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Fusarium Species Synthesize Alkaline Proteinases in Infested Barley
Journal of Cereal Science 37 (2003) 349±356 doi:10.1006/jcrs.2002.0512
Fusarium Species Synthesize Alkaline Proteinases in Infested Barley Anja I. Pekkarinen ²³, Tuija H. Sarlin³, Arja T. Laitila³, Auli I. Haikara³ and Berne L. Jones²§ ²Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706, USA; ³VTT Biotechnology, P.O. Box 1500, 02044 VTT, Finland and §Cereal Crops Research Unit, Agricultural Research Service, US Department of Agriculture, Madison, Wisconsin 53726, USA Received 5 June 2002 ABSTRACT Barley (Hordeum vulgare L.) that is infested with Fusarium head blight (FHB, `scab') is unsuitable for malting and brewing because it may contain mycotoxins and has unacceptable malting quality. Fungal proteinases are apparently often involved in plant-microbe interactions, where they degrade storage proteins, but very little is known about the enzymes that the fungi produce in the infected grain. We have shown previously that one plant pathogenic fungus, Fusarium culmorum, produced subtilisin- and trypsinlike enzymes when grown in a cereal protein medium. To establish whether these proteinases were also synthesized in FHB-infested barley in vivo, ®eld-grown barley was infested as the heads emerged. Extracts were prepared from the grain as it developed and matured and their proteolytic activities were measured with N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and N-benzoyl-Val-Gly-Arg p-nitroanilide. The heavily infested barleys contained both subtilisin- and trypsin-like activities. These enzymes reacted with antibodies prepared against each of the two F. culmorum proteinases, indicating that those produced in the laboratory cultures and in the ®eld-infested barley were the same. The presence of these proteinases correlated with the degradation of speci®c buffer-soluble proteins in the infested grains. These enzymes readily hydrolyzed barley grain storage proteins (C- and D-hordeins) in vitro. The presence of these Fusarium proteinases in the barley indicates that they probably play an important role in the infestation, but exactly how and when they function is not clear. # 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Fusarium, scab, proteinase, barley.
INTRODUCTION Fusarium head blight (FHB, scab) is a fungal disease of wheat and barley that is caused predominantly by Fusarium graminearum, F. culmorum and F. avenaceum1. Severe FHB infestations have occurred in humid ABBREVIATIONS USED: BVGRpNA N-benzoyl-Val-GlyArg p-nitroanilide; CBBR-250 Coomassie Brilliant Blue R-250; FAN free amino nitrogen; MH mechanically harvested; SAAPFpNA N-succinyl-AlaAla-Pro-Phe p-nitroanilide; SL subtilisin-like; TL trypsin-like.
Corresponding Author. Anja Pekkarinen, VTT Biotechnology, P.O. Box 1500, 02044 VTT, Espoo, Finland. Tel: 358±9-456-4461; Fax: 358±9-455-2103; E-mail: anja.pekkarinen@vtt.®
0733±5210/03/030349 08 $35.00/0
regions in the Americas, Asia and Europe and have often disastrously affected both producers and consumers2±4. Heavily infected cereals are unsuitable for use as either food or feed due to their poor processing properties and possible mycotoxin contamination. Infection by fusaria may lead to the production of toxins5 and hormone-like compounds6, as well as of various hydrolytic enzymes such as cutinase7,8, proteinases9±12, xylanases and cellulases12,13. These may be associated with pathogenesis but, so far, none of these have been considered as a clearly determining factor in virulence. The roles of proteinases that form during the FHB infection of cereals is of interest because protein degradation can strongly # 2003 Elsevier Science Ltd. All rights reserved.
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affect the baking, malting or brewing quality of the diseased grain10,14,15. Nightingale et al.10 showed that an extraneous alkaline proteinase occurred in wheat that was infested with F. graminearum and Schwarz et al.12 showed that there was an increase in the nonspeci®c proteinase activity of diseased barley grain. However, no one had previously identi®ed Fusarium proteinases in cereal grains. Recently, we puri®ed and described the characteristics of a subtilisin- and a trypsin-like proteinase that were produced by F. culmorum that was grown in a medium that contained wheat gluten16,17. For this study, arti®cially inoculated ®eld grown barley grain was used to study whether these enzymes were produced in vivo during Fusarium infection. Their presence in extracts prepared from the inoculated grain was established by using speci®c synthetic substrates to measure their activities and by using antibodies raised against these enzymes.
