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Interactions between cold hardening andMicrodochium nivaleinfection on expression of pathogenesis-related genes in winter wheat
Physiological and Molecular Plant Pathology (1998) 53, 301–310 Article No. : pp980182
Interactions between cold hardening and Microdochium nivale infection on expression of pathogenesis-related genes in winter wheat AH . E*, S. S. K and A. M. T Norwegian Crop Research Institute, Plant Protection Centre, Fellesbygget, N-1432 A| s, Norway (Accepted for publication September 1998 )
Cold hardening induces snow mould resistance in cereals and grasses. The mechanism for this induced resistance is not fully understood. The accumulation of transcripts encoding sucrose synthase and the pathogenesis-related proteins (PR-proteins) chitinase, β-1,3-glucanase, peroxidase and PR-1a were studied in hardened and non-hardened winter wheat at several time points after inoculation with the snow mould Microdochium niale. Sucrose synthase was induced by the hardening treatment itself. The PR-proteins were all induced by infection, this response was stronger and more rapid in plants that had been hardened prior to inoculation. The present results suggest that cold hardening has a conditioning effect on snow mould induced expression of PR-proteins. This may partly explain how cold hardening induces snow mould resistance. # 1998 Academic Press
INTRODUCTION
Microdochium niale (Fr.) Samuels & Hallet (pink snow mould, teleomorph Monographella nialis (Schaffnit) E. Mu$ ller) attacks winter cereals and grasses. The cold hardening that plants go through when temperatures decrease in autumn, is known to strongly increase the resistance of Poaceae species to snow moulds [4, 36, 37, 39, 42, 45 ] and other pathogenic fungi [38, 41 ]. Several changes occurring in plants during hardening could be responsible for the increase in snow mould resistance. Research has mainly been focused on the role of carbohydrate storage and metabolism and to some extent on the plant cell water potential as reviewed by Gaudet [15 ]. Tronsmo et al. [41 ] initiated investigation of the possible role of PR proteins. The synthesis of PR-proteins is a general and systemic response of plants to pathogen attack. Chemical substances and other environmental stresses [33, 34 ] can also induce the synthesis of these proteins. Induction of PR-proteins by low temperature has been shown in barley [41 ], rye [19 ], bermuda grass [13 ] and potato [44 ]. Many PR-proteins are encoded by small gene families, and different isoforms can be produced in different stress situations [8, 27, 34 ]. *To whom all correspondence should be addressed. Abbreviations used in the text : AFP, antifreeze protein ; OD, optical density ; PR-proteins, pathogenesisrelated proteins ; SAR, systemic acquired resistance ; UDP, uracil-diphosphate. 0885–5765\98\110301j10 $30.00\0
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Increased levels of the PR-proteins chitinase, β-1-3-glucanase, peroxidase and PR1a after inoculation has been reported, both at the mRNA and at the protein level, from several host-pathogen systems [3, 6, 9 ]. Several possible roles for chitinases and β1,3-glucanases in plant defence have been investigated [34 ]. Both groups of enzyme have antifungal activity, probably due to their ability to degrade chitin and β-1,3-glucan in fungal cell walls [2, 28 ]. Another function of these enzymes in host plant defence may be to release elicitor-active substances from plant or fungal cell walls [17, 18, 29, 34 ]. There are indications that transgenic plants with a constitutive expression of chitinase have increased resistance to fungal pathogens [34 ]. Peroxidases are implicated in a variety of defence-related processes, including lignification, cross-linking of phenolics and glycoproteins, suberization and phytoalexin production [30 ]. Little is known about the function of PR-1a, but it appears to have a deleterious effect on oomycetes, both in itro and in transgenic tobacco [1, 34 ]. Sucrose synthase is not generally regarded as pathogenesis-related, but was induced in barley following inoculation with Blumeria graminis f.sp. hordei (syn. Erysiphe graminis f.sp. hordei) [16 ]. Sucrose synthase catalyses the reaction sucrosejUDP(ADP) *- UDP(ADP)-glucosejfructose [12, 32 ]. Tronsmo et al. (41) found that sucrose synthase and chitinase were induced in barley by cold hardening. When infected with B. graminis, hardened plants had a much stronger expression of peroxidase than non-hardened plants. They suggested that increased resistance to fungal pathogens in cold hardened plants might be due to an enhanced expression of defence mechanisms in these plants. In order to investigate whether PR-proteins and sucrose synthase are involved in cold hardening induced snow mould resistance, we used cDNAS from barley encoding chitinase, β-1,3glucanase, peroxidase, PR-1a and sucrose synthase as probes to detect the expression of homologous sequences in hardened and non-hardened winter wheat (Triticum aestium L.) during infection with M. niale.
