Systemic expression of defense response genes in wheat spikes as a response to Fusarium graminearum infection

Systemic expression of defense response genes in wheat spikes as a response to Fusarium graminearum infection

Physiological and Molecular Plant Pathology (2001) 58, 1±12 doi:10.1006/pmpp.2000.0308, available online at http://www.idealibrary.com on Systemic ex...

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Physiological and Molecular Plant Pathology (2001) 58, 1±12 doi:10.1006/pmpp.2000.0308, available online at http://www.idealibrary.com on

Systemic expression of defense response genes in wheat spikes as a response to Fusarium graminearum infection C L A R A P R I T S C H 1{ , CA R ROL L P. VA N C E 1,2, W I L L IA M R . B U S H N E L L 3,4, D AVID A . S OM E R S 1, T HO M A S M . HO HN 5{ and G A RY J . M U E H L B A U E R 1* 1

Department of Agronomy and Plant Genetics, University of Minnesota, St Paul, MN 55108, U.S.A., 2Plant Science Research Unit, U.S. Department of Agriculture, Agricultural Research Service, St Paul, MN 55108, U.S.A., 3Cereal Disease Laboratory, U.S. Department of Agriculture, Agricultural Research Service, St Paul, MN 55108, U.S.A., 4Department of Plant Pathology, University of Minnesota, St Paul, MN 55108, U.S.A. and 5Mycotoxin Research Unit, USDA-ARS, NCAUR, Peoria, IL 61604, U.S.A. (Accepted for publication October 2000 and published electronically 22 December 2000) Wheat spikes infected by Fusarium graminearum result in Fusarium head blight, a devastating disease of wheat. The spikes respond to infection by inducing a set of defense response genes in infected spikelets much as has been shown in other plant-pathogen interactions. To determine whether defense response genes are expressed systemically within F. graminearum-inoculated wheat spikes, we examined transcript accumulation of four defense response genes ( peroxidase, PR-1, PR-3 and PR-5) in colonized and uncolonized portions of wheat spikes of resistant and susceptible genotypes. We determined whether sampled regions of point-inoculated wheat spikes were colonized or not using a Fusarium spp.-speci®c plate assay and an F. graminearum isolate expressing b-glucuronidase (GUS). The GUS-expressing F. graminearum isolate proved particularly useful for visualizing and following the spread of the fungus and for measuring the amount of fungal biomass in spike tissues. We found that transcripts of the four defense response genes accumulated in both colonized and uncolonized regions of spikes of resistant and susceptible genotypes at 48 h after inoculation. These results demonstrate that direct contact with the pathogen is not required for induction of defense response genes and that these genes are activated in both susceptible and resistant c 2001 Academic Press * genotypes. Keywords: Triticum aestivum L.; Fusarium graminearum; Fusarium head blight; defense response genes; systemic response; b-glucuronidase; SAR.

INTRODUCTION Fusarium head blight (FHB) has caused catastrophic damage in the north central wheat-growing regions of North America in recent years [27]. Ascospores of Fusarium graminearum (the principal head blight pathogen in North America) develop on soilborne crop residues and can infect wheat ¯orets from the time of anthesis until the soft dough stage of kernel development [48]. Visible lesions are produced within 3±4 days under conditions favorable for disease development. Although the pathways of ¯oret invasion have not been determined, most parts of the ¯oret can be colonized, including the lemma, palea, anther, stigma, and developing young * To whom all correspondence should be addressed. E-mail: [email protected] { Present address: GeneÂtica, Facultad de AgronomõÂ a, Universidad de la Republica, GarzoÂn 780, Montevideo, Uruguay. { Present address: Novartis Agribusiness Biotechnology Research, Inc., 3054 Cornwallis Road, Research Triangle Park, NC 27709-2257, U.S.A.

0885-5765/01/010001+12 $35.00/00

kernel [41]. From an infected ¯oret, the fungus can spread up or down the spike from one spikelet to another. This spread is an important component in the overall damage caused by the disease. Resistance to spread in the spike (designated as type II resistance) is provided by the cultivar Sumai 3, a principal source of FHB resistance widely used in breeding programs [1, 60]. To assist in understanding factors a€ecting initiation and spread of head blight in wheat spikes, we are investigating the possible role of defense response gene expression in limiting fungal infection. In previous experiments, we showed that transcripts of six typical defense response genes, POX ( peroxidase), PR-1, PR-2 (b-1,3-glucanase), PR-3 (chitinase), PR-4 and PR-5 (thaumatin-like protein) were induced in spray-inoculated heads of both the susceptible cv. Wheaton and the resistant cv. Sumai 3 [39]. Transcripts began to accumulate 6±12 h after inoculation with peak amounts at 36±48 h. Transcripts of PR-4 and PR-5 accumulated earlier and to a greater amount in Sumai 3 than in Wheaton. The rapid activation of defense response genes c 2001 Academic Press *