EXPERIMENTAL Field experiments Two-rowed malting barley (Hordeum vulgare L.) cultivars `A' and `B' were grown during the summer of 1998 at two experimental farms that were located at Hauho and Jokioinen in southern Finland. The plants were sprayed with conidia suspensions from Fusarium graminearum (VTT D-95470), F. culmorum (VTT D-80148) or F. poae (VTT D-82182) when the incipient kernels were emerging from the boot. Triplicate plots (each 10 m2) were treated with each of the three Fusarium species and three plots were left untreated to serve as controls. Thirty randomly chosen spikes were collected manually from each plot 18, 40 and 53 days (Hauho) or 21, 36, 49 and 63 days (Jokioinen) after inoculation and the samples from the triplicate plots were pooled. Fifteen spikes from each pool were freeze-dried and stored at ÿ20 C for the proteinase studies. The barleys that are called `mechanically harvested' (MH) were machine harvested 54 or 63 days after inoculation at Hauho and Jokioinen, respectively. Those samples were dried in warm air to moisture contents of approximately 12% and stored at 12±14 C. A replicate experiment with F. culmorum was carried out during the summer of 2000 with cv. A at Hauho. In this case, spike samples were collected manually 33, 46 and 55 days after inoculation and grain was machine harvested 55 days after inoculation.
Measuring infection rates The percentage of the grain that was infected by Fusarium was estimated by plating non-disinfected kernels on a selective CZID-agar plate18. In addition, samples of the 1998 MH grain and of the year 2000 grain were homogenized and analyzed for yeast and mold counts. Ten grams of grain were soaked in 90 mL of 09% NaCl for 30 min at 8 C and homogenized for 10 min with Stomacher LabBlender 400 BA.6021 (Seward Medical, London, UK). Dilutions of the homogenates of year 1998 and 2000 samples were plated on YM nutrient or potato dextrose (PD) agar, respectively. Both nutrient media were purchased from Difco (Detroit, MI, USA.) and supplemented with 001% chloramphenicol, 001% chlorotetracycline and 002% Triton X. The colonies formed by Fusarium species were counted after 7 days of incubation at room temperature. The results were expressed as a number of colony forming units (cfu) per gram of homogenized grain. Extracting protein from the barley grain Freeze-dried kernels and the MH grain were ground with a BuÈhler Universal Laboratory disc mill, type DLFU (BuÈhler-Miag GmbH, Braunschweig, Germany) that was set to `30'. Their proteins were extracted by incubating 20 g of ground grain with 50 mL of 50 mM Na acetate, pH 55, buffer at 22 C for 15 h. The mixtures were centrifuged at 1700 g for 5 min, after which the supernatants were transferred to microcentrifuge tubes and centrifuged at 11 600 g for 5 min. The supernatants were ®ltered with 13 mm wide, 045 mm Millex-HV ®lters (Millipore, Bedford, MA, USA) and stored at ÿ20 C until analyzed. Proteinase activity assays The proteinase activities of the grain extracts were measured using speci®c synthetic substrates as described previously16. The subtilisinlike (SL) activities were measured at 25 C with 50 mM N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA, Sigma, St Louis, MO, USA) in 180 mM Tris-HCl, pH 90, and the trypsin-like (TL) activities at 28 C with 05 mM N-benzoyl-Val-GlyArg pNA (BVGRpNA) in 4% dimethyl sulfoxide (DMSO) and 175 mM Tris-HCl, pH 90. The nonspeci®c proteinase activities were measured at 40 C
Fusarium proteinases in infested barley
