The endophytic Piriformospora indica reprograms to salt-stress tolerance, disease resistance, and higher yield

Frank Waller*†, Beate Achatz*†‡, Helmut Baltruschat*†,Jo´ zsef Fodor§, Katja Becker¶, Marina Fischer¶, Tobias Heier*, Ralph Hu¨ ckelhoven*, Christina Neumann*, Diter von Wettsteinʈ, Philipp Franken‡, and Karl-Heinz Kogel*,**

*Institute of Phytopathology and Applied Zoology, University of Giessen, D-35392 Giessen, Germany; ‡Institute for Vegetables and Ornamental Crops, D-14979 Grossbeeren, Germany; §Plant Protection Institute, Hungarian Academy of Sciences, H-1525 Budapest, Hungary; ¶Institute of Nutritional Biochemistry, University of Giessen, D-35392 Giessen, Germany; and ʈDepartment of Crop and Soil Sciences, Washington State University, Pullman, WA 99164-6420

Contributed by Diter von Wettstein, May 31, 2005 Disease resistance strategies are powerful approaches to sustain- nuclear DNA sequences from the D1͞D2 region of the large able agriculture because they reduce chemical input into the ribosomal subunit (12). In contrast to arbuscular environment. Recently, Piriformospora indica, a plant-root-coloniz- fungi, the fungus can be easily cultivated in axenic cultures, ing basidiomycete fungus, has been discovered in the Indian Thar where it asexually forms chlamydospores containing 8–25 nuclei desert and was shown to provide strong growth-promoting activ- (10). The fungus associates with roots of various plant species, ity during its symbiosis with a broad spectrum of plants [Verma, S. where it promotes plant growth. Hosts include the cereal crops et al. (1998) Mycologia 90, 896–903]. Here, we report on the rice, wheat, and barley as well as many Dicotyledoneae, including potential of P. indica to induce resistance to fungal diseases and Arabidopsis (13, 14). Interaction of the endophytic fungus with tolerance to salt stress in the monocotyledonous plant barley. The Arabidopsis roots is accompanied by a considerable requisition beneficial effect on the defense status is detected in distal leaves, of nitrogen from the environment (14). In the interaction with demonstrating a systemic induction of resistance by a root-endo- Arabidopsis and tobacco, the fungus stimulates nitrate reduction phytic fungus. The systemically altered ‘‘defense readiness’’ is (15), in contrast to the activity of arbuscular mycorrhiza fungi. associated with an elevated antioxidative capacity due to an We report here on the enormous agronomical potential of the activation of the glutathione–ascorbate cycle and results in an fungus. First, and most importantly, the growth-promoting ac- overall increase in grain yield. Because P. indica can be easily tivity of the fungus resulted in enhanced barley grain yield. propagated in the absence of a host plant, we conclude that the Second, P. indica amended tolerance to mild salt stress, and fungus could be exploited to increase disease resistance and yield third, P. indica conferred resistance in barley against root and in crop plants. leaf pathogens, including the necrotrophic fungus Fusarium culmorum (root rot) and the biotrophic fungus Blumeria grami- root ͉ powdery mildew ͉ symbiosis ͉ ascorbate ͉ glutathione nis. Thus, interaction of barley with P. indica constitutes a model system for systemic disease resistance in cereals. espite a worldwide intensification of agriculture and tre- Dmendous progress toward increasing yields in major crops Materials and Methods over the last decades, the goal to reduce the problems associated Plant and Fungal Material, Yield Experiments. Barley was grown in with hunger is far from being reached (1). Major causes for crop a 2:1 mixture of expanded clay (Seramis, Masterfoods, Verden, losses are abiotic and biotic stresses due to unfavorable climate Germany) and Oil-Dri (Damolin, Mettmann, Germany) in a and plant diseases and pests. Increased plant productivity, growth chamber at 22°C͞18°C day͞night cycle, 60% relative therefore, relies on a high chemical input and is achieved at the humidity, and a photoperiod of 16 h (240 ␮mol⅐mϪ2⅐sϪ1 photon expense of detrimental effects on the environment (2, 3). flux density) and fertilized weekly with 20 ml of a 0.1% Wuxal Abiotic-stress tolerance can be evoked in crops by the exploi- top N solution (Schering, N͞P͞K: 12͞4͞6). Hydroponic cultures tation of worldwide abundant endophytic arbuscular mycorrhiza contained expanded clay (Seramis, Masterfoods) as substrate. fungi, which live in reciprocally beneficial relationships with For inoculation with P. indica,2gofmycelium were added to Ϸ80% of land plants (4). However, mycorrhizal plants, albeit 300 g of substrate before sowing. P. indica was propagated in effective against many root diseases (5, 6), often show enhanced liquid Aspergillus minimal medium (14). For yield evaluations, susceptibility to biotrophic leaf pathogens (7, 8). On the other barley was sown in soil containing P. indica mycelium (4 g in hand, ascomycete have been frequently reported to 300 g of substrate) and grown for 4 weeks in the growth chamber. protect against plant pathogens and pests. Grasses (Poaceae) and Before transplantation to outdoor conditions, root samples were fungi of the family Clavicipitaceae have a long history of asso- checked for P. indica infestation. In the beginning of April 2004, ciations, ranging from mutualism to antagonism (9). These fungi when plants reached growth stage (GS) 30 (16), they were are strictly confined to upper parts of the plant, grow only transplanted into 6-liter Mitscherlich pots (Stoma, Siegburg, intercellularly, and exert a rather narrow host range. A critical Germany) (six plantlets per pot) and filled with a mixture of a review of the literature suggests that the beneficial action of loam (loess) soil and sand (1:2). The preceding crop grown in the these endophytes is based on direct antimicrobial and insecti- soil was potato. Soil nutrient additives were 0.25 g of N, 0.4 g of cidal activity due to alkaloid production. P, 1.6 g of K, and 0.2 g of Mg; N was applied a second time at We used the cereal model plant barley (Hordeum vulgare L.) to test whether growth-promoting activity of the recently dis- covered root-endophytic fungus Piriformospora indica (10) as- Abbreviations: GR, glutathione reductase; GSH, reduced glutathione. sociates with agronomically desirable traits. Discovered in the †F.W., B.A., and H.B. contributed equally to this work. Indian Thar desert in 1997 (11), P. indica has been recently **To whom correspondence should be addressed. E-mail: [email protected] related to the [ordo nov.] (form genus Rhizoctonia; giessen.de. Hymenomycetes, ) on the basis of an alignment of © 2005 by The National Academy of Sciences of the USA

