TY - BOOK
T1 - Plant-endophyte interactions as a source of bioactive compounds
T2 - Regulation of specialised metabolism during tomato and Serendipita indica interaction
AU - Ntana, Fani
PY - 2019
Y1 - 2019
N2 - Fungi are essential components of natural ecosystems, key players in the development of modern agriculture and important producers of compounds with pharmaceutical and industrial applications. In nature, fungi not only mediate crucial ecological processes like nutrient recycling, but also develop various interactions with plants. Fungal endophytes comprise a diverse group of plant-associated fungi that reside inside plant tissues without causing disease symptoms. Endophytic symbionts often benefit their hosts by promoting plant growth and resistance to biotic or abiotic stress. Although by definition, endophytic colonisation does not result in disease, interactions between the host and individual endophytic species are quite dynamic, ranging from beneficial to detrimental. Plants utilise a plethora of physical and chemical defence barriers to hinder or restrict fungal invasion and expansion, while endophytes employ mechanisms to overcome or suppress host defence. Plant-endophyte associations are mediated by a constant exchange of metabolic signals that finally determines the outcome of this interaction. This thesis deals with the transcriptional regulation of host and endophyte specialised metabolism during symbiotic associations and attempts to elucidate the role of specialised metabolites in the development of symbiosis as well as to identify compounds of ecological and commercial value. In Manuscript 1, we used the fungal root endophyte Serendipita indica and the domesticated tomato Solanum lycopersicum to determine the effect of endophytic colonisation on the expression of plant specialised metabolic pathways. Tissues (roots and leaves) from S. indica-inoculated and endophyte-free tomato seedlings were subjected to a whole-transcriptome sequencing analysis. Comparison between tissues from colonised and control plants showed that in roots S. indica-colonisation resulted in accumulation of transcripts involved in the production of lignin, defensive fatty acids and specific terpenes. On the contrary, root flavonoidrelated biosynthesis and the glycoalkaloid production pathway was almost left unaffected. In leaves, a similar regulation pattern is observed, except from the negatively affected glycoalkaloid biosynthesis and the activation of a previously undescribed terpene synthase. Changes observed in tomato metabolic pathways in the roots were read as the result of the constant signalling between the two symbionts, during which the endophyte suppresses host toxic metabolite production and the host balances extensive fungal colonisation by activating alternative chemical defence pathways. It is, however, unclear whether regulation of specialised metabolism in the leaves serves any ecological purpose or it is just a by-product of defence induction in roots. The ability of S. indica to produce bioactive compounds is studied in Manuscript 2. Here, S. indica and another root endophyte, Umbelopsis isabellina, were grown on synthetic medium inside glass vials. The volatile organic compounds (VOCs) accumulating in the headspace of the vials with the fungal cultures were extracted using the Solid Phase MicroExtraction (SPME) method and analysed with a Gas Chromatography- Mass Spectrometry (GC-MS) system. Most of the compounds produced by S. indica belonged to the class of eight-carbon metabolites, derivatives of fatty acids, while the headspace of U. isabellina cultures contained only a few short-chain hydrocarbons. To evaluate the effect of fungal VOCs on plant growth, tomato seeds were placed in plastic containers sharing the same headspace with S. indica or U. isabellina cultures, respectively. S. indica volatiles correlated with induction of plant growth, while the growth promotion effect observed in the plants sharing the headspace with U. isabellina was attributed to elevated amounts of carbon dioxide (CO2) produced from fungal respiration. Manuscript 3 deals with the specialised metabolism of S. indica and its ability to produce terpenoids, a specific class of bioactive metabolites. The genome of S. indica possesses a putative terpene synthase gene, but hitherto there is no evidence that the fungus actually produces terpenoids. However, in this study the specific gene was found to be upregulated after tomato root colonisation, indicating that it could be of significance for plant-endophyte association. The coding sequence of the fungal terpene synthase gene was isolated and expressed heterologously in Escherichia coli cells. In vivo and in vitro functional characterisation showed that the specific enzyme is catalysing formation of viridiflorol, a sesquiterpene (C15) with commercial applications in perfumery. To elucidate whether the viridiflorol synthase and its products contribute in the colonisation ability of S. indica, we constructed mutants over-expressing the gene and used them to inoculate tomato seedlings. Roots from tomato seedlings colonised by S. indica mutants and the wild type showed no significant difference in the colonisation ratio, demonstrating that over-expression of viridiflorol synthase does not affect the ability of the endophyte to colonise plant roots. Collectively, the results generated from this thesis provide preliminary steps in understanding the role of bioactive metabolites during plant and endophyte interactions.
