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Plant Physiol. ( 1 996) 1 1 1 : 735-739

Germin Gene Expression 1s lnduced in Wheat Leaves by Powdery Mildew lnfection

William J . Hurkman* and Charlene K. Tanaka United States Department of Agriculture, Agricultura1 Research Service, Western Regional Research Center,

Crop lmprovement and Utilization Research Unit, Albany, California 9471 O

Cermin gene expression i s induced in wheat (Triticum aestivum L.) leaves by powdery mildew (frysiphe graminis f. sp. triticr) infec- tion. Germin i s a protein marker for early cereal development and i s an oxalate oxidase, an enzyme that catalyzes the conversion of oxalate to CO, and H,O,. The induction of germin gene expression by powdery mildew infection is consistent with the importance of H,O, to plant defense and identifies a mechanism for the elevation of H,O, levels in wheat leaves. Cermin mRNA levels increased 2 d after inoculation of seedlings with powdery mildew and continued to increase throughout an 8-d time course. The increase in accu- mulation of germin mRNA was accompanied by an increase in the germin oligomer, which reached maximal levels by d 6. An increase in oxalate oxidase activity paralleled germin oligomer accumula- tion. Germin gene expression was induced in a relatively resistant cultivar (Bobwhite) as well as in a susceptible cultivar (Cheyenne), suggesting that the induction of germin gene expression i s an indi- cator of powdery mildew infection rather than cultivar resistance.

Germin is an approximately 130-kD homopentameric protein comprising subunits of approximately 26 kD (Mc- Cubbin et al., 1987) that increases significantly in the em- bryos of cereal seeds during germination (Grzelczak and Lane, 1984) and has been identified as an oxalate oxidase (Lane et al., 1993). Oxalate oxidase (oxa1ate:oxygen oxi- doreductase, EC 1.2.3.4) catalyzes the oxidation of oxalate by molecular oxygen, yielding CO, and H,O,. Because germin is localized in cell walls in wheat embryos (Lane et al., 1992) and H,O, is utilized by peroxidases in the oxida- tive cross-linking of cell-wall polymers (Olson and Varner, 1993), germin was postulated to have a role in initiation and termination of wall expansion (Lane, 1994). In addition to this developmental role in cereals, germin responds to environmental stress. In roots of barley seedlings, germin synthesis increases transiently during salt shock and accu- mulates when seedlings are grown on nutrient solution containing salt (Hurkman et al., 1991). An mRNA in the halophyte Mesembryanthemum crystallinum that is related to germin also responds to salt stress but decreases during salt treatment (Michalowski and Bohnert, 1992). The spherulins, which are related to germin, are putative cell- wall proteins in the slime mold Physarum polycephalum that increase during spherulation, a process brought on by var-

* Corresponding author; e-mail [email protected]; fax 1-510 -559 -5777.

ious environmental stresses, including drought (Lane et al., 1991).

A possible role for germin in plant defense, the resistance of wheat to rust infection, was mentioned (Lane et al., 1986) long before germin was shown to have oxalate oxidase activity (Lane et al., 1993). When germin was found to have oxalate oxidase activity, it was suggested (Lane, 1994) that the likely molecular basis for this resistance was the H,O,- directed signaling and lignification processes allied with PR responses (Aposto1 et al., 1989). Recently, Dumas et al. (1995) and Zhang et al. (1995), independently, provided experimental support for these views by showing that germin-like oxalate oxidase activity increases in barley leaves following infection with powdery mildew (Erysiphe graminis f . sp. hordei). In this paper we extend these studies to wheat leaves infected with powdery mildew ( E . graminis f. sp. tritici) and report the induction of germin gene ex- pression as well as the accumulation of germin and an accompanying increase in oxalate oxidase activity. We also demonstrate that the induction of germin gene expression by powdery mildew infection occurs in both relatively resistant and sensitive wheat cultivars.

MATERIALS A N D M E T H O D S

For initial experiments, wheat (Triticum aestivum L. cv Cheyenne) seeds were sown over moist sand in glass crys- tallizing dishes (approximately 60 seeds/ 190- x 100-mm dish), and the covered dishes were placed in a 4°C cold room with continuous light for 8 weeks. The vernalized plants were transplanted into potting mixture and trans- ferred to a greenhouse. During a powdery mildew infec- tion of plants that had been transferred to the greenhouse 19 d previously, leaves were harvested from uninfected and infected plants, frozen in liquid nitrogen, and stored at -70°C until used. In subsequent time-course experiments, seedlings were grown on moist sand, as described above, but dishes were maintained at room temperature for 4 d and transferred to a growth chamber (17°C day/l5"C night, 12-h photoperiod) for 2 d. Seedlings were then in- fected with powdery mildew (Erysiphe graminis f . sp. tritici) and placed in a growth chamber (20°C day/l6"C night, 12-h photoperiod) for 8 d, at which time the seedlings were covered with colonies of powdery mildew. For cv Bob- white, seeds were sown directly in soil mixture, and plants