MATERIALS AND METHODS
Plant and fungal materials Two Scandinavian cultivars of winter wheat, Rida and Folke, were used. The plants were treated in four different ways : (1) non-hardened, non-inoculated, (2) hardened, non-inoculated, (3) non-hardened, inoculated and (4) hardened, inoculated. Seeds were sown at 1 cm depth in soil in 9 cm pots (five plants per pot) and grown for two weeks in a controlled environment at 12 mC night\18 mC day, 12 h photoperiod with a light intensity of 200 µmol m−# s−". The plants were cold hardened for one week at 4 mC, 16 h photoperiod with a light intensity of 100 µmol m−# s−". Plants in the nonhardened treatments were sown one week later to ensure that they were approximately the same size as the hardened plants at the time of inoculation (3-leaf-stage, approximately 20 cm in height). The fungus used in this study was isolate 5\93 of Microdochium niale, originally isolated from Lolium perenne L. at Nordera/ s in 1986 (Plant Protection Centre, AH s, Norway). The inoculum was prepared as described by Tronsmo [40 ], except that the mycelial suspension was diluted to an OD of 0n5 at 430 nm. The plants were sprayed
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with 2n5 ml inoculum per pot and incubated under moist cellulose wadding and black plastic at 4 mC. Non-inoculated plants were incubated in the same way. Shoot tissue was sampled prior to inoculation (0 h), and at 3 h, 7 h, 21 h, 45 h, 4 days and 8 days following inoculation. Plants from three replicate pots within each treatment were pooled. The plants were cut 1–2 cm above soil level, immediately frozen in liquid nitrogen and stored at k80 mC. Mycelial growth was not observed until 3–4 weeks after inoculation, so no effort was made to remove mycelium from the leaf surface before sampling. Three pots from each treatment were incubated at 4 mC for a total of 6 weeks in order to establish the level of snow mould resistance. After incubation, the plants were cut 7 cm above soil level and allowed to regrow in a greenhouse (approximately 18 mC) for 9 days. Each plant was scored on a scale of 0 (dead or almost dead), 1 (severely affected) or 2 (apparently healthy). A value representing regrowth was then calculated for each pot using this formula : (number of plants with score 0)*0j(number of plants with score 1)*1j(number of plants with score 2)*2. The dry weight of all shoot tissue in each pot was also recorded. An analysis of variance (SAS Institute Inc., Cary, NC, U.S.A.) was performed on the dry weight data.
RNA isolation and northern blotting One to three grams of plant material were ground in liquid nitrogen with a mortar and pestle. Total RNA was isolated essentially as in Collinge et al. [7 ], but with 0n5 g l−" 8-hydroxyquinoline added to the phenol\chloroform. Fifteen micrograms of RNA from each sample was run on 1n2 % agarose gels containing 1n1 % formaldehyde, and blotted onto Zeta-Probe nylon membranes (BioRad) by using a capillary transfer system with 20iSSC. The RNA was bound to the membrane by ultraviolet radiation (700 µJ cm−#) followed by baking at 80 mC for 1 h. The membranes were stained with 0n02 % methylene blue in 0n3 sodium acetate in order to check the integrity and the amount of RNA in each lane. The inserts to be used as probes were isolated with GENECLEAN4 Kit (BIO 101, La Jolla, CA, U.S.A.) and labelled with αdCTP ($#P) using an Oligolabelling Kit (Pharmacia Biotech). Each probe was hybridised to a new membrane that had not been used previously. After being washed 2i15 minutes in 0n1iSSC, 0n5 % SDS at 68 mC, the membranes were prehybridised for 2 h at 68 mC in 8iDenhardt, 0n5 % SDS, 4iSSC, 1 m EDTA and 200 µg ml−" denatured salmon sperm DNA. Hybridisation was performed under the same conditions except that the concentration of Denhardt was 4i and 7 % dextran sulphate was added. Membranes were washed under low stringency in 1iSSC, 0n1 % SDS for 30 min at 42 mC and under medium stringency in 1iSSC, 0n1 % SDS for 30 min at 68 mC prior to autoradiography. The probes were prepared from five different cDNA clones kindly provided by Dr Hans Thordal-Christensen and Dr David Collinge, The Royal Veterinary and Agricultural University, Denmark. These cDNAs had been isolated from a cDNA library of barley leaves inoculated with B. graminis f.sp. hordei, and include pBH6-301 (accession No. X58396), encoding a putative peroxidase [35 ], pBH6-4, encoding a putative sucrose synthase [16 ], pHvPR-1a (accession No. X74939), encoding a basic PR-1 protein [4 ], N12, encoding a putative class II chitinase and I1, encoding a
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putative β-1,3-glucanase (The latter two from H. Thordal-Christensen). The PR-1a, chitinase and β-1,3-glucanase are all secreted into the apoplast of powdery mildew infected barley leaves [4, 10 ]. The whole experiment was conducted twice. RESULTS
Hardened plants had a significantly higher regrowth after incubation than the nonhardened plants (P l 0n0001 for dry weight data) (Table 1). The direct effects of T 1 Resistance to Microdochium nivale and the effect of hardening in two cultiars of winter wheat. Nonhardened (k) and hardened (j) plants were inoculated (j)or not inoculated (k) and incubated for 6 weeks under artificial snow coer. The plants were then cut down to 7 cm and allowed to regrow in a greenhouse. (a) Resistance expressed as dryweight after regrowth and regrowth score (means of three potspS.D.) as described in the text. Regrowth score 10 l no injury, regrowth score 0 l all plants dead. (b) Results from analysis of ariance on dry weight data (a) Cultivar Folke
Rida
Hardened
Inoculated
Dry weight (mg)
Regrowth score
k j k j k j k j
k k j j k k j j
112p38 160p36 0,3p0,6 123p36 90p12 192p11 19p26 137p36
10,0p0,0 10,0p0,0 0,7p0,6 5,7p0,6 9,0p1,7 10,0p0,0 2,3p3,2 6,3p2,3
DF
Mean square
1 1 1 1 1 1 1
0,000693 0,056940 0,028222 0,003151 0,000187 0,000900 0,001276
(b) Source Cultivar Hardening Inoculation Hardeningiinoculation Cultivariinoculation Cultivarihardening Cultivarihardeningiinoculation
F value P value 0,89 73,24 36,30 4,05 0,24 1,16 1,64
0,36 0,0001 0,0001 0,061 0,63 0,30 0,22
hardening on gene expression can be seen in the samples taken before incubation (0 h) in Fig. 1. The hardening treatment induced the expression of sucrose synthase in both cultivars. Effects of the incubation treatment Effects of the incubation treatment alone can be seen by comparing incubated samples that were non-hardened and non-infected with the non-hardened samples taken prior to inoculation (0 h). Expression of sucrose synthase was induced during incubation in both cultivars (Fig. 1e).
