The basidiomycete P. indica is a root endophyte, which has a beneficial impact on host plants. P. indica promotes growth in colonized host plants and mediates biotic stress resistance as well as abiotic stress tolerance such as drought or salt stress (Waller et al., 2005; Baltruschat et al., 2008). The fungus has a broad host spectrum and non-host plants have not been identified so far. During the establishment of a compatible interaction, the fungus is able to actively suppress root immune responses of the host plant including the root oxidative burst, induction of defense genes and callose deposition. Especially JA signaling seems to be influenced by P. indica during the active suppression of host basal defense. The colonization pattern of P. indica can be mainly divided in an initial biotrophic and a later cell death-associated phase (Schäfer et al., 2009; Jacobs et al., 2011). To reach compatibility with the host plant, P. indica is assumed to secrete a battery of effector proteins, especially during the biotrophic interaction phase. Effector proteins are secreted by pathogenic as well as beneficial microbes during plant colonization in order to suppress the plant innate immune system and to manipulate the host metabolism. Due to the broad host range which includes model plants like Arabidopsis, barley and tobacco, P. indica is an ideal model organism for the investigation of fungal derived effector proteins and their impact on plant innate immunity. The aim of this study was the identification and subsequent characterization of P. indica effector proteins. Therefore, the yeast signal sequence trap (YSST) was successfully applied. Eleven different secreted P. indica proteins could be isolated. Additionally, the P. indica effector candidate PIIN_00029 (PiALH43), which was previously identified (Khatabi, 2009), was included in this study. The in silico analyses revealed similarities of eight candidates to known or putative effector proteins identified in other biological systems. Furthermore, six of the candidates are probably associated with the cell membrane. The isolated effector candidates could be functioning in the adhesion of the fungus to the host surface or even in masking P. indica-derived MAMPs. In qPCR-based analyses, nine candidates showed the highest expression during biotrophic colonization (2 dai) of Arabidopsis roots in comparison to axenically grown P. indica or P. indica colonizing dead roots. Localization studies with GFP fusion proteins revealed a cytoplasmic expression pattern of the candidate PIIN_08513. This candidate was furthermore demonstrated to suppress MAMP-triggered oxidative burst upon transient overexpression in N. benthamiana leaves. The present work is a base for future studies on P. indica effectors and their impact on plant innate immunity and host metabolism. Functional studies on the RING E3 ligase PIIN_00029 (PiALH43) revealed that this enzyme had neither a proapoptotic nor antiapoptotic activity in yeast viability assays. Further, plant basal defense was not influenced in N. benthamiana leaves transiently expressing PIIN_00029. Arabidopsis plants stably expressing PIIN_00029 showed partly enhanced colonization by P. indica. PIIN_00029 might manipulate plant metabolism by the targeted labeling of proteins for degradation in order to enhance root colonization. The results of this work can be used as base for the characterization of further P. indica secreted proteins.
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