Abbreviations: PR, pathogenesis related; SA, salicylic acid. 73 5

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736 Hurkman and Tanaka Plant Physiol. Vol. 11 1 , 1996

were grown in the greenhouse for 6 weeks. Cheyenne and Bobwhite leaves were inoculated by shaking conidia from the leaves of heavily infected Cheyenne plants onto leaves of uninfected seedlings. Leaves were harvested from in- fected seedlings at the times indicated in "Results." Leaves harvested from uninoculated seedlings grown under iden- tical conditions served as controls.

Northern Analysis

Total RNA was isolated from frozen leaves based on a method described by Hurkman et al. (1989). Plant material, 300 to 500 mg fresh weight, was ground to a powder in liquid nitrogen with a prechilled mortar and pestle. An extraction reagent (Hurkman et al., 1989) was added in a leaf tissue to reagent ratio of 1:8 (fresh w / v ) . The powder was ground until a liquid homogenate was obtained. After the sample was centrifuged at 10,OOOg and 4°C for 15 min, the aqueous phase was re-extracted with an equal volume of pheno1:chloroform:isoamyl alcohol (25:24:1, v / v) and then with an equal volume of ch1oroform:isoamyl alcohol (24:1, v/v). An equal volume of cold (4°C) 4 M lithium acetate was added to the aqueous phase. Following incu- bation overnight at -2O"C, the solution was centrifuged at 12,OOOg and 4°C for 25 min. The RNA pellet was rinsed with cold 2 M lithium acetate and then with cold 75% ethanol. The dried pellet was solubilized in cold, sterile water. The RNA was purified further using a RNeasy Total RNA spin column' (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. The concentration of RNA was estimated spectrophotometrically. The RNA was pre- cipitated by addition of 0.1 volume of 2 M sodium acetate and 2.5 volumes of cold (-20°C) 100% ethanol. The sample was incubated overnight at -2O"C, and the precipitated RNA was collected by centrifugation at 12,OOOg and 4°C for 15 min. The precipitate was rinsed with cold (- 20°C) 75% ethanol, and the pellet was dried and then solubilized with cold (-4"C), sterile water to a concentration of approxi- mately 3 p g / p L . The final concentration of the RNA was determined spectrophotometrically.

Probes were labeled with digoxigenin according to pro- tocols supplied with the Genius System (Boehringer Mann- heim). An approximately 700-bp germin fragment (nucle- otides 157-858 of the 1-kb germin cDNA), containing most of the coding region and part of the 3' untranslated region, was subcloned into pSPT 19. The plasmid was linearized by digestion with EcoRI and transcribed with T7 polymer- ase to generate a digoxigenin-labeled RNA probe. The cDNA encoding P8.5 peroxidase from barley leaves was a generous gift from Dr. Shauna Somerville (Carnegie Insti- tution of Washington, Stanford, CA). The EcoRIlHindIII approximately 900-bp fragment of the peroxidase cDNA was labeled with digoxigenin using random priming.

RNA (10-13 pg/lane) was separated in 1.2% agarose/ 0.66 M formaldehyde gels and blotted by capillary transfer using 20x SSC onto a positively charged nylon membrane

' Mention of a specific product name by the U.S. Department of Agriculture does not constitute an endorsement and does not imply a recommendation over other suitable products.

(Boehringer Mannheim). The transferred RNA was UV linked to the membrane using a Stratalinker (Stratagene). Blots were hybridized with digoxigenin-labeled probes and visualized using the Genius System according to the manufacturer's instructions. Following hybridization at 62°C with the germin RNA probe, blots were washed twice with 2X SSC/O.l% SDS at room temperature for 5 min and twice with 0.1X SSC/O.l% SDS at 65°C for 15 min. Follow- ing hybridization at 42°C with the peroxidase DNA probe, blots were washed in 2X SSC/O.l% SDS, twice at room temperature for 5 min, and twice at 45°C for 15 min.