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nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h
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F. 1. Hybridisation on northern blots. Fifteen micrograms of RNA isolated from samples taken prior to inoculation (0 h) and at different time points after inoculation with M. niale were applied in each lane. Membranes were hybridised to DNA probes of barley chitinase (a), β-1,3-glucanase (b), peroxidase (c), PR-1a (d) and sucrose synthase (e). h : hardened, nh : non-hardened, i : inoculated, ni : noninoculated. A photograph of the membrane stained in methylene blue is shown under each autoradiogram.
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Interaction between hardening and the incubation treatment When comparing hardened, non-inoculated samples with non-hardened, noninoculated samples, it can be seen that hardening also had an enhancing effect on the expression of chitinase, β-1,3-glucanase and PR-1a during incubation (Fig. 1 a, b, d). Effects of inoculation Inoculated plants had a significantly lower regrowth after incubation than the noninoculated plants (P l 0n0001 for dry weight data) (Table 1). Effects of infection on gene expression can be studied by comparing the non-hardened, inoculated samples with the non-hardened, non-inoculated samples. Infection increased the expression of all the genes studied except for sucrose synthase (Fig. 1). Chitinase was enhanced early, while β-1,3-glucanase and PR-1a were enhanced later. The expression of these three genes increased steadily throughout the sampling period. Peroxidase was also induced by infection, but the expression did not increase throughout the sampling period. Folke appeared to have an expression peak around 4 days. Interaction between hardening and inoculation The analysis of variance revealed an interaction between hardening and inoculation on regrowth after incubation (P l 0n061 for dry weight data) (Table 1). Plants that had been hardened prior to inoculation were more resistant than those that had not been hardened. Expression of the M. niale induced genes was more rapid and\or stronger in plants that had been hardened before inoculation (Fig. 1a–d). As in non-hardened plants, the expression of chitinase, β-1,3-glucanse, and PR-1a increased throughout the sampling period and there was a peak in peroxidase expression around 45 h–4 days. Expression of these genes appeared to reach higher levels than what can be accounted for by a cumulative effect of hardening and infection, particularly around 45 h. Effects of cultiar There was no significant difference between cultivars in regrowth (P l 0n36) (Table 1), but there were some differences in gene expression (Fig. 1). Infected plants of Folke had a stronger expression of β-1,3-glucanase, peroxidase and PR-1a than infected plants of Rida. Multiple bands Hybridisation with the chitinase and β-1,3-glucanase probes produced two bands in addition to the main band (Fig. 1). The PR-1a and sucrose synthase probes also hybridised to one additional band visible on highly exposed films (data not shown). Additional bands were always weaker and of a larger molecular size than the main band. The signal strength of the bands relative to each other appeared to be the same in all lanes. DISCUSSION
We could not detect any difference between Folke and Rida in their resistance to M. niale, measured as regrowth, although Folke in field tests has been found to be the more resistant of the two [21, 22 ]. The level of replication in our study (n l 3) may
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simply be too low to reveal any regrowth differences. Results may also vary between studies due to experimental or environmental differences and cultivarienvironment interactions. Ho$ mmo$ [23 ] has shown that resistance at the whole plant level does not correlate tightly with resistance at the single leaf level, using a detached leaf testing system. The differences in gene expression that we observed between the two cultivars could be related to a type of resistance that is not detectable when measuring resistance as regrowth. Care should be taken when comparing the expression strength of the different genes at a given time. The probes are homologous with barley genes, and the % similarity of these genes with the corresponding wheat genes is unknown and could vary. There is also some variation in probe activity and exposure time. The cold hardening treatment induced expression of sucrose synthase in both cultivars. This is in agreement with earlier reports [5, 11, 25, 31 ] and supports a role for sucrose synthase in the cold hardening process. Hon et al. [20 ] found that apoplastic antifreeze proteins (AFPs) accumulating in rye cold hardened for 3 weeks at 5\2 mC (day\night) had sequence similarities to endochitinase and endo-β-1,3-glucanase and affinity to antibodies against these enzymes. They suggested that AFPs may have evolved from PR-proteins or that they are post-translationally modified PR proteins. The hardening treatment in our experiment was relatively mild (4 mC for 1 week) ; an induction of β-1,3-glucanase and chitinase might have been present in material hardened for a longer period or at a lower temperature. The effects of the incubation treatment, both in itself and in combination with hardening, could be responses to either the mechanical handling, the sudden darkness or the low temperature involved in