and pH 89 using an azogelatin method19, as modi®ed by Pekkarinen et al.16. The activities were expressed as nkat (synthetic substrates) or change in absorbance 100/min (azogelatin) per mL of grain extract. All of the analyses were duplicated. SDS-PAGE analysis of grain extracts Grain extracts (80 mL) prepared from the year 2000 samples were mixed with 5 SDS-sample buffer (20 mL) that contained 15% b-mercaptoethanol and held in a boiling water bath for 2 min. The proteins from 5 mL of the boiled samples were separated with a 12% SDS-PAGE gel20 and stained with 01% Coomassie Brilliant Blue R-250 (CBBR-250) that was dissolved in a 40% methanol±1% acetic acid solution. Antibody detection of F. culmorum proteinases The presence of the TL and SL enzymes in the year 2000 samples was examined using the western blotting method. Sixteen mL-samples of each grain extract and of the puri®ed TL proteinase17 (03 mg) were separated with SDS-PAGE as above and blotted, for 16 h, onto a nitrocellulose membrane at 8 C and in 25 mM Tris and 192 mM glycine, pH 83, with a voltage of 60 V. On a duplicate gel, 8 mL-samples of each grain extract and of the puri®ed SL proteinase16 (07 mg) were separated. The proteins were transferred onto the membrane for 24 h in pH 83-transfer buffer that contained 0035% SDS. Rabbit polyclonal antibodies that were raised against each of the puri®ed F. culmorum proteinases were used as primary antibodies (Animal Care Unit Polyclonal Antibody Service, University of Wisconsin Medical School, Madison, WI, USA) and the secondary antibody was a goat anti-rabbit horseradish peroxidase conjugate (Bio-Rad, Hercules, CA, USA). Hordein digestion with puri®ed enzyme The hordein hydrolyzing activity of the puri®ed F. culmorum SL proteinase16 was studied at pH 60 and 40 C using a previously described procedure17. The reaction suspension contained 31 mM Na citrate, pH 60, 4 mg/mL of the enzyme, 25% DMSO, and 25% (v/v) hordein extract that was prepared with 55% isopropanol and 2%
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b-mercaptoethanol21. The reactions were carried out in the presence or absence of a class speci®c inhibitor, chymostatin (40 mM, Sigma). A reaction mixture that contained no enzyme was prepared as a control for detecting any unspeci®c hordein degradation. Samples were removed from the suspensions after 0, 30 and 120 min of incubation and analyzed with a 12% SDS-PAGE gel. The gel was stained with CBBR-250 as above.
Hydrolysis of buffer-soluble proteins with puri®ed proteinases A sample of cv. A (grown in Finland in 1995) was ground with a U/D Cyclone Sample Mill (UDY, Ft. Collins, CO, USA) to pass a 05 mm screen and an extract was prepared in Na acetate, pH 55, buffer as described above. Seventy mL of extract was mixed with 3 mg of puri®ed F. culmorum SL or TL enzyme in a total volume of 350 mL of 130 mM Na citrate, pH 6, and incubated at 40 C for 0, 15, 30, 60 and 120 min. The reactions were stopped by mixing 50 mL of each sample with 13 mL of 5 SDS-sample buffer and heating in boiling water bath for 3 min. The hydrolysis products were analyzed with SDSPAGE on 12% acrylamide gels.
RESULTS AND DISCUSSION Infection rates Both the 1998 and 2000 summers were rainy, which provided excellent growth conditions for fusaria. These conditions caused a proliferation of the fungi, which resulted in the control barley also being contaminated with Fusarium. About 3 weeks after the treatments were applied in 1998, the noninoculated samples had a lower proportion of infected kernels than the Fusarium-inoculated ones, but the treated vs. control differences became smaller as the growing period progressed (Tables I and II). The data obtained by counting the colony forming units of the harvested samples indicated that the inoculated kernels generally contained more fungus than the controls. This implies that despite the high contamination level of all of the samples, the disease had proceeded further in the F. culmorum and F. graminearum-inoculated heads than in the nontreated or F. poae-inoculated ones.