Western Analysis

Wheat leaves (300-700 mg fresh weight) were ground to a powder in liquid nitrogen using a prechilled mortar and pestle. S16 buffer (100 mM potassium acetate, 3 mM magne- sium acetate, 20 mM Hepes, pH 7.5, 1 mM DTT; Grzelczak et al., 1985) was added (1.5 pL S16 buffer/mg fresh weight leaves), and the sample was ground until a liquid homoge- nate was obtained. The homogenate was centrifuged at 12,0009 and 4°C for 15 min, and an equal volume of Laemmli (1970) buffer was added to the supernatant. Protein was quantified by the procedure of Lowry et al. (1951) prior to addition of the Laemmli buffer, and samples containing 20 p g of protein were loaded onto 12.5% gels. In one set of experi- ments, germin oligomers were converted to monomers by heating samples at 100°C for 2 min (Lane et al., 1993). Polypeptides were separated by SDS-PAGE and transferred to nitrocellulose (0.45 pm; Schleicher & Schuell) essentially as described previously (Hurkman et al., 1991) except that the transfer buffer contained 25 mM Tris, 192 mM Gly, and 20% (v/v) ethanol, and electrotransfer was performed at 250 mA for 60 min. Germin was detected on the blots using phytohe- magglutinin-purified germin antibodies (Lane et al., 1992), a generous gift from Dr. Byron Lane (University of Toronto, Ontario, Canada), and goat anti-rabbit antibodies conjugated to alkaline phosphatase.

Oxalate Oxidase Activity Blots

Polypeptides were separated by SDS-PAGE and trans- ferred to nitrocellulose as described above, except that samples containing 60 pg of protein were loaded onto the gels. Oxalate oxidase activity was detected on the blots by the peroxidase-dependent staining method of Lane et al. (1993) that was adapted from an assay described by Sug- iura et al. (1979).

RESULTS

C e r m i n C e n e Expression 1s lnduced by Powdery M i l d e w lnfection

The effect of powdery mildew infection on germin gene expression was examined in leaves of vernalized, greenhouse-grown plants of Cheyenne, a hard, red, win- ter wheat that is relatively susceptible to powdery mil- dew infection. Germin mRNA levels from leaves that showed no visible signs of infection were compared with

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Germin and Powdery Mildew Infection 737

germin mRNA levels from leaves that had young colo- nies of powdery mildew on the upper surfaces. Germin mRNAs were not detectable in the "uninfected" leaves and were strongly induced in leaves infected with pow- dery mildew (Fig. 1). Since peroxidase (Kerby and Som- erville, 1992) and peroxidase mRNAs (Schweizer et al., 1989) are known to increase during powdery mildew infection, peroxidase mRNA levels were analyzed in wheat leaves using a cDNA encoding the P8.5 peroxi- dase from barley leaves. Like germin mRNA, peroxidase mRNA was not detectable in the uninfected leaves and was induced in leaves infected with powdery mildew (Fig. 1).

The effect of powdery mildew infection was examined in leaves of greenhouse-grown plants of Bobwhite, a hard, white, spring wheat that is relatively resistant to powdery mildew infection. Leaves were inoculated with conidia from infected leaves of Cheyenne plants, and 3 d after inoculation, germin mRNA and peroxidase mRNA levels from leaves of infected plants were compared with those from leaves of control plants. As found in Cheyenne, ger- min and peroxidase mRNAs were not detectable in control leaves and were induced in leaves infected with powdery mildew (Fig. 1). In contrast to Cheyenne leaves, germin and peroxidase mRNA levels appeared to increase much less in Bobwhite leaves following powdery mildew infection. However, this result is related to the length of time that leaves were infected with powdery mildew rather than to cultivar resistance. Comparison of the mRNA levels from leaves of Bobwhite 3 d after inoculation (Fig. 1) with those from Cheyenne 2 and 4 d after inoculation (Fig. 2) revealed that germin mRNA levels were also relatively low in Chey- enne leaves at this stage of powdery mildew infection.

Temporal expression of germin mRNA was examined during an 8-d time course following inoculation of 5-d-old Cheyenne seedlings with powdery mildew. Germin mRNA was detected in control leaves by d 4 and levels increased slightly during the 8-d time course, indicating that germin gene expression is developmentally regulated (Fig. 2). Al- though germin mRNA levels increased in control leaves during the time course, increases in germin mRNA levels were much greater in leaves inoculated with powdery mildew. In infected leaves, germin mRNA was first detect-

CHEYENNE BOBWHITE C PM C PM

GERMIN |0

PEROXIDASE ||| "***

Figure 1. Accumulation of germin and peroxidase mRNAs in leaves of two wheat cultivars, Cheyenne and Bobwhite, infected with pow- dery mildew. C, Control leaves; PM, leaves infected with powdery mildew. Infected leaves of 19-d-old Cheyenne plants, previously vernalized for 8 weeks, had powdery mildew colonies on their surfaces. Infected leaves of 6-week-old Bobwhite plants were ana- lyzed 3 d after inoculation with powdery mildew and had no visible symptoms of infection.

