by microbial signatures and by features signifying malfunctioning of plant processes.