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Identification of key determinants for broad-host compatibility and lifestyle switch in the Sebacinales

To establish and maintain a compatible interaction with different hosts, P. indica must respond and adapt to different host signals. Alternative lifestyles and colonization strategies in different host species may thus be a consequence of this adaptation to highly variable environments. We recently have shown by cytological studies and global investigations of fungal transcriptional responses to colonization of barley and Arabidopsis at two different symbiotic stages (early phase of communication, 3 dpi and late colonization stage, 14 dpi) that broad compatibility in P. indica-root symbioses is associated with host-dependent colonization strategies and with host-specifically-induced effector candidates. We demonstrated that in Arabidopsis, P. indica establishes and maintains biotrophic nutrition within living epidermal cells, while in barley the symbiont undergoes a nutritional switch to saprotrophy that is associated with the production of secondary thinner hyphae in dead cortex cells at 14 dpi. Consistent with a diversified trophic behaviour and with the occurrence of nitrogen limitation at the onset of saprotrophy in barley, fungal genes encoding hydrolytic enzymes were highly induced in this host but to a lesser extent in Arabidopsis (Fig. 1).
To obtain insights into the role played by host-derived nutritional cues on the lifestyle of P. indica during colonization of barley and Arabidopsis we performed a detailed analysis of predicted protein-coding genes for transporters and their expression in Arabidopsis and barley. This approach resulted in the identification of a high affinity ammonium transporter which was induced in barley at later colonization stages but to a lesser extent in Arabidopsis. After the establishment of an opposing promoter mediated RNAi system in P. indica, the PiAmt1 gene was silenced and the phenotype of the RNAi strains in barley and Arabidospsis was analyzed. We could show that upon silencing of PiAmt1, P. indica delayed the switch from the biotrophic to the saprotrophic lifestyle in barley. Consistent with low expression of the PiAMT1 gene in Arabidopsis at later interaction stage, analyses of the P. indica RNAi strains impaired in ammonium uptake and possibly ammonium sensing displayed no difference in their colonization patterns compared to the WT and empty vector controls. We therefore concluded that PiAmt1 is not required for biotrophic growth but involved in the lifestyle switch of P. indica to saprotrophic growth as observed in barley. Taken together, these results strongly suggest that host-related nutritional cues affect P. indica´s lifestyle and that the switch from biotrophic to saprotrophic nutrition during colonization of barley is affected by PiAmt1 and by nitrogen availability. This work strongly contributed to the finding that different host environments affect the colonization strategies in root endophytes.

Fig. 1. Schematic representation of P. indica colonization strategies at different symbiotic stages in Hordeum vulgare (barley) and in Arabidopsis thaliana. Upper panel: photograph showing invasive hyphae (IH) and secondary thin hyphae (SH) of P. indica in barley dead cells. Lower panel: photograph showing P. indica non-WGA-stainable biotrophic broad invasive hyphae (IH) in an Arabidopsis epidermal cell. Fungal structures were stained with WGA-AF488 (green), membranes were stained with FM4-64 (red). In H. vulgare roots cell death is initiated few days after germination by developmental RCD and is increased by P. indica colonization. Heatmap shows log2 fold expression changes of P. indica genes encoding hydrolytic enzymes. Significant (t-test P<0.05) log2 fold expression changes were calculated versus minimal medium control. A consistent divergence is observed at 14 dpi where a strong induction is visible for most of these genes during colonization of barley only (modified from Lahrmann et al., 2013).