13386–13391 ͉ PNAS ͉ September 20, 2005 ͉ vol. 102 ͉ no. 38 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504423102 Downloaded by guest on September 26, 2021 a rate of 0.25 g per pot, 2 weeks after planting (GS 32). The fungicide Opus Top (250 g⅐literϪ1 Fenpropimorph and 84 g⅐literϪ1 Epoxiconazole; BASF, Ludwigshafen, Germany) was sprayed at a rate of 1.5 liter⅐hectare (ha)Ϫ1 to control powdery mildew and Rhynchosporium secalis. Both systemic fungicides are transported acropetally via xylem and do not reach roots. Aphids and the cereal beetle Oulema spp. were controlled during anthesis by using the insecticide Karate (100 g⅐literϪ1 lambda- Cyhalothrin, Syngenta Agro, Basel) at a rate of 150 ml⅐haϪ1. The presence of P. indica was monitored microscopically throughout the vegetation period. Analysis of powdery mildew infections was done in a de- tached-leaf-segment assay on agar plates containing 0.4% benz- imidazole to inhibit leaf senescence. Plants were inoculated with 15 conidia⅐mmϪ2 (for macroscopic evaluation) or with 25 conidia⅐mmϪ2 (for microscopy) of B. graminis f.sp. hordei, race A6. For gene expression studies, leaves were inoculated with 80 conidia⅐mmϪ2. Colonies were counted at 7 days after inocula- tion. Microscopic inspection of powdery-mildew-infected leaves was done by determining the frequency of the three different interaction types. Cells showing a hypersensitive response were detected by their whole-cell autofluorescence. Successful pene- tration was ascertained by the detection of haustoria formation or the development of elongated secondary hyphae (17). ‘‘Non- penetrated cells’’ are those in which fungal penetration attempts were unsuccessful. For root inoculation with pathogens, oat kernels colonized by F. culmorum strain KF 350 or Cochliobolus sativus were used. Kernels (1 g) were added to 300 g of substrate before sowing. The control pots were amended with1gofautoclaved inoculum. For inoculum production, kernels were autoclaved (125°C, 25 min), inoculated with conidia of F. culmorum or C. sativus, and incubated for 1 week at room temperature.

