Sebacina vermifera Promotes the Growth and Fitness of Nicotiana attenuata by Inhibiting Ethylene Signaling

Sebacina vermifera , a growth-promoting endophytic fungus, significantly increases Nicotiana attenuata ’s growth but impairs both its herbivore resistance and its accumulation of the costly, jasmonate (JA)-regulated, defense protein, trypsin proteinase inhibitor (TPI). To determine if the fungi’s growth-promoting effects can be attributed to lower TPI-related defense costs, we inoculated transformed N. attenuata plants silenced in their ability to synthesize JA, JA-isoleucine (Ile), and TPI by antisense (as- lox3 and as- td ) and inverted repeat (ir- tpi ) expression, and found that inoculation promoted plant growth as in untransformed (WT) plants. Moreover, herbivore-elicited increases in JA and JA-Ile concentrations did not differ between inoculated and uninoculated WT plants. However, inoculation significantly reduced the morphological effect of 1-aminocyclopropane-1-carboxylic acid (ACC) on WT seedlings in a triple response assay, suggesting that ethylene signaling was impaired. Furthermore, S. vermifera failed to promote the growth of N. attenuata plants transformed to silence ethylene production (ir- aco ). Inoculating WT plants with S. vermifera decreased the ethylene burst elicited by applying Manduca sexta oral secretions (OS) to mechanical wounds. Accordingly, OS-elicited transcript levels of the ethylene synthesis genes NaACS3 , NaACO1 , and NaACO3 in inoculated plants were significantly lower compared to these levels in uninoculated WT plants. Inoculation accelerated germination in WT seeds; however, uninoculated WT seeds germinated as rapidly as inoculated seeds in the presence of the ethylene scrubber KMnO 4 . In contrast, neither inoculation nor KMnO 4 exposure influenced the germination of ir- aco seeds. We conclude that S . vermifera increases plant growth by impairing ethylene production independently of JA signaling and TPI production.


