Biochemical and molecular analyses of the biosynthesis pathway of the indole derivatives in Piriformospora indica
The mutualistic root endophyte Piriformospora indica has the ability to colonize a wide range of plants including the monocot barley (Hordeum vulgare) and the dicot model plant Arabidopsis thaliana. The colonization of both, Arabidopsis and barley is characterized by a biphasic colonization strategy...
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|Zusammenfassung:||The mutualistic root endophyte Piriformospora indica has the ability to colonize a wide range of plants including the monocot barley (Hordeum vulgare) and the dicot model plant Arabidopsis thaliana. The colonization of both, Arabidopsis and barley is characterized by a biphasic colonization strategy with an initial biotrophic interaction followed by a cell death associated phase. During both phases fungal inter- and intracellular growth is restricted to the root cortex cells and can never be observed in the endodermis or the central cylinder. The colonization of root cells by P. indica is often associated with beneficial effects to the host, such as growth promotion and changes in root morphology. The broad host range and the widely conferred benefits to the hosts suggest that the beneficial outcome could be based on general mechanisms and signaling pathways common to many different plant species. One such mechanism could be the recruitment of phytohormone pathways by P. indica. Beside their function in plant developmental processes, phytohormones were recently described to be involved in plant defence responses. Secretion of indole-3-acetic acid (IAA) by P. indica into the growth medium has already been reported. In this PhD study a tryptophan dependent IAA production pathway was identified in P. indica using biochemical and molecular methods. The main goals were focused on the functional analyses of the P. indica tryptophan dependent IAA pathway and how this may affect compatibility during the biotrophic interaction between P. indica and barley roots. For this, suitable molecular tools such as a PEG-mediated genetic transformation, a GFP reporter and an RNAi-mediated silencing system were established or optimized for use in P. indica. Time course transcriptional analyses after exposure to tryptophan designated the tryptophan aminotransferase piTam1 gene as the top candidate gene involved in the production of IAA in P. indica. A green fluorescence protein (GFP) reporter study and transcriptional analysis of colonized barley roots showed that piTam1 is induced during the biotrophic phase. Via an RNAi-mediated gene silencing piTam1 was identified as a key gene involved in the first step of auxin biosynthesis. RNAi transformants impaired in auxin production were characterized by a less compact colony growth phenotype and differences in their ability to utilize indole-3-acetaldehyde (IAD). Additionally, silencing of the piTam1 gene resulted in a reduced P. indica colonization of barley roots at 3 days after inoculation (dai) but the elicitation of growth promotion was not affected compared with barley colonized by the P. indica wild-type strain. Consistently an increased amount of free IAA and free indole-3-lactate (ILA), a byproduct of P. indica IAA biosynthesis pathway, could be detected in P. indica colonized barley roots compared to non colonized control plants at this time point Given the large amount of IAA detected in planta at 3 dai and the amount of IAA produced by P. indica in culture after tryptophan induction, it is unlikely that the differences in free IAA levels observed in planta are merely derived from fungal IAA. This suggests a local accumulation of both fungal and plant IAA during the initial biotrophic phase and is consistent with changes in IAA signaling and biosynthesis observed in barley transcriptome at this time point.|