Piriformospora indica, an endophytic fungus of the Sebacinaceae family, promotes growth of Arabidopsis and tobacco seedlings and stimulates nitrogen accumulation and the expression of the genes for nitrate reductase and the starch-degrading enzyme glucan-water dikinase (SEX1) in roots. Neither growth promotion nor stimulation of the two enzymes requires heterotrimeric G proteins. P. indica also stimulates the expression of the uidA gene under the control of the Arabidopsis nitrate reductase (Nia2) promoter in transgenic tobacco seedlings. At least two regions (؊470/؊439 and ؊103/ ؊89) are important for Nia2 promoter activity in tobacco roots. One of the regions contains an element, ATGATAGATAAT, that binds to a homeodomain transcription factor in vitro. The message for this transcription factor is up-regulated by P. indica. The transcription factor also binds to a CTGATAGATCT segment in the SEX1 promoter in vitro. We propose that the growthpromoting effect initiated by P. indica is accompanied by a co-regulated stimulation of enzymes involved in nitrate and starch metabolisms.Often nitrogen is the limiting source for plant growth and development. It is recruited by plants either as nitrate or ammonium or for a few species by nitrogen fixation with the help of rhizobia (1, 2). Mycorrhizal fungi also play an important role in delivering either nitrate or ammonium to the root cells. It is believed that mycorrhizal fungi preferentially recruit ammonium rather than nitrate from the soil and that amino acids represent the major compounds that serve to transfer nitrogen to the host plant (cf. Refs. 3 and 4). We studied Piriformospora indica, an endophytic fungus of the Sebacinaceae family, which colonizes the roots of a wide variety of plant species and promotes their growth (5-10). The interaction of the endophytic fungus with plant roots is accompanied by an enormous requisition of nitrogen from the environment. By analyzing the interaction of P. indica with Arabidopsis and tobacco roots we found that in contrast to mycorrhizal associations, nitrate reduction in the roots is stimulated by P. indica. A homeodomain transcription factor responds to the fungus and binds to promoter regions of the P. indica-responsive Nia2, SEX1, and 2-nitropropane dioxygenase genes. These results suggest that the expression of P. indica-responsive target genes may be controlled by common regulatory elements and trans-factors. MATERIALS AND METHODSTransgenic Tobacco-Transgenic seeds of Nicotiana tabacum L., var. Samsun NN were obtained from greenhouse-grown plants (6). They were sterilized and germinated on Murashige-Skoog medium (11) supplemented with 2% (w/v) sucrose and 0.8% (w/v) agar in temperaturecontrolled (25°C) growth chambers under a 16-h light/8-h dark cycle. 80 g of ml Ϫ1 (w/v) kanamycin was added to the medium. Four-week-old plantlets were transferred to soil to obtain seeds for the physiological experiments. The antisense lines for the heterotrimeric G protein subunit ␤ were described previously (12).Growth Conditions of Pla...

Summary• The endophytic fungus Piriformospora indica colonizes the roots of the model plant Arabidopsis thaliana and promotes its growth and seed production. The fungus can be cultivated in axenic culture without a host, and therefore this is an excellent system to investigate plant-fungus symbiosis.• The growth of etr1, ein2 and ein3 ⁄ eil1 mutant plants was not promoted or even inhibited by the fungus; the plants produced less seeds and the roots were more colonized compared with the wild-type. This correlates with a mild activation of defence responses. The overexpression of ETHYLENE RESPONSE FACTOR1 constitutively activated defence responses, strongly reduced root colonization and abolished the benefits for the plants.• Piriformospora indica-mediated stimulation of growth and seed yield was not affected by jasmonic acid, and jasmonic acid-responsive promoter b-glucuronidase gene constructs did not respond to the fungus in Arabidopsis roots.• We propose that ethylene signalling components and ethylene-targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis. The results show that the restriction of fungal growth by ethylene signalling components is required for the beneficial interaction between the two symbionts.

Piriformospora indica is an endophytic fungus that colonizes the roots of many plant species, including Arabidopsis. We exposed 18-day-old Arabidopsis seedlings, which were either cocultivated with the fungus or mock-treated for the last 9 days, to mild drought stress for 84 h. During the first 36 to 48 h, seedlings cocultivated with the fungus continued to grow, while the uncolonized controls did not. This results in a threefold difference in the fresh weight and a more than twofold difference in the chlorophyll content. The photosynthetic efficiency was only slightly reduced in the colonized (F variable/F maximum [Fv/Fm] at t(0 h) = 0.82 and t(36 h) = 0.79) and was severely impaired in the uncolonized (Fv/Fm at t(0 h) = 0.81 and (t)(36 h) = 0.49) seedlings, which also showed symptoms of withering. When seedlings exposed to drought stress for 72 or 84 h were transferred to soil, 10% (72 h) and none (84 h) of uncolonized seedlings reached the flowering stage and produced seeds, while 59% (72 h) and 47% (84 h) of the colonized seedlings flowered and produced seeds. After exposure to drought stress for 3 h, the message levels for RESPONSE TO DEHYDRATION 29A, EARLY RESPONSE TO DEHYDRATION1, ANAC072, DEHYDRATION-RESPONSE ELEMENT BINDING PROTEIN2A, SALT-, AND DROUGHT-INDUCED RING FINGER1, phospholipase Ddelta, CALCINEURIN B-LIKE PROTEIN (CBL)1, CBL-INTERACTING PROTEIN KINASE3, and the histone acetyltransferase (HAT) were upregulated in the leaves of P. indica-colonized seedlings. Uncolonized seedlings responded 3 to 6 h later, and the message levels increased much less. We identified an Arabidopsis ethylmethane-sulfonate mutant that is less resistant to drought stress and in which the stress-related genes were not upregulated in the presence of P. indica. Thus, P. indica confers drought-stress tolerance to Arabidopsis, and this is associated with the priming of the expression of a quite diverse set of stress-related genes in the leaves. Transfer to soil was again associated with a faster and stronger upregulation of the message levels for phospholipase Ddelta, CBL1, and HAT in P. indica-colonized seedlings, indicating that this response might also contribute to better survival on soil.

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