Introduction
Plants that associate with beneficial rhizosphere microorganisms, which include symbiotic and other endophytic and free-living rhizobacteria, often grow better than plants that don't (Glick, 1995;Varma et al., 1999;Strack et al., 2003;Barazani et al., 2005;Waller et al., 2005). The symbiotic associations of plants with arbuscular mycorrhizae (AM), ectomycorrhizal fungi, and nitrogen-fixing bacteria are referred to as mutualistic interactions. Symbiotic fungi or bacteria benefit from the plants' carbohydrates, while plants benefit when the supply of more stationary nutrients such as N, P, Ca, Mg, Zn, Cu, and Fe is increased. The sequence of events that leads to the development of symbiotic association involves regulating defenserelated genes, which have been characterized during the early establishment of AM symbiosis (Kapulnik et al., 1996;Garcia-Garrido and Ocampo, 2002;Liu et al., 2003;Balestrini and Lanfranco, 2006). In addition, phytohormones, usually associated with plants' responses to biotic stresses, were shown to play a role in mycorrhizal development. In Allium sativum, for example, treatment with jasmonic acid (JA) was shown to stimulate mycorrhizal development (Regvar et al., 1996), and JA and its conjugated form JA-isoleucine (Ile) accumulated in the roots of Hordeum vulgare colonized with G. intraradices (Hause et al., 2002). Furthermore, silencing the AOC gene, which encodes allene-oxide-cyclase, an enzyme of the JA biosynthesis pathway, suppressed AM colonization, suggesting that jasmonates are associated with the establishment of a strong carbon sink in the roots (Hause et al., 2002;Strack et al., 2003;Isayenkov et al., 2005). In contrast, the mycorrhization of tobacco with G. mosseae reduced salicylic acid (SA) levels in the plant, and colonization by the fungus was suppressed by constitutive SA synthesis (Medina et al., 2003).
In addition to establishing symbiotic associations, plants are associated with a diverse range of free-living microorganisms which increase plant performance(Glick, in grain yield and resistance among the pathogenic fungi of barley inoculated with Piriformospora indica to modifications in the anti-oxidative status of barley (Hordeum vulgare) (Waller et al., 2005). P. indica, a beneficial endophytic fungus (Sebacinales), was first isolated in India from the rhizosphere Prospis juliflora and Zizyphus nummularia (Verma et al., 1998). P. indica was shown to increase the survival of regenerated tobacco plantlets  and to increase the root and shoot biomass of Zea mays, Nicotiana tabacum, and Petroselinum crispum , as well as of Spilanthes calva and Withania somnifera (Rai et al., 2001).
Recently, we reported that P. indica and its genetically related species Sebacina vermifera increase the growth and fitness of Nicotiana attenuata (Barazani et al., 2005). However, the increased performance of inoculated N. attenuata came at the expense of the plant's resistance to attack from the larvae of one of the plant's most important lepidopteran insect herbivores, Manduca sexta. The decrease in herbivore resistance could be attributed to the down-regulation of trypsin protein inhibitor (TPI) activity (Barazani et al., 2005). Plants recognize that the specialist M. sexta is attacking when they are wounded and elicitors present in the larvae's oral secretions are introduced (OS) into the wounds during feeding (Halitschke et al., 2001). Applying OS to wounds is sufficient to induce a burst of two phytohormones, ethylene and JA, which activate a wide array of genes responsible for direct and indirect defenses, including the gene responsible for the accumulation of TPI.
Consequently, the specialist larvae grow more slowly, presumably because the protein digestion in their gut is inhibited (Zavala et al., 2004b). However, the resistance benefits of TPI expression come at a substantial fitness cost for the plant. N. attenuata plants expressing TPIs produce 20% fewer seeds than do isogenic plants transformed to silenced TPI production; restoring TPI production by transforming an ecotype of N. attenuata naturally deficient in TPI production reduces lifetime seed production by 20% (Zavala et al., 2004a). Hence we hypothesized that the increase in growth and seed production that N. attenuata realizes from associating with S. alterations in ethylene signaling. We show that: (i) increases in plant performance related to the fungus are independent of JA and TPI, but depend on the ability of the plant to produce ethylene; (ii) the beneficial effects of S. vermifera on seed germination and seedling growth are ethylene dependent; and (iii) the OS-induced ethylene emission and increased transcript accumulation of ethylene biosynthesis genes are reduced in S. vermifera-inoculated plants compared to uninoculated plants. Growth promotion by S. vermifera is unaffected in JA-, JA-Ile-, or TPI-silenced plants To determine whether the growth-promoting effect of S. vermifera resulted from the attenuation of the growth-related costs of TPI production or from the jasmonate signals that elicit TPI, we compared stalk lengths in inoculated and uninoculated WT plants and in plants transformed to silence JA and JA-Ile in antisense (as) orientation of lipoxygenase-3 (as-lox3), and threonine-deaminase (astd), respectively; and TPI levels by inverted repeat construct (ir-tpi). S. vermiferainoculation significantly increased stalk lengths of WT plants (ANOVA with repeated measures, F 1,23 =76.85; P<0.01). At the end of the growth phase, 56 d after germination, inoculated WT were 6.7% taller (t-test, F 1,28 =10.03; P<0.01) than uninoculated plants ( Fig. 2A). In addition, inoculated WT plants started to flower 1 day earlier than uninoculated plants, a difference which was highly significant ( Fig P<0.01; as-td: F 1,28 =12.39; P<0.01), and at the end of the growth phase, S. vermiferainoculated as-lox3 and as-td plants were 6.7 and 3.1% taller than uninoculated plants, respectively ( Fig. 2B,C; t-test, as-lox3: F 1,28 =21.14; P<0.01; as-td: F 1,25 =4.79; P=0.03). These results demonstrate that the growth-promoting effects are independent of the jasmonate signaling required to elicit herbivore defenses in N. attenuata.

