Systemic ectomycorrhizal defense induction in poplarDoctoral thesis
Date of Examination:2023-03-29
Date of issue:2023-05-25
Advisor:Prof. Dr. Andrea Polle
Referee:Prof. Dr. Andrea Polle
Referee:Prof. Dr. Guus Bakkeren
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Name:Steven Dreischhoff Doctoral Thesis.pdf
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EnglishEctomycorrhizal fungi (EMF) are important soil symbiotes in boreal and temperate forests. They form beneficial interactions (ectomycorrhizae) with the roots of tree species. Ectomycorrhizae facilitate plant nutrient supply and, in return, receive carbohydrates from the host. EMF also modulate plant stress resistance, for example, by negative effects on the fitness of leaf-feeding insects. In non-host interactions, the negative impact of EMF on herbivores is mediated by chitin signaling via the CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1). It is unknown whether this pathway is also involved in defense activation in EMF host plants. To gain new insights into the mechanisms of ectomycorrhizal-induced systemic resistance, leaf responses to the interaction of the symbiotic fungus Laccaria bicolor with its host species poplar (Populus x canescens) were investigated. Specifically, this thesis aimed to characterize early effects on the poplar leaf transcriptome while L. bicolor was colonizing the roots and to investigate the possible involvement of chitin and CERK1 in mediating the systemic defense responses. In addition, a bioassay for Botrytis cinerea-induced damage was developed and applied to test if L. bicolor affected the defenses of poplar against a necrotizing foliar pathogen. The initial transcriptional changes in poplar leaves after L. bicolor root inoculation were examined in an axenic setup and used to identify potential marker genes for systemic effects. Changes in the leaf transcriptome were not found after one or two days but occurred only four days after L. bicolor treatment. This result suggests that the presence of L. bicolor without direct interaction was not sufficient, but that initial root colonization was required for systemic changes. After seven days, genes involved in defense metabolism, including biosynthetic genes for phenolic compounds and flavonoids, had increased transcript abundances. Defense signaling components with increased transcript abundances included genes involved in the regulation and biosynthesis of jasmonate signaling and negative and positive regulators of ethylene signaling. KUNITZ TRYPSIN INHIBITOR (KTI) and MAJOR LATEX PROTEIN LIKE 423 (MLP423) were identified to have robustly increased transcript abundances after different ectomycorrhizal treatments and were selected as marker genes for further experiments. In addition, KTI was responsive toward methyl jasmonate treatment. This observation underpins the results of the transcriptome analysis that jasmonic acid signaling may play a role in ectomycorrhizal systemic defense induction. Summary 12 Using poplar cerk1 KO mutants, the role of CERK1 in systemic ectomycorrhizal defense induction was investigated. KTI and MLP423 served as marker genes for defense activation. The transcript abundances of KTI and MLP423 increased less after L. bicolor treatment in cerk1 KO lines than in wildtype poplars, suggesting that CERK1 is involved in ectomycorrhizal defense induction. However, the induction of KTI and MLP423 was not entirely abolished in the cerk1 KO lines, indicating that additional factors are likely implicated in the EMF-induced systemic induction of the defense marker genes. Since chitin did not induce the defense marker genes, a direct link between CERK1-mediated defense activation and EMF-induced systemic defense is unlikely. However, a global transcriptome analysis is required to find out if other defenses than the two selected marker genes were responding to chitin. Mycorrhizal colonization of the cerk1 mutants was reduced, suggesting that CERK1 might participate in the establishment of ectomycorrhizae, similarly as known for arbuscular mycorrhizae. If this can be further corroborated, then the reduced activation transcript levels of KTI or MLP423 in leaves of cerk1 mutants might be due to delayed mycorrhization and, thus, indicate only an indirect involvement of CERK1 in the systemic defense changes. This idea requires further analyses. Reliable bioassays to study the interaction of foliar pathogens and poplar are scarce. Therefore, a test system to quantify B. cinerea infections on poplar leaves was developed. Initial experiments with this new bioassay showed a positive relationship between mycorrhizal root colonization and the degree of B. cinerea infection. This result suggests that resistance by L. bicolor may not be broadly induced against various biological threats and opens the question of how jasmonate-dependent processes are modulated in ectomycorrhizal-induced systemic defense. In summary, this thesis suggests that initial root colonization by EMF may be required to induce systemic defense changes in the leaf transcriptome. The systemic signaling events include jasmonate-dependent and ethylene-responsive components. The responses of defense marker genes KTI and MLP423 suggest a contribution of CERK1, but the effect might be indirect through a potential involvement of CERK1 in symbiosis establishment. Based on the analyses of the marker genes, chitin is unlikely to play a role in the systemic induction process. The results further point toward ectomycorrhizal-mediated trade-off between herbivorous leaf insects and necrotrophic pathogens and thus may direct future research.
Keywords: Ectomycorrhiza; Poplar; Laccaria bicolor; Botrytis cinerea; Populus x canescens; Chitin