Biochemical Measurements. Ascorbate was determined by using SCIENCES the bipyridyl method (18). Dehydroascorbate reductase activity AGRICULTURAL was assayed spectrophotometrically at 265 nm as reduced glu- tathione (GSH)-dependent dehydroascorbate oxidation (19). The assay mixture contained 50 mM sodium phosphate buffer (pH 6.5), 0.1 mM Na2EDTA, 20 ␮M dehydroascorbate, 50 ␮M GSH, and 20- to 100-␮l extracts in a total volume of 2.3 ml. Fig. 1. Colonization pattern of P. indica in barley roots. (a) Fungal hyphae Glutathione concentrations ([GSH] and [oxidized glutathione]) enter roots via root hairs from 10-day-old plants. The fungus forms pear- were measured as described in ref. 20 with slight modifications. shaped chlamydospores within root hairs and proceeds into rhizodermis cells. (b) The fungus grows into the root cortex tissue. (c) Longitudinal section. The Briefly, 0.3 g of plant tissue was mixed with 3 ml of 2% fungus was not detected in the central part of the roots beyond the endoder- sulfosalicylic acid containing 0.15 g of ascorbic acid and 1 mM mis. Fungal structures were visualized by 0.01% acid fuchsin-lactic acid (28) Na-EDTA per 100 ml. The sample was centrifuged (10,000 ϫ g (red in a and c), or they were stained for mitochondrial respiratory activity by at 4°C for 10 min), and the supernatant was either used for the succinate dehydrogenase assay (29) (black in b). analysis directly or stored at Ϫ20°C. For glutathione determi- nation, 30 ␮l of supernatant was assayed in a total volume of 1 ml of phosphate buffer containing 0.6 mM 5.5Ј-dithiobis(2- barley plants as described in ref. 23. Blots were hybridized with nitrobenzoate) (DTNB), 0.5 units͞ml human glutathione reduc- 32P-labeled JIP-23 [a 1,100-bp fragment of nucleotide with tase (GR), and 0.3 mM NADPH. The reduction of DTNB to Entrez database accession no. X98124 (24)]-, BCI-1 [a 1,000-bp nitrothiobenzoate was determined spectrophotometrically at fragment of accession no. U56406 (25)]-, and barley PR-5 [a 412 nm and 25°C and was related to a calibration curve. 600-bp fragment of accession no. AJ276225 (26)]-specific probes Recombinant human GR was prepared as described in ref. 21. and, after removal of the probes, hybridized with a 28S rRNA To measure GR activity, 0.5 g of plant material was mixed with probe to confirm equal transfer of RNA onto the membrane. To 2 ml of 0.1 M Tris, 1 mM EDTA, and 7.5% wt͞vol polyvinylpyr- exclude the presence of P. indica in leaves from root-infested rolidone (pH 7.8). After centrifugation, protein was determined plants, RT-PCR reactions were performed with leaf material by in the supernatant according to Bradford (22) by using BSA as using primers specific for the P. indica tef gene (27). the standard. GR activity was measured spectrophotometrically Ϫ1 Ϫ1 Results and Discussion (for NADPH, ␧340 ϭ 6.22 mM ⅐cm ) in 47 mM potassium phosphate, 200 mM KCl, and 1 mM EDTA (pH 6.9) at 25°C (21). P. indica Colonizes Root Cortical Cells and Enhances Yield in Barley. A baseline was monitored in the presence of plant extract and Microscopic inspection of barley plants grown in P. indica- 100 ␮M NADPH to account for NADPH oxidase activity. The inoculated substrate showed that the fungus enters roots pri- reaction was then started with 1 mM glutathione disulfide. marily via root hairs (Fig. 1a) and, later, grows intracellularly in the root cortex (Fig. 1b). Hyphae were detected neither in the Northern Blot and RT-PCR Analysis. Northern blots were prepared central part of the roots beyond endodermis (Fig. 1c) nor in from 10 ␮g of total RNA extracted from leaves of 3-week-old stems or leaves. This observation was verified by performing