incubation. The incubation-induced expression of sucrose synthase indicates that this enzyme might also be induced by low temperature under very low light intensities. Infection with M. niale enhanced the expression of chitinase, β-1,3-glucanase, peroxidase and PR-1a. Hardened plants had a higher expression of these genes after inoculation than non-hardened plants had. This was also the case for peroxidase expression in barley infected with B. graminis [41 ]. Although the linearity between expression level and signal strength may be questionable, the combined effect of hardening and infection did not appear to be merely of a cumulative nature. Hardening prior to inoculation rather appeared to improve the plants’ ability to respond to snow mould attack in terms of PR-gene expression. Similar sensitising or conditioning effects have been reported in plants expressing systemic acquired resistance (SAR) [24, 26 ]. The state induced in plants by hardening could have similarities with SAR. A cold hardening induced expression of PR-genes and an induced ability to express these genes upon infection could explain, in part, why cereals and grasses are more resistant to M. niale and other fungal pathogens when hardened. The mechanism behind a conditioning effect of cold hardening on PR-gene expression is not known. Cold hardening might lead to a higher level of compounds that positively affect pathogen recognition, signalling or PR-gene expression. Ka$ stner et al. 1998 [26 ] presented evidence that a chemical compound inducing SAR (2,6dichloroisonicotinic acid, DCIA) and unknown fungal elicitors from Colletotrichum lagenarium can have a synergistic effect on chitinase expression in cucumber, and that the long-term influence of DCIA can lead to a developmentally regulated tuning-up
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of the elicitor-triggered chitinase expression machinery. Low levels of signals associated with one defence pathway have in several plant species been shown to positively affect the magnitude and kinetics of the defence responses associated with a different pathway, indicating that there is some cross-talk between different signalling pathways [43 ]. Similar phenomena could be involved in cold hardening conditioned PR-gene expression. It may also be that the hardened state of no or very low growth, with a large storage of photosynthetic products, makes plants more responsive to pathogen attack as well as to other stresses. It is unclear what the multiple bands observed might represent. They could represent either mRNAs at different steps in the processing or different members of the respective gene families, regulated in the same way as the gene behind the main band. In conclusion, we have shown that infection by M. niale induces expression of chitinase, β-1,3-glucanase, peroxidase and PR-1a, and that this induction is much stronger in hardened plants than in non-hardened plants. This may partly explain why cold hardened plants have an increased disease resistance. AH shild Ergon was supported by a grant from the Norwegian Research Council. We are grateful to Hans Thordal-Christensen and David Collinge, The Royal Veterinary and Agricultural University, Denmark for providing the DNA probes and to Linda Hjeljord for sharing her laboratory experience. Thanks also to Andre! Laroche, Denis A. Gaudet, Torbjørn Ergon and Grete Lund. REFERENCES 1. Alexander D, Goodman RM, Gut-Rella M, Glascock C, Weymann K, Friedrich L, Maddox D, Ahl-Goy P, Luntz T, Ward E, Ryals J. 1993. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proceedings of the National Academy of Sciences USA 90 : 7327–7331. 2. Boller T. 1993. Antimicrobial functions of the plant hydrolases, chitinase and β-1,3-glucanase. In : Fritig B, Legrand M, eds. Mechanisms of Plant Defence. Dordrecht : Kluwer Academic Publishers, 391–400. 3. Bowles D. 1990. Defence-related proteins in higher plants. Annual Reiew of Biochemistry 59 : 873–907. 4. Bryngelsson T, Sommer-Knudsen J, Gregersen PL, Collinge DB, Ek B, Thordal-Christensen H. 1994. Purification, characterisation, and molecular cloning of basic PR-1-type pathogenesisrelated proteins from barley. Molecular Plant-Microbe Interactions 7 : 267–275. 5. Calderon P, Pontis HG. 1985. Increase of sucrose synthase activity in wheat plants after a chilling shock. Plant Science 42 : 173–176. 6. Collinge DB, Slusarenko AJ. 1987. Plant gene expression in response to pathogens. Plant Molecular Biology 9 : 389–410. 7. Collinge DB, Milligan DE, Dow JM, Scofield G, Daniels MJ. 1987. Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv itians. Plant Molecular Biology 8 : 405–414. 8. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K. 1993. Plant chitinases. Plant Journal 3 : 31–40. 9. Collinge DB, Ek B, Thordal-Christensen H. 1994. The induction of gene expression in response to pathogenic microbes. In : Basra AS, ed. Mechanisms of Plant Growth and Improed Productiity : Modern Approaches and Perspectives. New York : Marcel Dekker, 391–433. 10. Collinge DB, Bryngelsson T, Gregersen PL, Smedegaard-Petersen V, Thordal-Christensen H. 1996. Resistance against fungal pathogens : its nature and regulation. In : Basra AS, Basra R, eds. Mechanisms of Enironmental Stress Resistance in Plants. Chur, Switzerland : Harwood Academic Publishers. 11. Crespi MD, Zabaleta EJ, Pontis HG, Salerno GL. 1991. Sucrose synthase expression during cold acclimation in wheat. Plant Physiology 96 : 887–891.
