On the role of amino acids in plant disease resistance: Interplay between pipecolic acid and salicylic acid in plant systemic acquired resistance

Summary: Recognition of microbes by plants leads to both local and systemic immune responses. Systemic acquired resistance (SAR) is a long-lasting, broad-spectrum disease resistance that occurs in uninfected parts of the plant. The establishment of SAR requires the accumulation of the phenolic compound salicylic acid (SA) in distal leaves, but SA itself is not the mobile signal. A number of potential SAR signals have recently been proposed in the last decade, such as methyl salicylate (MeSA), dehydroabietenal (DA), glycerol-3-phosphate (G3P), azelaic acid (AzA) and the lipid transfer protein DEFECTIVE IN INDUCED RESISTANCE 1 (DIR1), but the true identity of the mobile signal is still controversial. Our laboratory has recently identified the lysine (Lys)-derived non-proteinogenous amino acid pipecolic acid (Pip) as a novel important regulator of local and systemic acquired resistance, as well as defense priming, in Arabidopsis thaliana. In addition to Pip, massive changes in free amino acid levels were also observed upon pathogen recognition, revealing an unexpected role for these molecules in plant immunity. In this thesis, we investigated the role of free amino acids during plant defense, the mechanisms underlying Pip-induced resistance, and the relationship between Pip and SA during SAR and defense priming in Arabidopsis thaliana. We observed that the profile of amino acids changes was similar when plants were treated with virulent or avirulent strains of the bacterium Pseudomonas syringae pv. maculicola, or upon treatment with the bacterial pathogen-associated molecular pattern (PAMP) flg22. To test whether pathogen-induced free amino acid changes depend on immune hormone signaling pathways, we measured free amino acid levels in mutants affected in SA, jasmonic acid or ethylene biosynthesis and/or signaling. Interestingly, the lipase-like PHYTOALEXIN-DEFICIENT4 (PAD4) differentially regulated changes of distinct amino acids, revealing an unexpected uncoupling of amino acid induced biosynthesis during defense. To uncover the relationship between Pip and SA, we monitored amino acid levels and gene expression changes in distal leaves of the SA-deficient mutant sid2-1 during SAR. Surprisingly, we observed that it still exhibited a systemic increase in Pip levels, an increased expression of the genes encoding AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1; as an important Pip biosynthetic enzyme) and FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1; as a critical regulator of Pip-mediated resistance), and resistance induced by exogenous Pip treatment, albeit to lower levels than in wild-type distal leaves. Furthermore, we found that Pip and SA contributed additively to basal resistance, and that SA-deficient mutants exhibited a modest, but significant SAR response, which was otherwise absent in Pip-deficient mutants. Together, these results indicate an SA-independent role of Pip during SAR. To further study this novel SA-independent regulatory node of SAR, we analyzed transcriptional changes during SAR in wild-type, SA- and Pip-deficient plants. We observed a transcriptional reprogramming in distal leaves and found that SAR as a state with activated defense responses was further associated with decreased photosynthesis rates and anabolic metabolism. Interestingly, we identified a subset of SAR genes whose expression was partially SA-independent, and strikingly observed that the Pip-deficient mutant ald1 hardly mounted any transcriptional reprogramming during SAR, confirming that Pip is an SA-independent, central regulator of gene expression during SAR. We further wanted to characterize the role of Pip in the priming of defense responses by SAR. We found that defense priming is orchestrated by Pip and FMO1 in both an SA-dependent and -independent manner. Combined and single treatments with Pip and SA revealed that they employ two distinct pathways that lead to a synergistic effect on the priming of PR1 gene expression and disease resistance. Lastly, we sought to characterize the close ALD1 homolog, the diaminopimelate-aminotransferase ABERRANT GROWTH AND CELL DEATH 2 (AGD2) and found that agd2 accumulates an unknown compound that may partly explain the constitutive disease resistance observed in this mutant. To gain further insight in the enzymatic processes of the Pip biosynthetic pathway, we selected candidate genes with a potential role upstream and downstream of Pip biosynthesis based on expression patterns and homology in other organisms. Despite altered Pip levels, mutant lines in these genes did not show impaired SAR, suggesting potential functional redundancy and/or the involvement of other enzymes. Finally, we examined the sub-cellular localization of ALD1 and FMO1, which are required for Pip accumulation and signaling, respectively. We found that ALD1 localizes in the chloroplasts and FMO1 in the endoplasmic reticulum, suggesting that Pip biosynthesis and signaling act in different organelles. In summary, this thesis revealed Pip as a crucial regulator of local and systemic immunity and priming against bacteria, that acts via both SA-dependent and -independent pathways.