C PM

8 days

C PM

Figure 2. Temporal accumulation of germin mRNA in leaves of Cheyenne wheat inoculated with powdery mildew. Total RNA was isolated from leaves 2, 4, 6, and 8 d after inoculation of 6-d-old plants with powdery mildew (PM) and from leaves of control plants (C) of the same age.

able 2 d after inoculation and increased throughout the 8-d time course (Fig. 2).

Germin Accumulates following Powdery Mildew Infection

Germin is a homopentameric protein comprising 26-kD subunits (McCubbin et al., 1987) that are encoded by the germin mRNA. To determine whether the increase in ger- min mRNA during powdery mildew infection was accom- panied by an increase in the germin oligomer, germin levels were analyzed during an 8-d time course following inoculation of 5-d-old Cheyenne seedlings with powdery mildew. The oligomer was not detected on immunoblots of protein extracts from control leaves at any of the times examined (Fig. 3A). When leaves were inoculated with powdery mildew, the oligomer was first detectable 2 d after inoculation, reached maximal levels by d 6, and de- clined slightly by d 8 (Fig. 3A). One identifying character- istic of germin is that the oligomer, which is stable in SDS solubilization buffer, is reduced to monomers when heated (Lane et al., 1993). When protein samples were heated prior to SDS-PAGE, a 26-kD protein was recognized by the an- tibodies to germin (Fig. 3B). The analysis of germin sub- units during the 8-d time course confirmed the results obtained for the oligomer.

Oxalate Oxidase Activity Increases following Powdery Mildew Infection

Since the germin oligomer has oxalate oxidase activity (Lane et al., 1993), we wanted to determine whether the increase in germin oligomer during powdery mildew in- fection was accompanied by an increase in oxalate oxidase activity. Blots of the same protein extracts used for immu- noblot analysis of germin accumulation were analyzed for oxalate oxidase activity. This analysis revealed that an increase in oxalate oxidase activity paralleled the pattern of germin accumulation (Fig. 3C). Oxalate oxidase activity was not detectable in control leaves at any of the times sampled during the 8-d time course. In infected leaves, oxalate oxidase activity was first detectable 2 d after inoc- ulation, reached maximal levels by 6 d, and declined slightly by 8 d (Fig. 3C).

DISCUSSION

Inoculation of wheat leaves with powdery mildew in- duces germin gene expression. Germin mRNA, which en- codes the 26-kD subunit of the oligomer, increases substan-

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738 Hurkman and Tanaka Plant Physiol. Vol. 111, 1996

2 4 6 8 C PM C PM C PM C PM

2 4 6 8 2 4 6 8 days C PM C PM C PM C PM C PM C PM C PM C PM

Figure 3. Effect of powdery mildew infection on germin levels and oxalate oxidase activity in leaves of Cheyenne wheat. Protein extracts were prepared from leaves 2, 4, 6, and 8 d after inoculation of 6-d-old plants with powdery mildew (PM) and from leaves of control plants (C) of the same age. A, Immunoblot analysis of germin oligomer. The oligomer is approximately 116 kD. B, Immunoblot analysis of germin monomers. The monomers, approximately 26 kD, were produced by heating protein samples before separation by SDS-PAGE. C, Activity blot analysis of oxalate oxidase activity.

tially in leaves infected with powdery mildew. This increase in germin mRNA is accompanied by increases in the oligomer and in oxalate oxidase activity, neither of which are detectable prior to infection. Thus, germin is synthesized de novo in wheat leaves following powdery mildew infection. In contrast to wheat leaves, barley leaves contain significant amounts of a protein immunologically related to germin that does not have oxalate oxidase activ- ity (Zhang et al., 1995). Following powdery mildew infec- tion of barley leaves, increases in germin parallel increases in oxalate oxidase activity (Dumas et al., 1995; Zhang et al., 1995), and Zhang et al. (1995) concluded that germin is synthesized de novo in barley leaves following powdery mildew infection.