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in wheat spikes in response to F. graminearum parallels the many reports of pathogen-induced activation in both monocots and dicots [4, 6, 10, 15]. Given the importance of pathogen spread in wheat spikes to the development of FHB, we extended our investigations to learn if defense response genes are induced in uncolonized portions of partially-infected spikes. We postulated that systemic acquired resistance (SAR) might be induced in uncolonized portions of the spike in the same way that infection of a leaf is known to induce resistance in uninfected leaves [43, 44]. SAR is commonly associated with systemic expression of defense response genes [25, 62] as is well documented for dicotyledonous species such as tobacco [58], cucumber [30], and Arabidopsis [26]. Induced genes accompanying SAR include PR1, b-1,3-glucanase (PR-2), thaumatinlike protein (PR-5), chitinase (PR-3) and peroxidase [29, 50, 53, 62]. In monocots, pathogen-induced SAR has been reported for barley [18], pearl millet [23] and rice [51], but systemic activation of defense response genes was either not found [51] or was not investigated. Chemical activation of SAR by 2,6-dichloroisonicotinic acid (INA) or benzo (1,2,3) thiodiazol-7-carbothioic acid S-methyl ester (BTH) in barley [21], maize [33], rice [13] and wheat [13] was associated with activation of several defense response genes characteristic of SAR, although the genes activated in some cases di€ered among species. The biological and chemical induction of SAR in these cereals suggested that we might ®nd systemic induction of defense response genes in wheat spikes in response to localized F. graminearum infection. To determine if the defense response genes are expressed in uncolonized regions of wheat spikes, we measured the accumulation of defense response gene transcripts in colonized and uncolonized regions of the spike. We applied inoculum to ¯orets of two spikelets located in the middle region of spikes and later analysed separately the lower, middle and upper regions of the spikes for both fungal colonization and abundance of selected defense response gene transcripts. The results show clearly that defense response genes are induced systemically in infected spikes.

MATERIALS AND METHODS

Plant material Three wheat (Triticum aestivum L.) genotypes with contrasting levels of resistance to Fusarium head blight (FHB) were used: Wheaton, a hard red spring cultivar from the mid-western U.S.A. and highly susceptible to FHB; Bobwhite, a cultivar from CIMMYT that is slightly less susceptible than Wheaton (R. Dill-Macky, personal communication); and Sumai 3, a Chinese

cultivar known for its type II resistance which limits fungal spread within spikes [1, 60].

Plant and fungal growth conditions Wheat plants were grown in a growth chamber cabinet (Conviron PGV36, Pembina, ND, U.S.A.) as previously described by Pritsch et al. [39]. Wild-type F. graminearum isolate 3A-31 (gift from R. Dill Macky, University of Minnesota, U.S.A.) was collected from barley in Felton, MN in July 1995 and grown on Fusarium spp. selective pentachloronitrobenzene (PCNB) agar [34]. F. graminearum TEF-4, containing the Escherichia coli b-glucuronidase (GUS) gene and its parental wild-type isolate GZ3639 (see below) were grown in V-8 juice agar [9]. Media for growing TEF-4 was supplemented with hygromycin (300 g ml ÿ1). For spore production, all isolates were grown in mung bean agar plates as previously described by Pritsch et al. [39].

Development of b-glucuronidase expressing F. graminearum F. graminearum TEF-4, expressing GUS, was obtained by transformation of a wild-type isolate, GZ3639, with plasmid pTEFGUS1 as described by Proctor et al. [40]. GZ3639 was isolated from infected wheat in Kansas [2] and kindly provided by Dr R. Bowden (Kansas State University, U.S.A.) and maintained on V-8 juice agar slants. The pTEFGUS1 plasmid contains the translation elongation factor (TEF) promoter of Aureobasidium pullulans immediately upstream of the E. coli b-glucuronidase open reading frame and a chimeric HygB gene as the selectable marker [52]. For pTEFGUS1 construction, primers no. 1054 (reverse direction, 50 -GGACAGCGCTGGAAGATGGAGTGAAGTACG-30 ) and no. 1056 ( forward direction, 50 -GGCATCTAGAAACAGACCAGGCTAGA-30 ) were used to PCR amplify a 735 bp fragment corresponding to the Aureobasidium pullulans TEF promoter present on plasmid pTEFGFP [54]. Following PCR, the XbaI and Eco47III sites within the primers were digested and the fragment cloned into the compatible SpeI-SmaI sites of pGUS2-7 [17]. Singlespored cultures of F. graminearum transformants were grown for 18 h in a medium consisting of 2 % (w/v) glucose, 0.8 % (w/v) peptone (DIFCO), and 0.3 % (w/v) yeast extract (DIFCO) and tested for GUS activity as previously described by Hohn et al. [17].