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Table I Fusarium-infection rates of barley kernel samples from ®eld trials grown during the summer of 1998 in southern Finland
Location Hauho
Jokioinen
Control
Days after inoculation
A
18 40±53 54d 54d 21 36±63 63d 63d
4%c 80±96% 74% 10 103 cfu/ge 14% 86±100% 98% 550 cfu/g
a
F. culmorum b
F. graminearum
F. poae
B
A
B
A
B
A
B
8 58±66 94 550 18 76±100 100 63 103
76 96±100 100 40 104 46 96±100 100 40 104
24 100 100 20 104 64 100 100 16 105
96 100 100 40 105 96 100 100 20 106
74 100 100 63 105 98 100 99 40 106
30 68±98 86 32 103 32 92±100 100 550
32 82±90 93 40 103 44 66±100 96 10 105
a
Cultivar `A'. Cultivar `B'. c The percentage (%) of kernels that showed Fusarium contamination when whole kernels were incubated on CZID agar. d Mechanically harvested. Other samples were collected by hand. e The number of colony forming units per gram of homogenized grain that was analyzed using the YM agar plating method. b
Table II Fusarium-infection rates of control and F. culmorum-inoculated cv. `A' ®eld-grown barley kernels grown at Hauho during 2000 Days after inoculation 33 46 55 55c
Control
F. culmorum
93%a 550 cfu/gb 100% 55 103 cfu/g 97% 50 104 cfu/g 97% 50 102 cfu/g
100 22 105 100 17 106 100 24 106 100 41 105
a
The percentage (%) of kernels that showed Fusarium contamination when whole kernels were incubated on CZID agar. b The number of colony forming units per gram of homogenized grain that was analyzed using PD agar. c Mechanically harvested.
Fusarium proteinases were present in inoculated grains The SL activities of extracts prepared from barley samples that were grown in the ®eld during 1998 are presented in Table III. The activities of the inoculated and control extracts from both cultivars and both locations were quite similar after 18 days of inoculation. However, more pronounced differences were observed in the samples that were collected between 36 and 63 days after treatment. The activities of the extracts that were prepared from the F. culmorum and F. graminearum-inoculated
samples were generally 10±20 times higher than those of the corresponding controls. The differences between the inoculated and non-treated cv. A activities from the Jokioinen samples were, at most, eight-fold. This was partly due to the relatively low activities of the inoculated samples and partly because the non-treated samples of cv. A from Jokioinen contained slightly higher activities than the other controls. The activities of the F. poae inoculated barleys remained, in all cases, at the same level as those of the controls. Thus, the production of the alkaline proteinases was associated with higher pathogenicity, since both F. culmorum and F. graminearum are generally more pathogenic than F. poae1. However, whether the proteinase synthesis is important for cell wall penetration or whether it occurs only after penetration via other mechanisms, is not clear. When these three fungi were grown in a gluten medium, F. poae synthesized mostly acid proteinase(s) while the other two produced alkaline proteinase(s), implying that the synthesis of their proteinases were regulated differently22. Therefore, they may act in different ways in living organisms. However, when grown saprophytically on autoclaved barley, all three Fusarium species produced mainly alkaline proteinases. This implies that F. poae might produce alkaline proteinases in developing kernels if it could overcome the grain's defenses. The MH samples typically contained less SL activity than the ®nal manually picked samples, which were collected at nearly the same time. This difference may be due to the collection methods used since, during the mechanical harvesting, the lightest
Fusarium proteinases in infested barley
353
Table III The subtilisin-like activities (nkat/mL) of extracts of control and Fusarium-inoculated barleys that were grown during the summer of 1998 Control Location Hauho
Jokioinen
F. culmorum
F. graminearum
F. poae
Days after inoculation
Aa
Bb
A
B
A
B
A
B
18 40 53 54d 21 36 49 63 63d
077 020 009 013 036 029 062 090 078
054 013 009 008 025 029 026 024 016
182 295c 202 119 051 105 120 241 116
046 161 175 021 052 300 349 221 302
303 292 154 117 059 225 169 181 109
094 348 418 060 090 304 209 310 243
106 026 023 015 031 018 040 054 046
041 022 014 007 018 047 027 048 026
a