The accumulation of germin, an enzyme that generates H2O2, in response to powdery mildew infection is of par- ticular interest, since H2O2 is linked to plant defense re- sponses. Plant recognition of necrotizing pathogens results in a hypersensitive response, the rapid tissue death at the site of infection that restricts pathogen growth and spread. An oxidative burst, a rapid and transient release of active oxygen species, predominantly superoxide anions (O2~), H2O2, and hydroxyl radicals (OH-), is correlated with the hypersensitive response. The H2O2 generated during the oxidative burst has direct antimicrobial effects (Keppler and Baker, 1989), activates the biosynthesis of phytoalexins (Apostol et al., 1989), participates in oxidative cross-linking of specific cell-wall structural proteins (Bradley et al., 1992; Brisson et al., 1994), and functions as a diffusible signal in the induction of genes encoding enzymes, such as gluta- thione S-transferase, that are involved in cellular protection (Levine et al., 1994). Chen et al. (1993) isolated an SA- binding protein, an isoform of catalase, with activity that is

inhibited in vitro by SA. Based on the induced expression of PR-1 genes by relatively high levels of H2O2, Chen et al. (1993) suggested that H2O2 accumulated by this mecha- nism in vivo could act as a secondary messenger to activate defense gene expression. In testing this hypothesis, Neuen- schwander et al. (1995) found that H2O2 did not increase in tobacco leaves inoculated with tobacco mosaic virus and that PR-1 transcripts increased nevertheless. Neuen- schwander et al. (1995) found that injection of tobacco leaves with increasing concentrations of H2O2 caused a dose-dependent increase in total SA and suggested that it is SA that stimulates the induction of PR-1 gene expression.

For wheat and barley (Dumas et al., 1995; Zhang et al., 1995), the accumulation of germin and the parallel increase in oxalate oxidase activity suggest that oxalate oxidation could be a mechanism for the accumulation of H2O2 following powdery mildew infection. Peroxidase mRNA levels (Schweizer et al., 1989; Thordal-Christensen et al., 1992) and peroxidase (Kerby and Somerville, 1992) also increase in wheat and barley leaves following inoculation with powdery mildew. Peroxidase and H2O2 have been implicated in the cross-linking reactions of cell-wall structural components, e.g. the Hyp-rich glycoproteins (Varner and Lin, 1989; Bradley et al., 1992), and H2O2 has been localized in cells undergoing lignificarion (Olson and Varner, 1993). The timing of peroxi- dase gene induction in barley leaves coincides with formation of papillae, cell-wall appositions, at the site of the primary germ tube and the appressorial germ tube of the powdery mildew fungus (Thordal-Christensen et al., 1992).

Lignification, a widely recognized barrier to pathogen invasion, is H2O2 mediated. When germin, a cell-wall pro- tein (Lane et al., 1992), was found to be an oxalate oxidase, an enzyme that produces H2O2 from oxalate, it was sug-

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Germin and Powdery Mildew Infection 739

gested that germin might have a role in blocking fungal invasion (Lane, 1994). Thus, to explain the accumulation of germin following powdery mildew infection of barley leaves, Zhang et al. (1995) suggested that a possible func- tion of germin is to generate H,O, that is utilized by peroxidases to provide a barrier to cell-wall degradation and fungal penetration. It is interesting that accumulation of a PR-1 protein follows the increase in oxalate oxidase activity in barley leaves infected with powdery mildew (Zhang et al., 1995), but whether H,O, and SA have roles in the signal transduction pathway is yet to be determined.

In this study, we demonstrated that germin mRNA, ger- min, and oxalate oxidase activity increase in wheat leaves infected with powdery mildew. The response of wheat and also barley (Dumas et al., 1995; Zhang et al., 1995) to powdery mildew infection and the importance of H,O, in plant defense responses suggest a role for oxalate in cellu- lar protection of monocots against pathogen invasion. We also showed that the response occurs in a relatively resis- tant (Bobwhite) as well as a susceptible (Cheyenne) wheat cultivar, a result that is not altogether unexpected. In a study of the identification of markers for quantitative field resistance of winter wheat to powdery mildew, Kmecl et al. (1995) found no correlation between the degree of quantitative field resistance of wheat cultivars to powdery mildew infection and levels of accumulation of defense- related gene transcripts, including peroxidase, a thaumatin- like protein, and glutathione S-transferase. Thus, the accu- m u l a t i o n of germin transcripts, like other defense-related gene transcripts, in wheat seedlings inoculated with pow- dery mildew seems to be an indicator of infection rather than cultivar resistance. The relative resistance of Bobwhite to powdery mildew suggests that Bobwhite has mecha- nism(s) of resistance that are absent from Cheyenne.

Received December 26, 1995; accepted April 3, 1996. Copyright Clearance Center: 0032-0889/96/ 111 / 0735/ 05.

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