TPI activity and transcript accumulation is suppressed in OS
Similar results were found in trials with ir-tpi plants. Inoculation significantly increased the growth of the inoculated transformed plants ( Fig. 2D; ANOVA with repeated measures, F 1,23 =84.04; P<0.01) so that the final stalk lengths of S. vermiferainoculated ir-tpi plants were 6.5% taller than those of uninoculated plants (t-test, F 1,28 =13.40; P<0.01). The day flowering began did not differ between the two inoculation treatments ( Fig. 2D; t-test, F 1,28 =0.18; P=0.67). We conclude that the growth-promoting effects of S. vermifera can not be attributed to an alleviation of the fitness costs of TPI production.

S. vermifera inoculation does not affect the OS-elicited accumulation of JA and JA-Ile
Applying OS to wounded leaves elicits a dramatic JA burst which occurs in concert with a JA-Ile burst (Kang et al., 2006). These two factors have been shown to be responsible for most of the TPI transcript accumulation, as well as for the OSinduced increase in TPI activity (Halitschke and Baldwin, 2003;Kang et al., 2006).
To verify the conclusions obtained from our observations of plant growth in as-lox3 and as-td, we asked whether the JA and JA-Ile bursts were influenced by S. vermifera inoculation. No quantitative or qualitative differences were observed between the amounts of OS-elicited JA (ANOVA with repeated measures, F 1,7 =1.84; P=0.21) and JA-Ile (t-test, F 1,6 =0.29, P=0.61) accumulated in the two inoculation treatments (Fig.   3).

S. vermifera inoculation interferes with ethylene-signaling independently of ACC deaminase activity
The 'triple response assay' is a rapid means of estimating the sensitivity of plants to ethylene and has been successfully used to identify ethylene-insensitive mutants (Ecker, 1995). When dark-grown seedlings are exposed to ethylene, they display shortened root and hypocotyl growth, and a thickening of the hypocotyls, and the curvature of the apical hook becomes exaggerated. Since ACC synthase is frequently the rate-limiting step in ethylene biosynthesis, the germination media is often supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC) to accentuate the triple response phenotype. In the triple response assay of WT seedlings, root and hypocotyl growth were significantly inhibited by the presence of 5 µ M ACC in the media ( Fig. 4; ANOVA Student-Newman-Keuls multiple comparison test, P < 0.05). However, inoculating WT seeds with S. vermifera prior to the triple response assay significantly reduced the inhibitory effect of ACC on root and hypocotyl length ( Fig. 4; ANOVA Student-Newman-Keuls multiple comparison test, To determine whether the above effects are related to the ability of the fungus to degrade ACC by secreting ACC deaminase, the activity of the enzyme was assayed by measuring the amount of α -ketobutyrate produced during ACC cleavage (Penrose and Glick, 2003). By comparing the absorbance of α -ketobutyrate standard curve to the samples we found no evidence for ACC deaminase activity in cultures of S. vermifera, suggesting that the reduced inhibitory effect is not related to the fungus's use of ACC as a nitrogen source.