Waller et al. PNAS ͉ September 20, 2005 ͉ vol. 102 ͉ no. 38 ͉ 13387 Downloaded by guest on September 26, 2021 Fig. 2. Impact of P. indica on salt-stress tolerance and root infections by F. culmorum.(a) Shoot fresh weight of P. indica and control (noninfested) plants was determined in 5-week-old plants that had been grown for the final Fig. 3. Ascorbate, dehydroascorbate (DHA) content, and DHA reductase 2 weeks in the presence of 100 or 300 mM NaCl, in hydroponic culture. Data (DHAR) activity in P. indica-infested roots. Ascorbate content (a), DHA content points are representative of three independent experiments. Error bars, SD. (b), and DHAR activity (c) were measured in roots of 1-, 2-, and 3-week-old (b) Plant phenotypes demonstrating the protective potential of P. indica P. indica-infested (shaded columns) and control (free of P. indica, open toward F. culmorum.(c) Mean fold change of root and shoot weights relative columns) barley plants. Values are means of three or four samples. Similar to noninfested (no P. indica or F. culmorum) 4-week-old plants. CP, P. indica- results have been obtained with three independent sets of experiments. Error infested; CF, Fusarium-infected; PF, P. indica-infested, Fusarium-infected. Er- bars, SD. Within each frame, * and ** indicate statistically significant differ- ror bars, SD. Columns labeled with the same letter represent not-significantly- ences between roots of infested and noninfested plants (unpaired Student t different means, according to multiple unpaired Student t tests (P Ͻ 0.05), test; *, P Ͻ 0.05; **, P Ͻ 0.01). after ANOVA.

Mitscherlich pots in an open-air field station in spring͞summer RT-PCR reactions with leaf material using primers specific for 2004. P. indica-infested Annabell showed an increase in grain the P. indica tef gene (data not shown). During the first 4 weeks yield of 11%, mainly because of a higher number of ears per plant of barley development, shoot fresh weight of infested plants was (Table 1). In cultivar Ingrid, the grain yield increase was 5.5% up to 1.65 times higher compared with control plants (see Fig. (Table 1). A repetition of the complete set of yield experiments 2, first two columns). To assess whether this early increase in gave similar results, with elevated grain yields observed in the biomass would also lead to higher grain yields, two barley P. indica-infested plants. Notably, P. indica also increased grain cultivars, including the elite cultivar Annabell, were tested in yield in soils with a high nitrogen supply (data not shown).

Table 1. Effect of P. indica infestation on yield parameters in barley Cultivar P. indica Yield, g per potϪ Straw yield, g per potϪ Harvest index TGW, g Ears per pot Grains per ear

Ingrid Ϫ 50.3 Ϯ 1.56 52.4 Ϯ 3.14 0.96 Ϯ 0.04 48.0 Ϯ 0.90 47.8 Ϯ 2.22 22.4 Ϯ 0.93 ϩ 53.1 Ϯ 2.76 53.2 Ϯ 1.02 0.99 Ϯ 0.06 48.1 Ϯ 0.65 51.5 Ϯ 1.29* 22.5 Ϯ 1.86 Annabell Ϫ 53.9 Ϯ 3.61 46.4 Ϯ 3.46 1.17 Ϯ 0.11 50.6 Ϯ 1.28 47.3 Ϯ 3.50 23.1 Ϯ 1.41 ϩ 59.9 Ϯ 1.73** 48.0 Ϯ 1.35 1.25 Ϯ 0.04 50.6 Ϯ 0.96 50.0 Ϯ 2.45 23.7 Ϯ 0.98

To assess whether P. indica infestation affects yields, elite barley cultivar Annabell (Saatzucht Ackermann, Irlbach, Germany) and cultivar Ingrid were grown in soil containing mycelium inoculum of P. indica in Mitscherlich pots at an open-air field station near Marburg, central Hesse, Germany. Values given are means of six pots (with six plants per pot). TGW, thousand-grain weight; * and ** indicate statistically significant differences between infested and noninfested plants (unpaired Student t test; * P Ͻ 0.05; **, P Ͻ 0.01).

13388 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504423102 Waller et al. Downloaded by guest on September 26, 2021 Table 2. Glutathione content in P. indica-infested barley roots and leaves [Glutathione], nmol⅐gϪ1 f.wt. in roots [Glutathione], nmol⅐gϪ1 f.wt. in leaves Time after sowing, weeks Control P. indica % of control Control P. indica % of control

1 200 Ϯ 42 205 Ϯ 16 103 405 Ϯ 67 403 Ϯ 54 99.5 2 230 Ϯ 32 273 Ϯ 35 119 370 Ϯ 78 467 Ϯ 48 126.3 3 223 Ϯ 46 309 Ϯ 49 139 510 Ϯ 54 798 Ϯ 113 156.5*

Total glutathione (GSH and oxidized glutathione) was measured according to procedures described in ref. 20. Values are means of three samples. Similar results were obtained with three independent sets of experiments. *, P Ͻ 0.05; for intercohortal analyses the unpaired Student t test was applied. f.wt., fresh weight.