Demarcation of spike regions and fungal inoculation Wheat spikelets {Zadoks' stages 65±69; [65]} were sequentially numbered from the base of the spike upwards at anthesis (Fig. 1). Three regions within a spike were de®ned, including: the lower region (spikelets no. 1±4), the middle region (spikelets no. 5±10) and the

Systematic expression of defense response genes

3

with the transgenic isolate TEF-4 were incubated in a growth chamber (218C, continuous light, 85 % RH) after the inoculated spike was enclosed in a plastic bag to prevent the release of the transgenic F. graminearum isolate. Plants inoculated with GZ3639 were incubated in a growth chamber (218C, continuous light, 85 % RH). In all cases, plants were incubated for 48 h, at which point small necrotic lesions were evident in inoculated spikelets from all three wheat genotypes.

Analysis of GUS activity GUS activity of TEF-4-inoculated wheat glumes was detected histochemically with 3.33 mg ml ÿ1 of 5-bromo4-chloro-3-indolyl-b-D-glucuronide (X-Gluc) in 0.1 M sodium phosphate bu€er, pH 7.0, containing 0.5 mM potassium ferricyanide/ferrocyanide, 0.5 % (v/v) Triton X-100, and 20 % (v/v) methanol [19]. Glumes were sampled 3 days after inoculation and GUS-stained overnight followed by clearing in 70 % (v/v) ethanol. Cross-sections of glumes were obtained by hand-sectioning with a razor blade, mounted on slides in 100 % glycerol and observed in a bright ®eld microscope. For quantitative measurement of GUS activity, proteins were extracted from lower, middle, and upper regions of spikes, 48 h after inoculation (hai) [extraction bu€er: 0.1 M potassium phosphate bu€er, pH 7.8, 1 mM EDTA, 10 mM DTT, 5 % (v/v) glycerol, 0.1 % (v/v) Triton X100]. GUS enzymatic activity in the protein extracts was determined with a ¯uorometric assay which measures the rate of 4-methyl umbelliferone (MU) production using 4methyl umbelliferyl glucuronide (MUG) as the substrate [19]. Tissues from uninfected spikes, point-inoculated with water, were used to quantitate background GUS activity. Total protein concentration was determined according to Bradford [3] using BSA as a standard. F I G . 1. Demarcation of the lower (L), middle (M) and upper (U) regions in wheat spikes according to spikelet positions. Spikelets are numbered from the spike base upwards. Odd numbered spikelets are indicated on the left. The lower region (spikelets no. 1±4), middle region (spikelets no. 5±10), and upper region (spikelet no. 11Ðspike tip) are indicated. Arrows pointing towards spikelets no. 7 and 8 indicate the sites of point inoculation.

upper region (spikelet no. 11 and all distal spikelets). At anthesis, the middle region of the spike was pointinoculated by placing a droplet (5 ml) of F. graminearum spore suspension (2  105 conidia ml ÿ1) or 5 ml of water within the palea and lemma of the two basal ¯orets of spikelets no. 7 and 8 (Fig. 1). Thus, four ¯orets were inoculated in the middle region of each spike. Wheat plants inoculated with F. graminearum wild-type isolate 3A-31 were incubated in a dew chamber (218C, continuous light and 98 + 2 % RH). Plants inoculated

Pentachloronitrobenzene (PCNB) plate assay Point-inoculated spikes were sampled 48 hai. Individual spikelets from each spike were dissected, surface sterilized (1 min in 0.5 % (v/v) sodium hypochlorite and three rinses with sterile distilled water) and placed on a PCNB agar plate [34] according to their original position in the spike (Figs 1 and 2). Each PCNB plate contained spikelets no. 1±16 from a single spike. Similarly, individual spikelets dissected from water point-inoculated spikes were plated as controls. After 5 days of incubation at 218C in continuous light, the presence of visible hyphae on individual spikelets was scored.

RNA isolation and RNA gel blot analysis For two experiments, F. graminearum point-inoculated and water-inoculated controls of Sumai 3 and Wheaton spikes

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F I G . 2. Selective growth of F. graminearum on pentachloronitrobenzene (PCNB) plates indicating the extent of fungal spread in point-inoculated spikes at 48 hai. Individual spikelets from point-inoculated spikes (48 hai) were plated in order on a PCNB agar plate and incubated 5 days at 218C. (a) F. graminearum point-inoculated spike showing fungal growth from inoculated spikelets no. 7 and 8. (b) Water point-inoculated spikes (control) showing no fungal growth. Individual spikelets are numbered sequentially beginning with the spikelet no. 1 (see Fig. 1).

were sampled at 0, 24 and 48 hai, frozen in liquid nitrogen and stored at ÿ708C. In a third experiment, F. graminearum point-inoculated and water-inoculated controls of Sumai 3 and Bobwhite were sampled at 48 and 72 hai. Lower, middle and upper spike regions were separated and independently ground in liquid nitrogen. For all three experiments, the spike regions from two spikes were combined for each time of harvest. Total RNA from each combined sample was extracted as described by De Vries et al. [8], electrophoresed in 1 % (w/v) agarose/formaldehyde gels, blotted onto nylon membranes (Zeta-Probe, BIORAD, Hercules, CA, U.S.A.), and probed with a defense response gene cDNA as described below. Signal intensities of hybridizing bands were quanti®ed as arbitrary units using a PhosphoImager (Molecular Dynamic, Sunnyvale, CA, U.S.A.) equipped with the software ImageQuant. Resultant data were further normalized with respect to the amount of poly(A) ‡ RNA loaded in each lane by hybridization with a [g-32P] poly(U) ‡ oligonucleotide.