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Interactions between cold hardening and Microdochium nivale infection on expression of pathogenesis-related genes in winter wheat AH . E*, S. S. K and A. M. T Norwegian Crop Research Institute, Plant Protection Centre, Fellesbygget, N-1432 A| s, Norway (Accepted for publication September 1998 )
Cold hardening induces snow mould resistance in cereals and grasses. The mechanism for this induced resistance is not fully understood. The accumulation of transcripts encoding sucrose synthase and the pathogenesis-related proteins (PR-proteins) chitinase, β-1,3-glucanase, peroxidase and PR-1a were studied in hardened and non-hardened winter wheat at several time points after inoculation with the snow mould Microdochium niale. Sucrose synthase was induced by the hardening treatment itself. The PR-proteins were all induced by infection, this response was stronger and more rapid in plants that had been hardened prior to inoculation. The present results suggest that cold hardening has a conditioning effect on snow mould induced expression of PR-proteins. This may partly explain how cold hardening induces snow mould resistance. # 1998 Academic Press
INTRODUCTION
Microdochium niale (Fr.) Samuels & Hallet (pink snow mould, teleomorph Monographella nialis (Schaffnit) E. Mu$ ller) attacks winter cereals and grasses. The cold hardening that plants go through when temperatures decrease in autumn, is known to strongly increase the resistance of Poaceae species to snow moulds [4, 36, 37, 39, 42, 45 ] and other pathogenic fungi [38, 41 ]. Several changes occurring in plants during hardening could be responsible for the increase in snow mould resistance. Research has mainly been focused on the role of carbohydrate storage and metabolism and to some extent on the plant cell water potential as reviewed by Gaudet [15 ]. Tronsmo et al. [41 ] initiated investigation of the possible role of PR proteins. The synthesis of PR-proteins is a general and systemic response of plants to pathogen attack. Chemical substances and other environmental stresses [33, 34 ] can also induce the synthesis of these proteins. Induction of PR-proteins by low temperature has been shown in barley [41 ], rye [19 ], bermuda grass [13 ] and potato [44 ]. Many PR-proteins are encoded by small gene families, and different isoforms can be produced in different stress situations [8, 27, 34 ]. *To whom all correspondence should be addressed. Abbreviations used in the text : AFP, antifreeze protein ; OD, optical density ; PR-proteins, pathogenesisrelated proteins ; SAR, systemic acquired resistance ; UDP, uracil-diphosphate. 0885–5765\98\110301j10 $30.00\0
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Increased levels of the PR-proteins chitinase, β-1-3-glucanase, peroxidase and PR1a after inoculation has been reported, both at the mRNA and at the protein level, from several host-pathogen systems [3, 6, 9 ]. Several possible roles for chitinases and β1,3-glucanases in plant defence have been investigated [34 ]. Both groups of enzyme have antifungal activity, probably due to their ability to degrade chitin and β-1,3-glucan in fungal cell walls [2, 28 ]. Another function of these enzymes in host plant defence may be to release elicitor-active substances from plant or fungal cell walls [17, 18, 29, 34 ]. There are indications that transgenic plants with a constitutive expression of chitinase have increased resistance to fungal pathogens [34 ]. Peroxidases are implicated in a variety of defence-related processes, including lignification, cross-linking of phenolics and glycoproteins, suberization and phytoalexin production [30 ]. Little is known about the function of PR-1a, but it appears to have a deleterious effect on oomycetes, both in itro and in transgenic tobacco [1, 34 ]. Sucrose synthase is not generally regarded as pathogenesis-related, but was induced in barley following inoculation with Blumeria graminis f.sp. hordei (syn. Erysiphe graminis f.sp. hordei) [16 ]. Sucrose synthase catalyses the reaction sucrosejUDP(ADP) *- UDP(ADP)-glucosejfructose [12, 32 ]. Tronsmo et al. (41) found that sucrose synthase and chitinase were induced in barley by cold hardening. When infected with B. graminis, hardened plants had a much stronger expression of peroxidase than non-hardened plants. They suggested that increased resistance to fungal pathogens in cold hardened plants might be due to an enhanced expression of defence mechanisms in these plants. In order to investigate whether PR-proteins and sucrose synthase are involved in cold hardening induced snow mould resistance, we used cDNAS from barley encoding chitinase, β-1,3glucanase, peroxidase, PR-1a and sucrose synthase as probes to detect the expression of homologous sequences in hardened and non-hardened winter wheat (Triticum aestium L.) during infection with M. niale.