S. vermifera inoculation increases plant performance by inhibiting ethylene production
The altered growth performance of S. vermifera-inoculated seedlings observed in the triple response assay may be due to changes either in ethylene biosynthesis or in its perception. To examine how inoculation affects ethylene biosynthesis, we first compared the performance of inoculated and uninoculated N. attenuata plants transformed to silence ACC oxidase expression in inverted repeat constructs (ir-aco).
S. vermifera did not increase the performance of inoculated ir-aco plants as it did with WT plants. Stalk lengths of ir-aco plants were also not influenced by inoculation with S. vermifera ( Fig. 5A; ANOVA with repeated measures P>0.05). However, at the end of the growth phase, uninoculated ir-aco plants were 31.8% taller than uninoculated WT plants (compare Fig. 2A and Fig. 5A; t-test, F 1,27 =161.85; P<0.01 ). We therefore hypothesized that S. vermifera's ability to reduce ethylene synthesis in inoculated WT, as-lox3, as-td, and ir-tpi plants was the reason for the increased growth performance of inoculated plants.
In the triple response assay, ACC significantly inhibited the root and hypocotyl growth of ir-aco seedlings by 79.5 and 75.7%, respectively ( Fig. 5B; root: t-test, F 1,6 =21.04; P<0.01; hypocotyl: t-test, F 1,6 =3.43; P<0.01). This demonstrates that ir-aco plants still harbor sufficient ACO activity to induce a triple response. However, the inhibitory effect of ACC on both roots and hypocotyls was significantly reduced by pre-inoculation with S. vermifera ( To learn how the fungus inhibits ethylene production, we measured the transcript accumulation of N. attenuata's ethylene biosynthetic genes by quantitative RT-PCR. OS elicitation in both uninoculated and inoculated plants resulted in the rapid accumulation of NaACS3a transcripts, the first committed step of ethylene biosynthesis. Maximum transcript levels attained were not influenced by inoculation (t-test, F 1,8 =0.01; P=0.90). Six hours after OS elicitation, NaACS3a levels began to decrease; final transcript levels of inoculated plants were significantly lower than those of uninoculated plants ( Fig. 6; t-test, F 1,6 =7.39; P=0.03). In addition, we measured the transcripts levels of three ACO genes, which are involved in the second committed step of ethylene biosynthesis. Compared to transcript levels in uninoculated plants, those in inoculated plants of NaACO1 and NaACO3 were significantly reduced following OS elicitation: by 1.9-fold after 2.5 h and 3.0-fold after 6 h ( Fig. 6; NaACO1 at 2.5 h 1: t-test, F 1,7 =16.72; P<0.01; NaACO3 at 6 h: ttest, F 1,8 =8.34; P=0.02). No differences between the two inoculation treatments were measured in NaACO2 transcripts (Fig. 6). Unlike levels of ethylene biosynthetic genes, levels of the ethylene receptor gene NaETR1 were not affected by either OS elicitation or fungal inoculation ( Supplementary Fig. 1). Whereas the germination of WT seeds on S. vermifera-inoculated media was significantly higher (85%) (F 1,5 =9.54; P=0.03) than on uninoculated media (53%) (Fig. 7), the germination rate (70%) of ir-aco seeds was not influenced by the presence of the fungi (Fig. 7). The presence of the ethylene scrubber, KMnO 4, increased the germination rates of uninoculated WT seeds to the level found in S.

Inoculation accelerates seed germination
vermifera-inoculated seeds (t-test, F 1,6 =5.85; P=0.05). KMnO 4 had no effect on the germination of inoculated and uninoculated ir-aco seeds (Fig. 7). These results are consistent with the hypothesis that reducing ethylene in the headspace of germinating seeds accelerates germination and that inoculating plants with S. vermifera inhibits how much ethylene seeds produce during germination.