AB - Fungi are essential components of natural ecosystems, key players in the development of modern agriculture and important producers of compounds with pharmaceutical and industrial applications. In nature, fungi not only mediate crucial ecological processes like nutrient recycling, but also develop various interactions with plants. Fungal endophytes comprise a diverse group of plant-associated fungi that reside inside plant tissues without causing disease symptoms. Endophytic symbionts often benefit their hosts by promoting plant growth and resistance to biotic or abiotic stress. Although by definition, endophytic colonisation does not result in disease, interactions between the host and individual endophytic species are quite dynamic, ranging from beneficial to detrimental. Plants utilise a plethora of physical and chemical defence barriers to hinder or restrict fungal invasion and expansion, while endophytes employ mechanisms to overcome or suppress host defence. Plant-endophyte associations are mediated by a constant exchange of metabolic signals that finally determines the outcome of this interaction. This thesis deals with the transcriptional regulation of host and endophyte specialised metabolism during symbiotic associations and attempts to elucidate the role of specialised metabolites in the development of symbiosis as well as to identify compounds of ecological and commercial value. In Manuscript 1, we used the fungal root endophyte Serendipita indica and the domesticated tomato Solanum lycopersicum to determine the effect of endophytic colonisation on the expression of plant specialised metabolic pathways. Tissues (roots and leaves) from S. indica-inoculated and endophyte-free tomato seedlings were subjected to a whole-transcriptome sequencing analysis. Comparison between tissues from colonised and control plants showed that in roots S. indica-colonisation resulted in accumulation of transcripts involved in the production of lignin, defensive fatty acids and specific terpenes. On the contrary, root flavonoidrelated biosynthesis and the glycoalkaloid production pathway was almost left unaffected. In leaves, a similar regulation pattern is observed, except from the negatively affected glycoalkaloid biosynthesis and the activation of a previously undescribed terpene synthase. Changes observed in tomato metabolic pathways in the roots were read as the result of the constant signalling between the two symbionts, during which the endophyte suppresses host toxic metabolite production and the host balances extensive fungal colonisation by activating alternative chemical defence pathways. It is, however, unclear whether regulation of specialised metabolism in the leaves serves any ecological purpose or it is just a by-product of defence induction in roots. The ability of S. indica to produce bioactive compounds is studied in Manuscript 2. Here, S. indica and another root endophyte, Umbelopsis isabellina, were grown on synthetic medium inside glass vials. The volatile organic compounds (VOCs) accumulating in the headspace of the vials with the fungal cultures were extracted using the Solid Phase MicroExtraction (SPME) method and analysed with a Gas Chromatography- Mass Spectrometry (GC-MS) system. Most of the compounds produced by S. indica belonged to the class of eight-carbon metabolites, derivatives of fatty acids, while the headspace of U. isabellina cultures contained only a few short-chain hydrocarbons. To evaluate the effect of fungal VOCs on plant growth, tomato seeds were placed in plastic containers sharing the same headspace with S. indica or U. isabellina cultures, respectively. S. indica volatiles correlated with induction of plant growth, while the growth promotion effect observed in the plants sharing the headspace with U. isabellina was attributed to elevated amounts of carbon dioxide (CO2) produced from fungal respiration. Manuscript 3 deals with the specialised metabolism of S. indica and its ability to produce terpenoids, a specific class of bioactive metabolites. The genome of S. indica possesses a putative terpene synthase gene, but hitherto there is no evidence that the fungus actually produces terpenoids. However, in this study the specific gene was found to be upregulated after tomato root colonisation, indicating that it could be of significance for plant-endophyte association. The coding sequence of the fungal terpene synthase gene was isolated and expressed heterologously in Escherichia coli cells. In vivo and in vitro functional characterisation showed that the specific enzyme is catalysing formation of viridiflorol, a sesquiterpene (C15) with commercial applications in perfumery. To elucidate whether the viridiflorol synthase and its products contribute in the colonisation ability of S. indica, we constructed mutants over-expressing the gene and used them to inoculate tomato seedlings. Roots from tomato seedlings colonised by S. indica mutants and the wild type showed no significant difference in the colonisation ratio, demonstrating that over-expression of viridiflorol synthase does not affect the ability of the endophyte to colonise plant roots. Collectively, the results generated from this thesis provide preliminary steps in understanding the role of bioactive metabolites during plant and endophyte interactions.
UR - https://rex.kb.dk/permalink/f/h35n6k/KGL01012069459
M3 - Ph.D. thesis
BT - Plant-endophyte interactions as a source of bioactive compounds
PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
ER -