MATERIALS AND METHODS
Plant and fungal materials Two Scandinavian cultivars of winter wheat, Rida and Folke, were used. The plants were treated in four different ways : (1) non-hardened, non-inoculated, (2) hardened, non-inoculated, (3) non-hardened, inoculated and (4) hardened, inoculated. Seeds were sown at 1 cm depth in soil in 9 cm pots (five plants per pot) and grown for two weeks in a controlled environment at 12 mC night\18 mC day, 12 h photoperiod with a light intensity of 200 µmol m−# s−". The plants were cold hardened for one week at 4 mC, 16 h photoperiod with a light intensity of 100 µmol m−# s−". Plants in the nonhardened treatments were sown one week later to ensure that they were approximately the same size as the hardened plants at the time of inoculation (3-leaf-stage, approximately 20 cm in height). The fungus used in this study was isolate 5\93 of Microdochium niale, originally isolated from Lolium perenne L. at Nordera/ s in 1986 (Plant Protection Centre, AH s, Norway). The inoculum was prepared as described by Tronsmo [40 ], except that the mycelial suspension was diluted to an OD of 0n5 at 430 nm. The plants were sprayed
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with 2n5 ml inoculum per pot and incubated under moist cellulose wadding and black plastic at 4 mC. Non-inoculated plants were incubated in the same way. Shoot tissue was sampled prior to inoculation (0 h), and at 3 h, 7 h, 21 h, 45 h, 4 days and 8 days following inoculation. Plants from three replicate pots within each treatment were pooled. The plants were cut 1–2 cm above soil level, immediately frozen in liquid nitrogen and stored at k80 mC. Mycelial growth was not observed until 3–4 weeks after inoculation, so no effort was made to remove mycelium from the leaf surface before sampling. Three pots from each treatment were incubated at 4 mC for a total of 6 weeks in order to establish the level of snow mould resistance. After incubation, the plants were cut 7 cm above soil level and allowed to regrow in a greenhouse (approximately 18 mC) for 9 days. Each plant was scored on a scale of 0 (dead or almost dead), 1 (severely affected) or 2 (apparently healthy). A value representing regrowth was then calculated for each pot using this formula : (number of plants with score 0)*0j(number of plants with score 1)*1j(number of plants with score 2)*2. The dry weight of all shoot tissue in each pot was also recorded. An analysis of variance (SAS Institute Inc., Cary, NC, U.S.A.) was performed on the dry weight data.
RNA isolation and northern blotting One to three grams of plant material were ground in liquid nitrogen with a mortar and pestle. Total RNA was isolated essentially as in Collinge et al. [7 ], but with 0n5 g l−" 8-hydroxyquinoline added to the phenol\chloroform. Fifteen micrograms of RNA from each sample was run on 1n2 % agarose gels containing 1n1 % formaldehyde, and blotted onto Zeta-Probe nylon membranes (BioRad) by using a capillary transfer system with 20iSSC. The RNA was bound to the membrane by ultraviolet radiation (700 µJ cm−#) followed by baking at 80 mC for 1 h. The membranes were stained with 0n02 % methylene blue in 0n3 sodium acetate in order to check the integrity and the amount of RNA in each lane. The inserts to be used as probes were isolated with GENECLEAN4 Kit (BIO 101, La Jolla, CA, U.S.A.) and labelled with αdCTP ($#P) using an Oligolabelling Kit (Pharmacia Biotech). Each probe was hybridised to a new membrane that had not been used previously. After being washed 2i15 minutes in 0n1iSSC, 0n5 % SDS at 68 mC, the membranes were prehybridised for 2 h at 68 mC in 8iDenhardt, 0n5 % SDS, 4iSSC, 1 m EDTA and 200 µg ml−" denatured salmon sperm DNA. Hybridisation was performed under the same conditions except that the concentration of Denhardt was 4i and 7 % dextran sulphate was added. Membranes were washed under low stringency in 1iSSC, 0n1 % SDS for 30 min at 42 mC and under medium stringency in 1iSSC, 0n1 % SDS for 30 min at 68 mC prior to autoradiography. The probes were prepared from five different cDNA clones kindly provided by Dr Hans Thordal-Christensen and Dr David Collinge, The Royal Veterinary and Agricultural University, Denmark. These cDNAs had been isolated from a cDNA library of barley leaves inoculated with B. graminis f.sp. hordei, and include pBH6-301 (accession No. X58396), encoding a putative peroxidase [35 ], pBH6-4, encoding a putative sucrose synthase [16 ], pHvPR-1a (accession No. X74939), encoding a basic PR-1 protein [4 ], N12, encoding a putative class II chitinase and I1, encoding a
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putative β-1,3-glucanase (The latter two from H. Thordal-Christensen). The PR-1a, chitinase and β-1,3-glucanase are all secreted into the apoplast of powdery mildew infected barley leaves [4, 10 ]. The whole experiment was conducted twice. RESULTS
Hardened plants had a significantly higher regrowth after incubation than the nonhardened plants (P l 0n0001 for dry weight data) (Table 1). The direct effects of T 1 Resistance to Microdochium nivale and the effect of hardening in two cultiars of winter wheat. Nonhardened (k) and hardened (j) plants were inoculated (j)or not inoculated (k) and incubated for 6 weeks under artificial snow coer. The plants were then cut down to 7 cm and allowed to regrow in a greenhouse. (a) Resistance expressed as dryweight after regrowth and regrowth score (means of three potspS.D.) as described in the text. Regrowth score 10 l no injury, regrowth score 0 l all plants dead. (b) Results from analysis of ariance on dry weight data (a) Cultivar Folke
Rida
Hardened
Inoculated
Dry weight (mg)
Regrowth score
k j k j k j k j
k k j j k k j j
112p38 160p36 0,3p0,6 123p36 90p12 192p11 19p26 137p36
10,0p0,0 10,0p0,0 0,7p0,6 5,7p0,6 9,0p1,7 10,0p0,0 2,3p3,2 6,3p2,3
DF
Mean square
1 1 1 1 1 1 1
0,000693 0,056940 0,028222 0,003151 0,000187 0,000900 0,001276
(b) Source Cultivar Hardening Inoculation Hardeningiinoculation Cultivariinoculation Cultivarihardening Cultivarihardeningiinoculation
F value P value 0,89 73,24 36,30 4,05 0,24 1,16 1,64
0,36 0,0001 0,0001 0,061 0,63 0,30 0,22
hardening on gene expression can be seen in the samples taken before incubation (0 h) in Fig. 1. The hardening treatment induced the expression of sucrose synthase in both cultivars. Effects of the incubation treatment Effects of the incubation treatment alone can be seen by comparing incubated samples that were non-hardened and non-infected with the non-hardened samples taken prior to inoculation (0 h). Expression of sucrose synthase was induced during incubation in both cultivars (Fig. 1e).
