Plants have evolved a sophisticated two-layered immune system to protect themselves from microbial invaders. However, in order to promote infection, plant associated microbes including pathogens as well as mutualists secrete molecules called effectors to suppress the plant s immune response. Mutualistic fungi including the arbuscular mycorrhiza (AM) Glomus intraradices and the ectomycorrhiza (ECM) Laccaria bicolor secretes effector proteins which contribute to the establishment and maintenance of mutualistic symbiosis by suppressing host immunity during root colonization.The root endophytic fungus, Piriformospora indica is capable of colonizing the roots of a wide variety of different plant species including the monocot barley (Hordeum vulgare) and the dicot Arabidopsis thaliana and establishes mutualistic symbiosis during colonization. The colonization pattern of P. indica is mainly divided into two phases, an initial biotrophic phase and a later cell death-associated colonization phase. Colonization by the fungi which is mostly limited to the root cortex cells leads to several beneficial effects to the host such as growth promotion, increased resistance to biotic and abiotic stresses. The recently released genome of P. indica helped to identify hundreds of small secreted proteins (SSPs) coding for candidate effectors and provides an opportunity to investigate the role of these effectors during the interaction between this endophytic fungus with plants. It has been reported that P. indica actively suppresses PTI to colonize and establish a mutualistic relationship with plants. However, the deployment of effectors and their role during the interaction of P. indica with plants has not been reported.In this study, PIIN_08944, an effector candidate of P. indica was functionally characterized in- planta. PIIN_08944 encode a 120 amino acid protein with a predicted 23 amino acid N-terminal signal peptide. Expression profile analysis revealed that PIIN_08944 was induced in in-vitro germinated P. indica chlamydospore as well as in-planta during colonization of Arabidopsis roots by P. indica. To investigate the functional role of PIIN_08944 in mutualistic symbiosis, transgenic P. indica with reduced production or complete loss of PIIN_08944 were generated. RNAi silencing as well as deletion of the gene coding for PIIN_08944 by homologous recombination resulted in delayed colonization of Arabidopsis roots by P. indica. On the hand transgenic Arabidopsis and barley plants expressing PIIN_08944 show enhanced colonization by P. indica compared to wild type. Heterologous expression of PIIN_08944 in barley suppresses flg22 and chitin triggered ROS production. Meanwhile expression of PIIN_08944 in Arabidopsis interfered with the transcription of marker genes for PTI, AtWRKY22 and salicylic acid, CBP60g. However, the expression of PIIN_08944 in Arabidopsis and barley did not affect the plant s response to the necrotrophic fungal pathogens Botrytis cinerea and Fusarium graminearum respectively. Yeast two hybrid screen revealed that PIIN_08944 interacts with CSN5A and CSN5B, components COP9 signalosome protein complex, in Arabidopsis and N. benthamiana and with SINA3, a member of the SEVEN IN ABSENTIA family of E3 protein ligase in tobacco. Subcellular localization assays performed on N. benthamiana leaves revealed that CSN5A, CSN5B and PIIN_08944 localized to the nucleus and cytoplasm. In summary, the fact that PIIN_08944 targets the evolutionarily conserved eukaryotic proteins, CSN5A and CSN5B and interfere with basal defense, indicate that P. indica has evolved strategies involving the use of effectors as general compatibility determinants to establish and/or maintain a mutualistic relationship during interaction with plants. Moreover, this might further explain the capability of P. indica to colonize a multitude of different plant species.
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