S. vermifera (Sebacinales) increases the performance of N. attenuata plants by
down-regulating ethylene production. Previously we showed that the association of S.
vermifera increased the performance of inoculated N. attenuata. This growth benefit was accompanied by a decreased resistance to attack from Manduca sexta larvae, which could be attributed to the down-regulation of trypsin protein inhibitors (TPIs) (Barazani et al., 2005). Here we show that the association with S. vermifera also reduces the transcript levels of NaTPI ( Fig. 1 Inset). Since the production of defense compounds provides a fitness benefit when plants are exposed to herbivores, but In addition to their effect on the nutritional status of a plant, its primary metabolism, and the plants' tolerance to stress, beneficial microorganisms can increase plant growth by modifying endogenous phytohormone levels in the plant (Smith and Read, 1997;Arkhipova et al., 2005;Ryu et al., 2005;Wang et al., 2005;Madhaiyan et al., 2006). Moreover, JA and its conjugated form, JA-Ile, were shown to be involved in the establishment of AM fungi (Hause et al., 2002;Isayenkov et al., 2005). To understand whether the increase in plant performance caused by S.
vermifera is related to changes in phytohormone signaling, we measured the performance of S. vermifera-inoculated transgenic lines that had been independently silenced in two steps of the oxylipin pathways. Silencing the expression of lipoxygenase-3 (NaLOX3) (as-lox3) and threonine-deaminase (NaTD) (as-td) lowers TPI expression and increases plants' vulnerability to herbivores (Halitschke and Baldwin, 2003;Kang et al., 2006). The plant-growth-promoting effects of S.
vermifera were as evident in these jasmonate-impaired transgenic lines as they are in WT lines (Fig. 2), demonstrating that the growth-promoting effects and downregulation of TPIs in inoculated plants (Fig. 1) are not mediated by alterations in JA signaling by S. vermifera inoculation. Further support for this hypothesis was found in measurements of the JA and JA-Ile concentrations, which did not differ between the two inoculation treatments (Fig. 3). Similarly, P. indica (Sebacinales), which is closely related to S. vermifera, had no effect on the regulation of JA-and SA-related related genes on plant growth and fitness of N. attenuata has been previously discussed (Zavala and Baldwin, 2006). Here we show that inoculated as-lox3 and astd plants flowered earlier than uninoculated plants, which was not the case in ir-aco plants (Fig. 2, 5A). The fact that TPI is constitutively down-regulated in all the three transgenic lines is consistent with the hypothesis that ethylene signaling, rather than TPI production, mediates the growth promotion of S. vermifera-inoculated N.
We further hypothesized that plants decrease their ethylene production when inoculated with S. vermifera. When ACC was added to germinating seedlings, the fungus inhibited the triple response of inoculated seedlings of both WT and ir-aco plants (Fig. 4, 5B). In addition, oxidizing ethylene with a KMnO 4 ethylene scrubber mimicked the effect of S. vermifera on the germination of WT seeds (Fig. 7), attenuata's natural habitat. cycle. Ten-day-old seedlings were transferred to Teku pots and 10 d later transferred to 1 L pots filled with B410 pot-soil mixture consisting of 95% turf and 5% clay, including 70 mg L -1 N, 35 mg L -1 P, and 75 mg L -1 K with a pH between 5.5 and 6 (Stender, Lukau, Germany). Each of the genotype comparisons of uninoculated and S.

Seeds of an inbred line of
vermifera-inoculated plants consisted of 10 to 15 pots with a single plant in each pot.
About 1 month after germination, when had plants reached the elongation stage, stalk length was measured every second day and the start of flowering was recorded for each plant. About sixty days after germination, when plants stopped elongating, final stalk length was measured.

OS-elicitation treatment
Creating standardized puncture wounds and immediately applying Manduca sexta larvae oral secretions (OS) to the puncture wounds precisely mimics the transcriptional (Roda et al., 2004), proteomic (Giri et al., 2006 and metabolic (Halitschke et al., 2001) responses of N. attenuata to M. sexta attack. Moreover, with this method, the timing of the elicitation can be standardized precisely. To elicit TPI activity, transcript accumulation, and ethylene emission, puncture wounds on the leaf blade were created with a pattern wheel on each side of the midrib and diluted OS was immediately applied to the wounds. OS were collected from M. sexta larvae reared on N. attenuata leaf diet, diluted 1:5 (v/v) with water prior to each experiment.

PI-activity assay
To determine trypsin proteinase inhibitor (TPI) activity, leaf samples were harvested 3 d after OS elicitation, frozen in liquid nitrogen, and stored at -80°C until further processing. Samples were analyzed for TPI activity in an agar diffusion assay as described in (van Dam et al., 2001). Levels of TPI are expressed in nmol of inhibited trypsin proteinase molecules per milligram of total soluble protein, calculated by the clear zone of inhibitor-proteinse complex of the tested samples in reference to a standard soybean proteinase inhibitor curve (Jongsma et al., 1994).
Protein concentration was determined according to Bradford (Bradford, 1976