P. indica-Inoculated Plants Are Tolerant to Salt Stress and More P. indica infestation. Ascorbate levels were consistently higher 1, Resistant to Root Pathogens. We analyzed the fungus’ potential to 2, and 3 weeks after root infestation (Fig. 3a), whereas levels of protect barley from salt stress. When noninfested barley seed- dehydroascorbate were reduced (Fig. 3b). At the same time, the lings were exposed for 2 weeks to moderate (100 mM NaCl) and activity of ascorbate recycling dehydroascorbate reductase in- high (300 mM NaCl) salt concentrations in hydroponic culture, creased during root infestation, reaching 2.2-fold higher levels 3 they showed increasing leaf chlorosis and reduced growth (Fig. weeks after infestation with P. indica (Fig. 3c). Concomitantly, 2a). The detrimental effect of moderate salt stress was com- we found slightly (but not significantly) enhanced total gluta- pletely abolished by P. indica, as shown by the fact that infested thione concentrations (Table 2). Corresponding GR activity was plants produced higher biomass than did nonstressed control only slightly enhanced early after infestation (1 week; Table 3). plants under these conditions. However, under high salt-stress It can be reasoned that higher antioxidant levels protect roots conditions, both noninfested and infested plants exhibited a from cell death provoked by root pathogens F. culmorum and C. severe biomass reduction. sativus. Because production of reactive oxygen species and We addressed the question of whether P. indica-infested plants host-cell killing is a prerequisite for successful fungal develop- would also be more resistant to biotic stress. Barley was grown ment and pathogenesis of necrotrophic nourishment (32), we in soil containing macroconidia of the necrotrophic fungal speculate that elevated ascorbate levels could be the reason for pathogen F. culmorum, and root and shoot biomass was re- the observed control of necrotrophic pathogens in barley roots. corded. We found that P. indica-infested plants are more resis- tant to root diseases (Fig. 2b). Fusarium root infection caused a P. indica Induces Systemic Disease Resistance. We wanted to know Ͼ12-fold decrease in root and shoot fresh weight of 4-week-old whether root infestation by P. indica protects barley leaves from plants, compared with control plants, which were infested with fungal infections. Because the fungus does not infest leaves, pro- neither P. indica nor F. culmorum (Fig. 2c). In the presence of tection against leaf pathogens would require a systemic response

P. indica, this devastating effect of F. culmorum infection was emanating from the root that has not been described for a basid- SCIENCES strongly diminished. Root and shoot fresh weight was reduced iomycete in cereals. We recorded the outcome of P. indica infes- AGRICULTURAL only 2-fold in P. indica-infested plants, compared with the tation on leaf infections by the biotrophic barley powdery mildew 12-fold decrease in controls with F. culmorum alone. Similar fungus, B. graminis f.sp. hordei, which belongs to the order Ery- results were obtained when we tested resistance to the root- siphales, comprising pathogenic fungi of a wide range of crop plants. pathogenic fungus C. sativus (data not shown), which shows a We found a reduction in powdery mildew infection in P. indica- hemibiotrophic nourishment strategy. In axenic culture, P. indica infested plants (Fig. 4a). The relative decrease of disease, assessed did not exhibit antifungal activity to F. culmorum or C. sativus by powdery mildew colony numbers, was 48% in the second- (data not shown), indicating that the protective potential of the youngest leaves (data not shown) and 58% in the youngest leaves endophytic fungus does not rely on antibiosis. These results show of 3-week-old P. indica-infested plants. Importantly, by microscopic that P. indica exerts beneficial activity against two major cereal analysis, we uncovered higher frequencies of a hypersensitive pathogens that cause enormous worldwide economic losses (30). reaction, including a host-cell-death response, as well as a cell-wall- associated defense, confirming that the pathogen is arrested by an P. indica-Infested Roots Show Higher Antioxidant Capacity. The active plant response (Fig. 4b). Because of these plant responses, the beneficial protective activity exerted by P. indica against root biotrophic fungus cannot establish nutrition organs (haustoria). pathogens with necrotrophic nourishment strategies prompted Together with the biochemical data (see Tables 2 and 3), these data us to ask whether the antioxidant status of the infested roots was substantiate that the systemic plant response, leading to a reduction altered by the endophyte. Because ascorbic acid is a major of powdery mildew infection, is the result of induced resistance antioxidant buffer and free-radical scavenger (31), we recorded rather than being caused by antibiotics secreted by P. indica. root concentrations of this compound during the first 3 weeks of We determined systemically elevated antioxidants in leaves from