Defense response gene probes Four defense response gene cDNAs were used as probes in RNA gel blot analyses: wheat POX ( peroxidase POX381) [42]; wheat PR1-2 [32]; barley acidic PR-3 (chitinase; HvCht2a) [14]; and barley acidic PR-5 (thaumatin-like protein, pBH72-C6) [14]. The coding region of these genes was used as probes. Therefore, each probe indicates the expression of all highly-related genes that may be present within a family. The probes were labeled with [a-32P]dCTP by random priming [12].

RESULTS

Localization of F. graminearum in point-inoculated spikes using the PCNB plate assay To assess the extent of F. graminearum colonization in point-inoculated spikes (Fig. 1), we inoculated Sumai 3 (type II resistance to fungal spread) and Wheaton (susceptible) with wild-type isolate 3A-31. Spikes were harvested at 48 hai and individual spikelets were plated on PCNB medium (Fusarium spp. selective) {Fig. 2; [34]}. A total of 18 spikes (nine from each cultivar) were analysed. Results with Sumai 3 and Wheaton were similar. In both cultivars, the fungus was found in all spikelets directly inoculated (spikelets no. 7 and 8). In addition, the fungus had spread to spikelet no. 5 (in nine spikes) and to spikelet no. 6 (in eight spikes). Of these, the fungus was in both spikelets no. 5 and no. 6 in ®ve spikes. The results showed that at 48 hai, F. graminearum spread was localized in spikelets no. 5±8, within the middle region of the spike as de®ned in Fig. 1.

Characterization of F. graminearum TEF-4 and its localization in point-inoculated spikes Assessment of in planta b-glucuronidase (GUS) activity of GUS-transformed phytopathogenic fungi provides an alternative approach to detect and quantify fungal biomass during plant infection [7, 16, 37]. We developed a stable F. graminearum transformant, isolate TEF-4, expressing the E. coli b-glucuronidase (GUS) gene, driven by the constitutive A. pullulans transcription elongation factor (TEF) promoter. We used this isolate as a tool to assess further the extent of fungal spread in pointinoculated wheat spikes. The TEF-4 isolate produced

Systematic expression of defense response genes

5

F I G . 3. Ex planta and histochemical detection of b-glucuronidase (GUS) activity in F. graminearum TEF-4. Fungal spores and inoculated wheat glumes (3 days after inoculation) were stained for GUS activity. (a) GUS-staining F. graminearum macroconidia. (b) GUS-staining hyphae on the abaxial glume surface. (c) Cross section of F. graminearum-infected wheat glume, showing abundant colonization of parenchyma by GUS-staining hyphae. (d) F. graminearum hypha-expressing GUS activity within a glume parenchyma cell. Scale bar: (a) 50 mm; (b) 50 mm; (c) 30 mm; (d) 15 mm.

high levels of GUS activity (10.1 nmol MU min ÿ1 mg ÿ1 protein) when grown in a rich synthetic medium (see Materials and Methods; T. M. Hohn, unpublished results). Furthermore, macroconidia harvested from TEF-4 growing on mung bean agar plates readily stained for GUS [Fig. 3(a)]. The parental wild-type isolate GZ3639 did not exhibit in vitro GUS activity (data not shown). To test the pathogenicity of TEF-4 in relation to its wildtype parent GZ3639 and to wildtype 3A-31, three spikes of Sumai 3 and ®ve spikes of Wheaton were pointinoculated separately with each isolate. Fungal development was analysed by the PCNB plate assay at 48 hai. Table 1 shows the extent of fungal spread in Sumai 3 and Wheaton for the three strains. The inoculated spikelets (no. 7 and 8) were colonized as in the earlier experiment with wild type 3A-31 (except for spikelet no. 7 in one spikelet each for Sumai 3 and Wheaton inoculated with TEF-4). Again, spread beyond spikelets no. 7 and 8 was limited. In another experiment (data not shown), TEF-4 was found only in spikelets no. 7 and 8 in all of six tested

spikes of Sumai 3 and six of Wheaton. Together, the results indicate that TEF-4 was able to infect inoculated spikelets, and like its wildtype parental strain (GZ3639), was generally limited to the inoculated spikelet in both Sumai 3 and Wheaton. With both isolates, the extent of fungal spread was somewhat less than the spread of wildtype 3A-31. None of the isolates spread beyond the middle region of the spike. GUS activity in TEF-4 was evident histologically in hyphae on the abaxial surface of glume tissues [Fig. 3(b)] and in cross-sections of colonized glumes [Fig. 3(c) and (d)]. Neither the parental wildtype GZ3639 nor waterinoculated spikes exhibited any GUS activity histologically. Clearly the TEF-4 isolate was able to colonize glume tissue and to express GUS in hyphae on and within plant tissues. To quantify F. graminearum spread along the spike, wheat spikes of cv. Sumai 3 and cv. Wheaton, were again point-inoculated at spikelet no. 7 and 8 with isolate TEF4 (Fig. 1). At 48 hai, spikes were sampled and lower, middle, and upper spike regions (Fig. 1) were analysed

