Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H2O2 and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H2O2 formation is even reduced by the fungus. Importantly, phospholipase D (PLD)α1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

SUMMARYCalcium (Ca 2+ ), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca 2+ ] elevations are early events in the interaction between the plant growth-promoting fungus Piriformospora indica and Arabidopsis thaliana. A cell wall extract (CWE) from the fungus promotes the growth of wild-type seedlings but not of seedlings from P. indica-insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca 2+ signalling-related functions. The CWE induces a transient cytosolic Ca 2+ ([Ca 2+ ] cyt ) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum) plants, as well as in BY-2 suspension cultures expressing the Ca 2+ bioluminescent indicator aequorin. Nuclear Ca 2+ transients were also observed in tobacco BY-2 cells. The Ca 2+ response was more pronounced in roots than in shoots and involved Ca 2+ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca 2+ response by staurosporine and the refractory nature of the Ca 2+ elevation suggest that a receptor may be involved. The CWE does not stimulate H 2 O 2 production and the activation of defence gene expression, although it led to phosphorylation of mitogen-activated protein kinases (MAPKs) in a Ca 2+ -dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica. Thus, Ca 2+ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco.

SummaryPiriformospora indica, an endophyte of the Sebacinaceae family, promotes growth and seed production of many plant species, including Arabidopsis. Growth of a T-DNA insertion line in PYK10 is not promoted and the plants do not produce more seeds in the presence of P. indica, although their roots are more colonized by the fungus than wild-type roots. Overexpression of PYK10 mRNA did not affect root colonization and the response to the fungus. PYK10 codes for a root-and hypocotyl-specific b-glucosidase/myrosinase, which is implicated to be involved in plant defences against herbivores and pathogens. Expression of PYK10 is activated by the basic helix-loop-helix domain containing transcription factor NAI1, and two Arabidopsis lines with mutations in the NAI1 gene show the same response to P. indica as the PYK10 insertion line. PYK10 transcript and PYK10 protein levels are severely reduced in a NAI1 mutant, indicating that PYK10 and not the transcription factor NAI1 is responsible for the response to the fungus. In wild-type roots, the message level for a leucine-rich repeat protein LRR1, but not for plant defensin 1.2 (PDF1.2), is upregulated in the presence of P. indica. In contrast, in lines with reduced PYK10 levels the PDF1.2, but not LRR1, message level is upregulated in the presence of the fungus. We propose that PYK10 restricts root colonization by P. indica, which results in the repression of defence responses and the upregulation of responses leading to a mutualistic interaction between the two symbiotic partners.

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