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nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h nh h
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F. 1. Hybridisation on northern blots. Fifteen micrograms of RNA isolated from samples taken prior to inoculation (0 h) and at different time points after inoculation with M. niale were applied in each lane. Membranes were hybridised to DNA probes of barley chitinase (a), β-1,3-glucanase (b), peroxidase (c), PR-1a (d) and sucrose synthase (e). h : hardened, nh : non-hardened, i : inoculated, ni : noninoculated. A photograph of the membrane stained in methylene blue is shown under each autoradiogram.
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Interaction between hardening and the incubation treatment When comparing hardened, non-inoculated samples with non-hardened, noninoculated samples, it can be seen that hardening also had an enhancing effect on the expression of chitinase, β-1,3-glucanase and PR-1a during incubation (Fig. 1 a, b, d). Effects of inoculation Inoculated plants had a significantly lower regrowth after incubation than the noninoculated plants (P l 0n0001 for dry weight data) (Table 1). Effects of infection on gene expression can be studied by comparing the non-hardened, inoculated samples with the non-hardened, non-inoculated samples. Infection increased the expression of all the genes studied except for sucrose synthase (Fig. 1). Chitinase was enhanced early, while β-1,3-glucanase and PR-1a were enhanced later. The expression of these three genes increased steadily throughout the sampling period. Peroxidase was also induced by infection, but the expression did not increase throughout the sampling period. Folke appeared to have an expression peak around 4 days. Interaction between hardening and inoculation The analysis of variance revealed an interaction between hardening and inoculation on regrowth after incubation (P l 0n061 for dry weight data) (Table 1). Plants that had been hardened prior to inoculation were more resistant than those that had not been hardened. Expression of the M. niale induced genes was more rapid and\or stronger in plants that had been hardened before inoculation (Fig. 1a–d). As in non-hardened plants, the expression of chitinase, β-1,3-glucanse, and PR-1a increased throughout the sampling period and there was a peak in peroxidase expression around 45 h–4 days. Expression of these genes appeared to reach higher levels than what can be accounted for by a cumulative effect of hardening and infection, particularly around 45 h. Effects of cultiar There was no significant difference between cultivars in regrowth (P l 0n36) (Table 1), but there were some differences in gene expression (Fig. 1). Infected plants of Folke had a stronger expression of β-1,3-glucanase, peroxidase and PR-1a than infected plants of Rida. Multiple bands Hybridisation with the chitinase and β-1,3-glucanase probes produced two bands in addition to the main band (Fig. 1). The PR-1a and sucrose synthase probes also hybridised to one additional band visible on highly exposed films (data not shown). Additional bands were always weaker and of a larger molecular size than the main band. The signal strength of the bands relative to each other appeared to be the same in all lanes. DISCUSSION
We could not detect any difference between Folke and Rida in their resistance to M. niale, measured as regrowth, although Folke in field tests has been found to be the more resistant of the two [21, 22 ]. The level of replication in our study (n l 3) may
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simply be too low to reveal any regrowth differences. Results may also vary between studies due to experimental or environmental differences and cultivarienvironment interactions. Ho$ mmo$ [23 ] has shown that resistance at the whole plant level does not correlate tightly with resistance at the single leaf level, using a detached leaf testing system. The differences in gene expression that we observed between the two cultivars could be related to a type of resistance that is not detectable when measuring resistance as regrowth. Care should be taken when comparing the expression strength of the different genes at a given time. The probes are homologous with barley genes, and the % similarity of these genes with the corresponding wheat genes is unknown and could vary. There is also some variation in probe activity and exposure time. The cold hardening treatment induced expression of sucrose synthase in both cultivars. This is in agreement with earlier reports [5, 11, 25, 31 ] and supports a role for sucrose synthase in the cold hardening process. Hon et al. [20 ] found that apoplastic antifreeze proteins (AFPs) accumulating in rye cold hardened for 3 weeks at 5\2 mC (day\night) had sequence similarities to endochitinase and endo-β-1,3-glucanase and affinity to antibodies against these enzymes. They suggested that AFPs may have evolved from PR-proteins or that they are post-translationally modified PR proteins. The hardening treatment in our experiment was relatively mild (4 mC for 1 week) ; an induction of β-1,3-glucanase and chitinase might have been present in material hardened for a longer period or at a lower temperature. The effects of the incubation treatment, both in itself and in combination with hardening, could be responses to either the mechanical handling, the sudden darkness or the low temperature involved in incubation. The incubation-induced