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T A B L E 1. Location of F. graminearum isolates 3A-31, GZ3639 and TEF-4 as detected by the PCNB plate assay of spikelets from Sumai 3 and Wheaton, 48 hai of spikelets no. 7 and 8 3A-31 Spikelet combinationa 5, 6, 5, 7, 7, 7, 8

6, 7, 7, 8 8, 8,

7, 8 8 8 9 10

Total a b

GZ3639

TEF-4

Sumai 3

Wheaton

Sumai 3

Wheaton

Sumai 3

Wheaton

0b 0 0 2 0 1 0

1 2 0 2 0 0 0

0 0 0 2 1 0 0

0 0 0 5 0 0 0

0 1 0 1 0 0 1

0 0 0 4 0 0 1

3

5

3

5

3

5

Combination of spikelets, numbered as shown in Fig. 1. The number of spikes with the fungus in the indicated combination of spikelets.

T A B L E 2. Location of F. graminearum isolate TEF-4a by ¯uorometric assay of GUS activity in three spike regions of pointinoculated spikes of Sumai 3 and Wheaton, 48 hai Mean GUS activityb ( pmol MU min ÿ1 mg ÿ1 protein) Sumai 3 Spike regionc Upper Middle Lower

Wheaton

Inoculatedd

Controle

Inoculated

Control

4.9 + 0.9 515.3 + 95.8 8.3 + 1.4

12.12 + 2.29 16.76 + 5.48 11.33 + 3.46

4.0 + 1 177.6 + 14.8 17.6 + 2

3.35 + 0.08 3.88 + 1.85 6.93 + 0.94

a

GUS-expressing isolate. Means + SE of spikes. Six, 12, ®ve and six spikes were examined from Sumai 3 inoculated, Sumai 3 control, Wheaton inoculated and Wheaton control, respectively. c As de®ned in Fig. 1. d Spikelets no. 7 and 8 (in the middle region) were inoculated with F. graminearum TEF-4 spore suspension. e Spikelets no. 7 and 8 (in the middle region) were inoculated with water. b

for in vitro GUS activity. Similarly, corresponding spike regions of water point-inoculated spikes were analysed as controls. Low endogenous GUS activity was observed in all control treatments (Table 2). In inoculated spikes, high levels of GUS activity were observed in the middle (inoculated) spike region, while low background GUS activity was found in lower and upper spike regions. Interestingly, GUS activity in the middle regions of TEF4-inoculated spikes was more than twice as high in Sumai 3 compared to Wheaton. The results of Table 2 clearly indicate that, in both genotypes at 48 hai, F. graminearum biomass in point-inoculated spikes was restricted to the middle region of the spike, in agreement with results from the PCNB plate assay.

Temporal and spatial assessment of transcript accumulation of defense response genes in point-inoculated wheat spikes To determine whether defense response genes were expressed in uncolonized regions of point-inoculated

spikes, we assessed the transcript accumulation of four defense response genes in both middle (infected) and lower and upper (uninfected) spike regions from both resistant and susceptible genotypes. To this end, two experiments were conducted in which F. graminearum 3A-31 and water control point-inoculated spikes from cv. Sumai 3 (resistant) and cv. Wheaton (susceptible) were sampled at 0, 24 and 48 hai and total RNA was extracted from lower, middle and upper spike regions. Transcripts of defense response genes encoding POX ( peroxidase), PR-1, PR-3 (chitinase) and PR-5 (thaumatin-like protein) were detected by RNA gel blot analysis and signal intensities quanti®ed with PhosphoImager analysis. Transcript abundance at 0 and 24 hai was negligible. Since transcript abundance showed no change at 24 hai compared to 0 hai, only data from 48 hai are presented. In contrast to the absence of induced change at 24 hai, pathogen-induced transcript levels increased

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T A B L E 3. Means of defense response transcript levels from two independent experiments with F. graminearum and water control point-inoculated spikes for Sumai 3 and Wheaton at 48 hai Signal intensitiesb Sumai 3