Table 3. GR activity in P. indica-infested barley roots and leaves GR activity in leaves, milliunits͞mg GR activity in roots, milliunits͞mg protein protein Time after sowing, weeks Control P. indica % of control Control P. indica % of control

1 24.7 Ϯ 11.0 32.6 Ϯ 15.9 132 11.5 Ϯ 9.2 22.8 Ϯ 9.0 198* 2 44.3 Ϯ 9.8 44.6 Ϯ 11.5 101 7.9 Ϯ 4.2 22.4 Ϯ 6.0 284** 3 47.5 Ϯ 7.5 37.5 Ϯ 12.2 79 9.5 Ϯ 5.0 16.2 Ϯ 6.0 171

Total GR activity was measured according to procedures described in ref. 20. Values are means of three samples. Similar results were obtained with two independent sets of experiments. *, P Ͻ 0.05; **, P Ͻ 0.005; for intercohortal analyses, the unpaired Student t test was applied.

Waller et al. PNAS ͉ September 20, 2005 ͉ vol. 102 ͉ no. 38 ͉ 13389 Downloaded by guest on September 26, 2021 Fig. 5. Expression of potential marker genes for known resistance pathways. Shown are Northern blots of 10 ␮g of total RNA from leaves of 3-week-old barley plants with roots infested (ϩ) or not infested (Ϫ) with P. indica. Plants were harvested either directly or at 12 or 24 h after inoculation with powdery mildew (B. graminis f.sp. hordei, race A6). Blots were hybridized with radio- labeled JIP-23, BCI-1, and PR-5 probes. After removal of the probe, they were subsequently hybridized with a 28S rRNA probe to confirm equal transfer of RNA onto the membrane.

disease resistance is not associated with elevated foliar steady- Fig. 4. Systemic disease resistance conferred by P. indica.(a) Severity of state mRNA levels of the barley chemically induced gene 1 powdery mildew infection (disease index) was calculated as colonies produced by B. graminis on the youngest leaf of 3-week-old barley plants (cultivar (BCI-1), encoding a 13-lipoxygenase (25). BCI-1 and its cereal Ingrid), with roots either not infested (Control) or infested (P. indica). (b) orthologues are strongly responsive to SA, to resistance-inducing Cellular responses to powdery mildew attack were evaluated by counting cells chemicals mimicking SA, such as acibenzolar-S-methyl, and to showing an active defense response, a hypersensitive response of the whole JA (25, 37, 38). Consistently, the jasmonate-induced protein cell (Hypersensitive reaction), a local defense stopping a penetration attempt gene 23 (JIP-23), expression of which is sensitive to exogenous (Nonpenetrated cell), or a successful penetration (Haustoria), visible as the jasmonate and elevated levels of endogenous jasmonate (24), did successful formation of a fungal haustorium in the cell. Error bars, SD. * and not respond to P. indica infestation. Moreover, the pathogenesis- ** indicate statistically significant differences between leaves of infested and related gene 5 (PR-5), indicative for pathogen infections (26, 39), Ͻ Ͻ noninfested plants (unpaired Student t test; *, P 0.05; **, P 0.01). was strongly expressed 24 h after powdery mildew inoculation, although essentially to the same extent in both control and P. indica-infested plants. These results indicate that P. indica might P. indica-infested plants. Initially, we measured ascorbate but did induce systemic disease resistance by an as yet unknown signal- not detect consistent major changes due to P. indica infestation. ing pathway. However, when we analyzed the glutathione pool (GSH and Together, we show here that P. indica-infested barley is more oxidized glutathione), constituting a major cellular thiol-disulfide resistant to abiotic and biotic stress and that the reprogrammed redox buffer, an enhanced foliar antioxidant capacity was found metabolic state, which includes an enhanced antioxidant capac- (Table 2). Strikingly, GR activity (Table 3) was also enhanced in ity and an activation of the glutathione–ascorbate cycle, does not leaves during the first 3 weeks of P. indica infestation, corroborating negatively affect grain yield. Our findings challenge the notion systemic induction of antioxidant capacity by P. indica. Notably, that plant resistance to pathogens and pests induced by micro- enhanced GSH concentrations are also associated with resistance organisms necessarily involves internal, physiological, and eco- to powdery mildew infections in barley, as mediated by major logical costs resulting in yield losses (40, 41). Because P. indica, resistance genes (33). Hence, P. indica might cause systemic resis- unlike arbuscular mycorrhiza fungi, can easily be propagated on tance by ameliorating the antioxidative capacity of barley plants. a large scale in axenic culture in the absence of a host plant (13), Consistently, it has been shown recently that fungal and algal we suggest consideration of this endophyte as a tool for sustain- partners in a lichen mutually benefit from the interaction by an able agriculture. Exploitation of P. indica may not only comple- enhanced glutathione-based antioxidative capacity (34), enabling a ment crop-growing strategies, but may also serve as a model higher protection against stress. system to study molecular traits affecting disease resistance and To determine whether any of the known cereal defense grain yields in cereals. pathways was activated by P. indica, we tested expression of selected marker genes in barley leaves. Initial experiments using We thank Ajit Varma for discussions and support in the initial stage of RT-PCR and cDNA microarrays indicated that P. indica does the project, Markus Kolmer and Wolfgang Friedt for support in the yield not induce expression of large sets of pathogenesis-related or experiments at the Rauischholzhausen experimental station, Sachin Deshmukh for performing RT-PCR to detect P. indica in barley jasmonic-acid (JA)-induced genes (data not shown). Northern tissues, Carin Jansen for help in preparing microscopic images, and blot experiments confirmed that gene markers indicative for B. Harrach for support in antioxidant measurements. This work was salicylic acid (SA) (35) and JA (36) accumulation were not supported by Deutsche Forschungsgemeinschaft Grants FOR 343 and expressed. As displayed in Fig. 5, P. indica-induced systemic SFB 299.