Cultivar `A'. Cultivar `B'. c The activities that are between 5 and 10 times higher than those of the corresponding controls are shaded and those that are more than 10 times higher are bolded and shaded. d Mechanically harvested. b
8 Activity, AU or nkat/mL
(most shrivelled) kernels were separated away from the grain pool by the harvesting machine. During hand harvesting, all of the grains were retained. When malts were produced from the 1998 MH grains and their malting qualities were analyzed, those from the F. culmorum- or F. graminearuminoculated cv. B barleys contained more wort soluble nitrogen and/or free amino nitrogen (FAN) than those made from the non-treated and F. poaeinoculated samples (A. Haikara, personal communication). With these two particularly virulent species, the highest SL activities were detected in the corresponding barley samples, especially at Jokioinen. This implies that the proteinase was probably at least partially responsible for the elevated protein degradation. The soluble nitrogen and FAN levels were also elevated in all of the inoculated cv. A malts23. These data do not indicate whether the increased protein hydrolysis occurred in the ®eld, during malting, or both. Similar results, i.e. increased levels of wort FAN and of neutral and alkaline proteinase activities in Fusarium-inoculated barley, have also been obtained in other studies12,14. In addition, the concentrations of the fungal toxins deoxynivalenol and zearalenone in the cv. B samples correlated strongly with their SL proteinase activities23. The non-speci®c (measured using the azogelatin substrate) and the SL and TL proteinase activities of the cv. A samples of the summer 2000 experiment showed the same trend as those from 1998 (Fig. 1). The differences between the inoculated and control samples were consistent for all three proteinase
6 4 2 0 33
C, non-sp.
46 55 Days after inoculation C, SL
C, TL
Fc, non-sp.
MH, 55 Fc, SL
Fc, TL
Figure 1 The proteinase activities of extracts that were prepared from control (C) and F. culmorum-inoculated (Fc) barley (cv. `A') in Hauho, Finland, in 2000. The activities were measured at pH 9 using the substrates N-succinyl-Ala-Ala-ProPhe pNA (subtilisin-like, SL), N-benzoyl-Val-Gly-Arg pNA (trypsin-like, TL) or azogelatin (non-speci®c proteinase).
analyses. The activities of the inoculated grains increased with time and 55 days after inoculation they were about 30 (non-speci®c), 70 (SL) or 20 (TL) times higher than those of the controls. The activities in the MH grain were once again very low compared to those of the manually collected samples, but they were still about 10 times higher in the inoculated than in the control samples, and were roughly the same as those of the corresponding cv. B samples from Jokioinen in 1998. Both the SL and TL proteinases were detected in extracts of inoculated grain, using antibodies that were raised against the puri®ed enzymes (Fig. 2). The TL enzyme antibodies did not cross react with
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A
kD -100 - 50 - 37 - 25
- 15
C Fc 33 d
C
Fc 46 d
C Fc C Fc_ TL 55 d MH 55d
C Fc 33 d
C Fc C Fc M C Fc_ 46 d 55 d MH, 55d
Figure 3 SDS-PAGE separations of buffer soluble proteins from barley (cv. `A') grown at Hauho during 2000. C, controls; Fc, F. culmorum inoculated; M, molecular weight standard. The samples were as in Figure 2. The protein bands that had disappeared or clearly diminished in the inoculated sample are indicated with arrows.
B
C Fc 33 d
C
Fc 46 d
C Fc 55 d
C Fc_ SL MH 55d
Figure 2 Western blots of control (C) and F. culmoruminoculated (Fc) barley (cv. `A') extracts. The barley was grown at Hauho, Finland, in 2000. Antibodies against A) the TL or B) the SL enzymes were used to detect the proteinases. The samples were collected by hand 33, 46, or 55 days after inoculation, or mechanically (MH) after 55 days. The right hand lanes were loaded with puri®ed TL (03 mg), or SL (07 mg) enzymes. Sixteen and 8 mL extract samples were loaded onto gels A and B, respectively.
the SL ones, and vice versa. The responses with both of the antibodies were strongest in the manually collected 55-d sample. Both the activity (Fig. 1) and antibody reactions (Fig. 2) of the inoculated MH samples were subtle, indicating that their low activity levels were not the result of enzyme inactivation, but that the lightest, most heavily infected, grains were apparently removed by the mechanical harvester.