Wheaton I±Cc

Inoculated

Control

1.46 9.22 4.55

10.7 11.33 9.03

5.2 10.68 6.8

5.5 0.65 2.23

2.83 3.52 3.2

0.83 5.94 6.2

3.17 6.1 4.4

1.98 3.65 2.02

1.19 2.45 2.38

14.48 21.24 14.19

10.8 9.28 9.88

3.68 11.96 4.31

1.57 8.86 4.55

1.17 2.36 1.33

0.4 6.5 3.22

24.23 23.79 15.73

19.71 14.54 12.26

4.52 9.25 3.47

17.24 21.18 15.4

12.26 15.02 7.67

4.98 6.16 7.73

Gene

Spike regiona

Inoculated

Control

PR1

Lower Middle Upper

15.14 25.12 17.31

13.68 15.9 12.76

PR3

Lower Middle Upper

3.66 9.46 9.4

PR5

Lower Middle Upper

POXd

Lower Middle Upper

I±C

a

As de®ned in Fig. 1. Hybridization data are the mean of two independent experiments for each genotype. Signal intensities are in 105 arbitrary units. c Indicates the subtraction of the hybridization intensities of the control spikes (water control) from values for inoculated spikes inoculated with F. graminearum. d Peroxidase. b

dramatically by 48 hai in the middle spike region (inoculated) for all genes analysed (Table 3). In both Sumai 3 and Wheaton, this increase in transcript accumulation coincided with the appearance of small necrotic areas on the inoculated spikelets. In both genotypes, transcripts also accumulated in the lower and upper portions of F. graminearum-inoculated spikes (compared to transcript levels in control spikes). To test the signi®cance of di€erences between inoculated spikes and water controls in the three regions, we combined the data from the two genotypes and performed a paired-t test between inoculated and control treatments from data collected at 48 hai (Fig. 4). In the middle region of the spike, all defense response genes exhibited signi®cantly greater transcript accumulation in the inoculated spikes than in the water controls. In the lower region of the spike, PR-3 and POX exhibited signi®cant increases in transcript accumulation in the inoculated spikes over the water controls. PR-1 and PR-5 transcripts also showed an increase in the lower region, although the increase was not statistically signi®cant. In the upper region of the spike, transcripts of PR-1, PR-5 and POX increased signi®cantly in inoculated spikes over the water controls. PR-3 transcripts also increased in the upper region, but not signi®cantly. As shown in Table 3, transcript levels in water controls were high in several gene-cultivar combinations (Sumai 3 with PR1, PR5 and POX; Wheaton with PR1 and POX). This apparently resulted from mock inoculation

with water injected into spikelets, since transcript levels in controls were generally low in samplings taken at the time of inoculation (data not shown). To con®rm that the pathogen-induced transcript levels are higher than in controls, we conducted a third point-inoculation experiment, this time involving Sumai 3 and the susceptible cultivar, Bobwhite. Lower, middle and upper regions of spikes were sampled at both 48 and 72 hai. Although we did not measure the extent of fungal colonization in Bobwhite, the fungus was probably con®ned to the central spike region as shown earlier for Sumai 3 and Wheaton, since Bobwhite is known to be slightly less susceptible than Wheaton (R. Dill-Macky, personal communication). By 48 hai, transcripts of all four defense response genes were especially abundant in middle inoculated regions of both Sumai 3 and Bobwhite and were consistently higher than in controls in all three regions (Table 4). Generally, transcript levels were lower in controls than in earlier experiments and di€erences in middle regions between inoculated and control spikes were greater (compare Tables 3 and 4). Likewise, di€erences in lower and upper regions between inoculated and control spikes were more consistent than the earlier experiments. At 72 hai, transcript levels were similar to those at 48 hai for both Sumai 3 and Bobwhite (data not shown). Taken together, our results (Tables 3 and 4, Fig. 4) show that by 48 hai, spikes from both resistant and susceptible genotypes responded to point-inoculation with enhanced

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Signal intensities 105 arbitrary units

6

PR1

PR3

*

4

* *

2

*

0

8

PR5

POX

*

**

6 4

**

**

*

2 0 0

48 0 Hours after inoculation

48

F I G . 4. Data from Table 3 for defense response transcript levels for lower, middle, and upper regions of inoculated and control spikes. Values combined for Sumai 3 and Wheaton. Background transcript levels for control samples have been subtracted from values for inoculated samples. Each value is the mean of four separate analyses, two for Sumai 3 and two for Wheaton. Black, pale gray, and white boxes correspond to lower, middle and upper spike regions, respectively. Single and double asterisks indicate signi®cance at the 0.10 and 0.05 probability levels, respectively, based on paired t-tests of values for inoculated vs. control spikes. Signal intensities are in 105 arbitrary units. POX, peroxidase.

T A B L E 4. Defense response gene transcript levels from a single experiment with F. graminearum and water control point-inoculated spikes of Sumai 3 and Bobwhite at 48 hai Signal intensitiesb Sumai 3

Bobwhite

Gene

Spike regiona

Inoculated

Control

I±Cc

Inoculated

Control

I±C

PR1

Lower Middle Upper

2.1 9.4 4.0

1.3 1.6 1.7

0.8 7.8 2.3

0.3 5.2 0.9

0.2 0.5 0.6

0.1 4.7 0.3

PR3

Lower Middle Upper

7.1 40.5 9.5

4.2 2.9 2.3

2.9 37.6 7.2

3.5 38.2 8.6

1.1 2.1 3.8

2.4 36.1 4.8

PR5

Lower Middle Upper

4.5 94.9 11.7

2.2 1.5 1.3

2.3 93.4 10.4

7.4 116.3 22.3

0.9 4.2 14.2

6.5 112.1 8.1

POXd

Lower Middle Upper

6.7 51.5 10.1

4.2 3.1 2.7

2.5 48.4 7.4

4.3 52.1 7.6

1.4 2.6 3.4

2.9 49.5 4.2

aAs

de®ned in Fig. 1. Signal intensities are in 105 arbitrary units. c Indicates the subtraction of the hybridization intensities of the control spikes (water-inoculated) from inoculated (F. graminearuminoculated) spikes. d Peroxidase. b

Systematic expression of defense response genes transcript accumulation of defense response genes throughout colonized and uncolonized regions of the spike.