Phytohormone measurements
Leaf samples for hormone analysis were harvested at the indicated time points following OS elicitation. Approximately 300 mg of harvested leaf tissue were homogenized in 1 mL ethyl acetate spiked with 200 ng mL -1 [ 13 C 2 ] jasmonic acid and para chlorogenic acid, as internal standards for JA and JA-Ile, respectively. After centrifugation at 13000 rpm for 20 min at 4°C, extraction was repeated with 1 mL ethyl acetate. The supernatants were combined and evaporated until dryness. The dried residue was re-dissolved in 500 µL 70% (v/v) methanol. Prior to analysis the samples were centrifuged for 10 min at 13000 rpm and 15 µL of the supernatant was analyzed using a Varian 1200L triple quadrupole MS (Varian, Darmstadt, Germany).
For the high-performance liquid chromatography, a Pursuit C8 column (150 mm x 2.0 mm, 3 µm particle size) was used and a gradient of water and methanol, both including 0.05% (v/v) formic acid, was the mobile phase with a flow rate of 0.2 mL min -1 . The mass spectrometer was operated in negative electro-spray ionization

Seedling performance assays
We used the triple response assay to measure the effect of 1aminocyclopropane-1-carboxylic acid (ACC) supplementation and hence, ethylene, on the growth of uninoculated and S. vermifera-inoculated WT and ir-aco seedlings.
Square (12 cm 2 ) Petri dishes were filled with 80 mL of GB5, with or without 5 ACC (Fluka, Sigma, Taufkirchen, Germany), and the solidified agar was portioned out into two plates. Seeds (sterile or pre-inoculated with S. vermifera) were placed on the agar to germinate. The plates were stored vertically in an incubator (26°C with an 11 h:13 h day/night cycle); after 3 days, when the radicles emerged, the light was turned off and seedlings were grown in the dark. Each inoculation and ACC treatment consisted of 4 plates each with 15 seedlings. After 10 d, the lengths of roots and hypocotyls were measured.
An ethylene scrubber (KMnO 4 ) was used to test the role of ethylene in S.
vermifera-mediated effects (Jayaraman and Raju, 1992). Seeds of WT and ir-aco plants were germinated on S. vermifera pre-inoculated or sterile GB5 media in round Petri dishes (r = 4.5 cm) as described above. The open plates containing the seeds were placed in the center of a larger Petri dishes (r = 7 cm). Measurements of enzyme activity were conducted on two separate cultures as described by Penrose and Glick (2003).

RNA isolation and mRNA expression
Fully mature leaves (at nodal position +1) of rosette-stage plants were elicited with OS as described above. Leaves were collected at different time points after the elicitation (for ethylene biosynthesis genes: 0 non-elicited, 60,120,180,240,300,and 360 min;for NaTPI: 0,6,12,24,48,and 72  Taufkirchen, Germany). cDNA was synthesized from 20 ng of total RNA as described by (Schmidt et al., 2005) using the Taqman reverse transcription reagent kit (Applied Biosystems, Darmstadt Germany). Analysis of the relative expression of ethylene biosynthesis and perception genes was performed using primer pairs and fluorescent dye-labeled probes for NaACS3a (AY426752), NaACO1 (AY426756), NaACO2 (EF123109, NaACO3 (EF123111), and NaETR1 (EF203416), as described by (von Dahl et al., 2007). Analysis of NaTPI (AF542547) was performed using primers and fluorescent dye-labeled probes as described by (Zavala et al., 2004a). For each analysis, a linear standard curve, threshold cycle number (Ct) vs. Log (designated transcript level), was constructed using a series dilutions of a specific cDNA standard; the levels of the transcript in all unknown samples were determined according to the standard curve. A N. attenuata sulfite reductase (ECI), which is a house-keeping gene involved in plant sulfur metabolism and has been shown to have constant levels of transcript by both northern blotting and q-PCR, after W+W and W+OS treatments (Wu et al., 2007), was used as an internal standard for normalizing cDNA concentration variations. Real-time PCR was performed on a SDS7700 (Applied Biosystems, Darmstadt, Germany) using the qPCR TM reagent kit (Eurogentec, Seraing, Belgium); for a detailed description see (Schmidt et al., 2005).