Plants have developed diverse strategies for protection against the threat of invading pathogens. In order to improve their performance as well as to evade abiotic and biotic stresses, one strategy of plants is to establish associations with beneficial microbial organisms. Piriformospora indica is a root interacting fungus, which transfers several benefits to colonized plants like a better tolerance to various biotic and abiotic stresses, as well as an improved plant growth and yield. P. indica colonizes a broad range of monocot and dicot plants. This broad host range indicates that P. indica has developed efficient strategies to overcome innate immune responses and to manipulate the metabolism in different plants. This is even more intriguing as the fungus was shown to follow an initial biotrophic colonisation strategy at which penetrated cells are living. Plant colonizing microbes are known to secrete proteins (also called effectors) in order to modify host physiology and modulate plant defense mechanisms and, hence, confer compatibility. The aim of this study was to identify P. indica effector proteins as well as plant compatibility factors that are involved in the manipulation of those processes required for successful fungal establishment in planta. Therefore, two different strategies were followed. In the first approach, the so-called yeast signal sequence trap (YSST) assay was established. As the result of YSST, several plant genes were identified that are known to be involved in stress responses and cell wall development. These genes were shown to have a specific expression in barley roots during P. indica colonisation. In addition, a fungal gene was identified that does not show any similarities to other sequences deposited in public databases. The identified P. indica protein (PIALH43) carries a signal peptide and was shown to be induced during barley root colonisation. Interestingly, PIALH43 harbours a highly conserved C-terminal RING finger motif. In silico protein modelling of PIALH43 confirmed a 3D structural overlap and verified the accurate conformation of the E2 binding residues when compared with known human and plant ubiquitin ligases. Moreover, E3 ligase activity of PIALH43 was confirmed in vitro. Currently, PIALH43 is overexpressed in planta and in P. indica in order to study its function in mutualistic root colonisation. In a second approach, a simplified subtraction-based assay, designated Transcript Subtractive Hybridization (TSH), was established to identify and study plant compatibility factors in the barley-P. indica interaction. The subtraction assay delivered various differentially regulated genes. These genes are known to be involved in stress responses, phytohormone- and secondary metabolism, autophagy, and protein processing. Among the up-regulated candidates was a gene encoding S-adenosylmethionine synthetase 2, which is thought to be involved in the synthesis of ethylene. De novo synthesis of ethylene during root colonization was verified by quantifying the ethylene precursor 1-aminocyclopropane 1-carboxylic acid (ACC) in barley and by cytologically monitoring GUS accumulation in ACC synthase reporter plants of Arabidopsis. In addition, the effects of ethylene precursor ACC or ethylene antagonist 1-methylcyclopropene (MCP) was determined. In these pharmacological experiments, barley plants were about 40% less colonised by P. indica after application of MCP while treatment with ACC resulted in significant increase (~ 60%) in colonization. To further elucidate the impact of ethylene on plant root colonization by P. indica, genetic analyses were performed with Arabidopsis mutants altered in ethylene synthesis and signaling at early biotrophic (~ 3 dai) and later cell death-associated colonization phases (~ 14 dai). In accordance with the studies in barley, Arabidopsis mutants ctr1-1 (constitutive ethylene signaling) and eto1-1 (ethylene overproducer) exhibited a significant increase in fungal colonization (especially at later interaction stages), while a reduced colonization was observed in ein2-1 (ethylene insensitive). In summary, ethylene might function as general plant compatibility factor in the plant-P. indica system.
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