1. Food and Agriculture Organization of the United Nations (2004) The State of Reynolds, H. L., Hooper, D. U., Lavorel, S., Sala, O. E., Hobbie, S. E., et al. Food and Agriculture 2003–04, www.fao.org͞documents͞show࿝cdr. (2000) Nature 405, 234–242. asp?url࿝fileϭ͞docrep͞006͞Y5160E͞Y5160E00.HTM. 3. Parmesan, C. & Yohe, G. (2003) Nature 421, 37–42. 2. Chapin, F. S., III, Zavaleta, E. S., Eviner, V. T., Naylor, R. L., Vitousek, P. M., 4. Newman, E. I. & Reddell, P. (1987) New Phytol. 106, 745–751.

13390 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504423102 Waller et al. Downloaded by guest on September 26, 2021 5. Azco´n-Aguilar, C. & Barea, J. M. (1996) Mycorrhiza 6, 457–464. 24. Hause, B., Demus, U., Teichmann, C., Parthier, B. & Wasternack, C. (1996) 6. Borowicz, V. A. (2001) Ecology 82, 3057–3068. Plant Cell Physiol. 37, 641–649. 7. Gernns, H., von Alten, H. & Poehling, H.-M. (2001) Mycorrhiza 11, 25. Besser, K., Jarosch, B., Langen, G. & Kogel, K.-H. (2000) Mol. Plant Pathol. 237–243. 1, 277–286. 8. Shaul, O., Galili, S., Volpin, H., Ginzberg, I., Elad, Y., Chet, I. & Kapulnik, Y. 26. Hahn, M., Jungling, S. & Knogge, W. (1993) Mol. Plant–Microbe Interact. 6, (1999) Mol. Plant–Microbe Interact. 12, 1000–1007. 745–754. 9. Schardl, C. L., Leuchtmann, A. & Spiering, M. J. (2004) Annu. Rev. Plant Biol. 27. Bu¨tehorn, B., Rhody, D. & Franken, P. (2000) Plant Biol. 2, 687–692. 55, 315–340. 28. Kormanik, P. P. & McGraw, A.-C. (1982) in Methods and Principles of 10. Verma, S., Varma, A., Rexer, K.-H., Hassel, A., Kost, G., Sarabhoy, A., Bisen, Mycorrhizal Research, ed. Schenck, N. C. (Am. Phytopathol. Soc., St. Paul, MN), P., Bu¨tenhorn, B. & Franken, P. (1998) Mycologia 90, 896–903. pp. 37–45. 11. Varma, A., Singh, A., Sudha, S., Sharma, J., Roy, A., Kumari, M., Rana, D., 29. Weber, R. W. S., Wakley, G. E. & Pitt, D. (1998) Mycologist 12, 174–179. Thakran, S., Deka, D., Sahay, N. S., et al. (2001) in Mycota IX, ed. Hock, B. 30. Kumar, J., Scha¨fer, P., Hu¨ckelhoven, R., Langen, G., Baltruschat, H., Stein, E., (Springer, Heidelberg), pp. 125–150. Nagarajan, S. & Kogel, K.-H. (2002) Mol. Plant Pathol. 3, 185–195. 12. Weiss, M., Selosse, M.-A., Rexer, K.-H., Urban, A. & Oberwinkler, F. (2004) 31. Pignocchi, C. & Foyer, C. H. (2003) Curr. Opin. Plant Biol. 6, 379–389. Mycol. Res. 108, 1003–1010. 