DISCUSSION Our ®ndings have extended the understanding of Fusarium head blight of wheat by showing that accumulation of four defense response gene transcripts ( peroxidase, PR-1, PR-3 and PR-5) are induced in adjacent (uncolonized) spikelets of point-inoculated spikes. This systemic response occurs concurrently with a similar but local response in inoculated regions of spikes. The systemic molecular response is expected because similar responses have been observed in vegetative organs of dicots expressing pathogen-activated SAR as well as in organs of both monocots and dicots responding to chemical activators of SAR. We also show that activation of defense response gene expression in uninfected tissues is found in both resistant and susceptible cultivars. In addition, we report the characterization and the use of a F. graminearum isolate expressing GUS to study the colonization of wheat by the fungus. This strain exhibits nearly comparable fungal spread in wheat spike tissues to wild-type isolates and is potentially useful for detailed studies of wheat-F. graminearum interaction.

Defense response gene transcripts accumulate systemically in F. graminearum-inoculated wheat spikes Our results with F. graminearum point-inoculated wheat spikes show that at 48 hai, induction of peroxidase, PR-1, PR-3 and PR-5 genes occurs not only in the middle (inoculated) spike region but also in uncolonized lower and upper adjacent locations of the spike. The systemic nature of the response in wheat spikes, i.e. response in the absence of a direct contact with the pathogen, was deduced in part from results showing the restricted development of F. graminearum colonization in pointinoculated spikes. Both the PCNB plate assay and assessment of GUS activity in wheat spikes inoculated with a GUS-transformed F. graminearum isolate indicated that the fungus was restricted to the middle (inoculated) region of the spike. It is notable that both local and systemic activation of defense response genes occurred between 24 and 48 hai, which suggests that the signal perception that activates gene expression in both local and distal spike regions may be similar. In agreement with our results, wheat leaves attacked by the Russian wheat aphid (Diuraphis noxia) showed a systemic induction of intercellular b-1,3-glucanase, chitinase and peroxidase proteins in unattacked leaves, 48 h after infestation [55, 56]. In this case however, the systemic response was observed in the resistant wheat genotype but

9

not in the isogenic susceptible genotype. Such results indicate that wheat plants can respond to either pathogen or pest attacks with rapid systemic expression of defense response genes.

Defense response genes and resistance Patterns of transcript accumulation were similar in resistant cv. Sumai 3 and susceptible cultivars Wheaton and Bobwhite. The resistant cv. Sumai 3 exhibits type II resistance, i.e. resistance to fungal spread in the spike. Type II resistance is one of the major types of resistance mechanisms to FHB in wheat [28, 48, 60]. Type II resistant plants are not immune to F. graminearum but instead, initially respond to infection by forming necrotic lesions in infected spikes, much as occurs in susceptible plants. Further spread of the pathogen in the spike is restricted [1]. Although Sumai 3 di€ers from both Wheaton and Bobwhite in resistance to FHB, our results indicate that within the ®rst 48 hai, both resistant and susceptible genotypes exhibit similarities in activation of defense response genes in response to F. graminearum infection. The resistance to fungal spread in Sumai 3 was not yet evident in the samples we harvested at 48 hai of spikelets no. 7 and 8. At that time, the fungus had spread to other spikelets in only a few inoculated spikes of either Sumai 3 or Wheaton, and then usually to only one spikelet adjacent to an inoculated spikelet (see results in text and in Table 1). The factors that limit spread in Sumai 3 apparently come into play later than 48 h, including activation of any defense responses involved in limiting spread of the fungus from spikelet to spikelet. Such gene activation would necessarily be superimposed on the general response that occurred in all the cultivars we used [Sumai 3 (resistant), Wheaton (susceptible) and Bobwhite (susceptible)]. Results similar to ours were reported by Kombrick et al. [22] and SchroÈder et al. [47] for systemic expression of b-1,3-glucanase and chitinase in potato in response to Phytophtora infestans, in that patterns were similar in compatible and incompatible interactions. These authors concluded that for the potato-P. infestans interaction, the systemic defense response was not causally related to the resistant phenotype but was part of a general host defense response to infection. Also, Gregersen et al. [14] observed that, at the site of barley-Erysiphe graminis f. sp. hordei interaction, the expression pattern of several defense response genes including peroxidase, PR-1, PR-2, PR-3, PR-4 and PR-5 at both the attacked epidermal cells and unattacked, subjacent mesophyll cells was similar in compatible and incompatible interactions, indicating that these genes are also part of a general defense response to infection. When entire spikes were spray-inoculated with F. graminearum, transcripts of peroxidase, PR-1,