expression of sucrose synthase indicates that this enzyme might also be induced by low temperature under very low light intensities. Infection with M. niale enhanced the expression of chitinase, β-1,3-glucanase, peroxidase and PR-1a. Hardened plants had a higher expression of these genes after inoculation than non-hardened plants had. This was also the case for peroxidase expression in barley infected with B. graminis [41 ]. Although the linearity between expression level and signal strength may be questionable, the combined effect of hardening and infection did not appear to be merely of a cumulative nature. Hardening prior to inoculation rather appeared to improve the plants’ ability to respond to snow mould attack in terms of PR-gene expression. Similar sensitising or conditioning effects have been reported in plants expressing systemic acquired resistance (SAR) [24, 26 ]. The state induced in plants by hardening could have similarities with SAR. A cold hardening induced expression of PR-genes and an induced ability to express these genes upon infection could explain, in part, why cereals and grasses are more resistant to M. niale and other fungal pathogens when hardened. The mechanism behind a conditioning effect of cold hardening on PR-gene expression is not known. Cold hardening might lead to a higher level of compounds that positively affect pathogen recognition, signalling or PR-gene expression. Ka$ stner et al. 1998 [26 ] presented evidence that a chemical compound inducing SAR (2,6dichloroisonicotinic acid, DCIA) and unknown fungal elicitors from Colletotrichum lagenarium can have a synergistic effect on chitinase expression in cucumber, and that the long-term influence of DCIA can lead to a developmentally regulated tuning-up
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of the elicitor-triggered chitinase expression machinery. Low levels of signals associated with one defence pathway have in several plant species been shown to positively affect the magnitude and kinetics of the defence responses associated with a different pathway, indicating that there is some cross-talk between different signalling pathways [43 ]. Similar phenomena could be involved in cold hardening conditioned PR-gene expression. It may also be that the hardened state of no or very low growth, with a large storage of photosynthetic products, makes plants more responsive to pathogen attack as well as to other stresses. It is unclear what the multiple bands observed might represent. They could represent either mRNAs at different steps in the processing or different members of the respective gene families, regulated in the same way as the gene behind the main band. In conclusion, we have shown that infection by M. niale induces expression of chitinase, β-1,3-glucanase, peroxidase and PR-1a, and that this induction is much stronger in hardened plants than in non-hardened plants. This may partly explain why cold hardened plants have an increased disease resistance. AH shild Ergon was supported by a grant from the Norwegian Research Council. We are grateful to Hans Thordal-Christensen and David Collinge, The Royal Veterinary and Agricultural University, Denmark for providing the DNA probes and to Linda Hjeljord for sharing her laboratory experience. Thanks also to Andre! Laroche, Denis A. Gaudet, Torbjørn Ergon and Grete Lund. REFERENCES 1. Alexander D, Goodman RM, Gut-Rella M, Glascock C, Weymann K, Friedrich L, Maddox D, Ahl-Goy P, Luntz T, Ward E, Ryals J. 1993. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proceedings of the National Academy of Sciences USA 90 : 7327–7331. 2. Boller T. 1993. Antimicrobial functions of the plant hydrolases, chitinase and β-1,3-glucanase. In : Fritig B, Legrand M, eds. Mechanisms of Plant Defence. Dordrecht : Kluwer Academic Publishers, 391–400. 3. Bowles D. 1990. Defence-related proteins in higher plants. Annual Reiew of Biochemistry 59 : 873–907. 4. Bryngelsson T, Sommer-Knudsen J, Gregersen PL, Collinge DB, Ek B, Thordal-Christensen H. 1994. Purification, characterisation, and molecular cloning of basic PR-1-type pathogenesisrelated proteins from barley. Molecular Plant-Microbe Interactions 7 : 267–275. 5. Calderon P, Pontis HG. 1985. Increase of sucrose synthase activity in wheat plants after a chilling shock. Plant Science 42 : 173–176. 6. Collinge DB, Slusarenko AJ. 1987. Plant gene expression in response to pathogens. Plant Molecular Biology 9 : 389–410. 7. Collinge DB, Milligan DE, Dow JM, Scofield G, Daniels MJ. 1987. Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv itians. Plant Molecular Biology 8 : 405–414. 8. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K. 1993. Plant chitinases. Plant Journal 3 : 31–40. 9. Collinge DB, Ek B, Thordal-Christensen H. 1994. The induction of gene expression in response to pathogenic microbes. In : Basra AS, ed. Mechanisms of Plant Growth and Improed Productiity : Modern Approaches and Perspectives. New York : Marcel Dekker, 391–433. 10. Collinge DB, Bryngelsson T, Gregersen PL, Smedegaard-Petersen V, Thordal-Christensen H. 1996. Resistance against fungal pathogens : its nature and regulation. In : Basra AS, Basra R, eds. Mechanisms of Enironmental Stress Resistance in Plants. Chur, Switzerland : Harwood Academic Publishers. 11. Crespi MD, Zabaleta EJ, Pontis HG, Salerno GL. 1991. Sucrose synthase expression during cold acclimation in wheat. Plant Physiology 96 : 887–891.
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