32. Govrin, E. M. & Levine, A. (2000) Curr. Biol. 10, 751–757. 13. Varma, A., Verma, S., Sudha, Sahay, N., Bu¨tehorn, B. & Franken, P. (1999) 33. Vanacker, H., Carver, T. L. & Foyer, C. H. (2000) Plant Physiol. 123, Appl. Environ. Microbiol. 65, 2741–2744. 1289–1300. 14. Peškan-Bergho¨fer, T., Shahollari, B., Giong, P. H., Hehl, S., Markert, C., 34. Kranner, I., Cram, W. J., Zorn, M., Wornik, S., Yoshimura, I., Stabentheiner, Blanke, V., Kost, G., Varma, A. & Oelmu¨ller, R. (2004) Physiol. Plant 122, E. & Pfeifhofer, W. (2005) Proc. Natl. Acad. Sci. USA 102, 3141–3146. 465–477. 35. Ryals, J., Lawton, K. A., Delaney, T. P., Friedrich, L., Kessmann, H., 15. Sherameti, I., Shahollari, B., Venus, Y., Altschmied, L., Varma, A. & Oel- Neuenschwander, U., Uknes, S., Vernooij, B. & Weymann, K. (1995) Proc. mu¨ller, R. (2005) J. Biol. Chem., in press, 10.1074͞jbc.M500447200. Natl. Acad. Sci. USA 92, 4202–4205. 16. Zadoks, J. C., Chang, T. T. & Konzak, C. F. (1974) Weed Res. 14, 415–421. 36. Verhagen, B. W. M., Glazebrock, J., Zhu, T., Chang, H.-S., van Loon, L. C. & 17. Hu¨ckelhoven, R. & Kogel, K.-H. (1998) Mol. Plant–Microbe Interact. 11, Pieterse, C. M. J. (2004) Mol. Plant–Microbe Interact. 17, 895–908. 292–300. 37. Go¨rlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, 18. Kno¨rzer, O. C., Durner, J. & Bo¨ger, P. (1996) Physiol. Plant 97, 388–396. K.-H., Oosterdorp, M., Staub, T., Ward, E., Kessmann, H., et al. (1996) Plant 19. Klapheck, S., Zimmer, I. & Cosse, H. (1990) Plant Cell Physiol. 31, 1005–1013. Cell 8, 629–643. 20. Becker, K., Gui, M., Traxler, A., Kirsten, C. & Schirmer, R. H. (1994) 38. Schaffrath, U., Freydl, E. & Dudler, R. (1997) Mol. Plant–Microbe Interact. 10, Histochemistry 102, 389–395. 779–783. 21. Nordhoff, A., Bu¨cheler, U. S., Werner, D. & Schirmer, R. H. (1993) Biochem- 39. Valle´lian-Bindschedler,L., Me´trauxJ.-P. & Schweizer, P. (1998) Mol. Plant– istry 32, 4060–4066. Microbe Interact. 11, 702–705. 22. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. 40. Brown, J. K. M. (2003) Trends Genet. 19, 667. 23. Logemann, J., Schell, J. & Willmitzer, L. (1987) Anal. Biochem. 163, 16–20. 41. Heil, M. (2002) Curr. Opin. Plant Biol. 5, 345–350. SCIENCES AGRICULTURAL

Waller et al. PNAS ͉ September 20, 2005 ͉ vol. 102 ͉ no. 38 ͉ 13391 Downloaded by guest on September 26, 2021