10

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PR-2, PR-3, PR-4 and PR-5 accumulated in both FHB resistant and susceptible cultivars, although transcripts of PR-4 and PR-5 accumulated earlier and to a greater amount in Sumai-3 [39]. Our results support the conclusion that the systemic molecular response in uninfected spike regions of F. graminearum point-inoculated wheat spikes is not causally related to the type II resistance mechanism(s) but represents a general host response to infection expressed at both infected and adjacent, uninfected tissues. The role of defense response genes in resistance is still unclear and needs further investigation. In wheat, for example, induced resistance activated by application of BTH did not induce expression of PR-1 and PR-5 [32, 45]. It is notable that several reports from monocots and dicots indicate that systemic induced resistance activated by di€erent microorganisms or wounding is not always associated with a systemic activation of defense response genes [38, 49, 51, 57]. Systemic expression of defense response genes has been previously reported as a response to a number of inducers including pathogen infection [53], chemical treatments [13, 20, 21, 24, 63] and abiotic stresses such as wounding [59]. However, in rice, no systemic induction of peroxidase, PR-5, and other defense response genes was observed as a response to wounding in leaf tissue [49]. In our experiments, a certain amount of disturbance occurred during the point-inoculation procedure because the ¯orets of the selected spikelets were forced open in order to apply the inoculum. This was the probable cause of gene activation in water-inoculated controls, although for unknown reasons, activation was much greater in the experiments of Table 3 than Table 4. In all cases, however, transcript abundance in inoculated spikes was greater than in controls, and the di€erence was statistically signi®cant in nine of 12 possible comparisons in the experiments of Fig. 4. Thus, we conclude that defense response transcript accumulation was due to a combination of infection and inoculation procedures, but that infection induced a portion of the accumulation in all cases.

The transgenic F. graminearum isolate carrying a constitutively-expressed b-glucuronidase reporter gene is a useful tool for studying the infection process We were successful in using a F. graminearum isolate (TEF4) carrying a constitutively-expressed b-glucuronidase (GUS) reporter gene to visualize the fungus histologically and to determine amounts of the fungus using an in vitro assay. GUS-expressing hyphae were readily detected histologically in TEF-4-infected glumes of infected spikes (Fig. 3). The presence of hyphae within glume parenchyma cells con®rmed previous observations that this tissue is abundantly colonized by F. graminearum [39, 41]. Colonization of spikelets by TEF-4 as measured by fungal spread using a PCNB plate assay was similar to

colonization by its parental wild-type isolate GZ3639 and only slightly less than the wild-type 3A-31 (Table 1), although data were obtained only for 48 hai. Colonization of glume tissues and ability to infect and begin to spread from spikelet to spikelet indicate that TEF-4 can be used as an experimental tool to investigate pathogen development in wheat spikes. An in vitro ¯uorometric assay was used to measure relative amounts of TEF-4 in inoculated and in adjacent uninoculated regions of wheat spikes (Table 2), con®rming that the fungus was restricted to inoculated regions. Unexpectedly, fungal biomass in inoculated regions of spikes was greater in Sumai 3 than Wheaton (Table 2), as if resistance to spread in Sumai 3 was associated with enhanced fungus development in inoculated spikelets. Additional studies are needed to verify and explain these data because the number of spikes analysed was small. The GUS gene has been used successfully as a reporter to track the spread and quantity of fungal growth for several plant pathogens including Cladosporium fulvum and Leptosphaeria maculans [37], Pseudocercosporella herpotrichoides [7], F. oxysporum [5], F. culmorum [11] and F. moniliforme [64]. Other methods to detect and quantify F. graminearum in plants include measurement of ergosterol [31, 61] and polymerase chain reaction-based assays [35, 36, 46]. However, using GUS as a reporter for the presence of the fungus o€ers the advantage of high sensitivity and the possibility for both in planta detection and quantitative analysis. Moreover, treatment of tissues to detect GUS is simple. Background GUS activity is low in wheat spike tissues, therefore increasing the sensitivity of fungal detection. The TEF-4 isolate is a useful tool for visualizing F. graminearum development in planta during infection and for assessing the amount of fungal colonization. This work was supported by a grant to G.J.M. and D.A.S. from the Minnesota State Legislative Scab Initiative. C.P. was partially supported by BIDCONICYT (URUGUAY). We thank R. Dill-Macky (University of Minnesota) for providing F. graminearum isolate 3A-31, R. Dudler (University of Zurich) for the wheat peroxidase clone, H. Thordal Christensen (KVL, Denmark) for the barley PR-3 and PR-5 clones and L. Friedrich (NOVARTIS) for the wheat PR1-2 clone. We also thank Ruth Dill-Macky and Corby Kistler for critical reading of the manuscript.

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