The effect of inoculation on buffer soluble grain proteins When extracts of the various year 2000 grain samples were separated by SDS-PAGE, their
protein compositions differed (Fig. 3). The approximately 62, 32, 30 and 25 kD proteins that were present in the 55-d control samples had disappeared from the inoculated grain and a new protein of about 44 kD had formed. These changes were most apparent in the 55-d samples, that contained the highest alkaline proteinase activities, but some differences were also visible in the earlier samples. On the other hand, when an extract of these soluble proteins was prepared and treated with each of the puri®ed proteinases in vitro under conditions similar to those used in the hordein hydrolysis experiment, no hydrolysis was observed (results not shown). This may have been due to the added enzyme being bound to proteinase inhibitors that occur in barley grains24. The apparent protein decomposition shown in Figure 3 may have occurred either during the fungal invasion or during the protein extraction process (15 h at 22 C), due to the `excess' proteinase that was not bound by the endogenous inhibitors. The soluble protein patterns of the control and experimental MH barleys were identical (Fig. 3), probably because they contained little proteinase activity (Fig. 1). Fusarium proteinases hydrolyze hordeins in vitro The F. culmorum TL proteinase hydrolyzed the D hordein and all but one of the C hordeins of Morex barley in vitro17 and the SL proteinase hydrolyzed all of the C and D hordeins (Fig. 4). None of
Fusarium proteinases in infested barley
kD 10075-
-D
50-
C
25B 151
2
3
4
5
6
7
8
Figure 4 The in vitro hydrolysis of a hordein extract by the puri®ed F. culmorum subtilisin-like enzyme. Lanes 2±4: Enzyme, no inhibitor reactions were stopped at 0, 30 and 120 min, respectively; lanes 5±6: Enzyme plus chymostatin, 0 and 120 min; lanes 7±8: hordein, no enzyme, no chymostatin. A molecular weight standard sample was loaded in lane 1. The letters B, C and D indicate the positions of the different classes of hordeins as in reference 25.
the hordeins were degraded in the absence of the puri®ed enzyme, and chymostatin inhibited the hydrolysis, indicating that no nonspeci®c protein degradation occurred in the reaction mixtures. The C and D hordeins comprise 10±20% and 2±4% of the barley prolamins, respectively25. No degradation of the major, B, hordein fraction was detected under these conditions. Whether or not these enzymes contribute to the protein matrix degradation that occurs in vivo10,15,26,27, remains to be determined with immunomicroscopy. What roles do the Fusarium proteinases play in grain invasion? The synthesis of one particular enzyme is normally not crucial for the invasion of a fungus; the pathogens generally produce a multitude of enzymes that work together to decompose the host plant structures28±30. The roles of proteinases in different plantmicrobe interactions may differ: in some cases, the expression of proteinases has been suggested as being a primary pathogenesis factor31,32, but a fungal proteinase has also been detected in a symbiotic fungal-plant interaction33. A subtilisin-like proteinase was produced by F. oxysporum f. sp. lycopersici in tomato roots and stems during infection, but disruption of its gene did not affect the pathogenicity of the fungus11. Regardless of how essential the proteinases may be for pathogenicity, it has been hypothesized that certain proteinase inhibitors
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protect the host plant from pathogen invasion34. Both the SL and TL proteinases were produced by Fusarium in the grain, indicating that the interaction between the fungus and barley involves fungal extracellular proteinases at some stage(s) of disease development. The highest activities were detected in grains that were inoculated with the most pathogenic species (Table III), implying that the proteinase production is associated with pathogenicity. However, because the activities were found only in heavily infected grain, it seems that they were produced (or at least detected) only after a successful invasion had occurred and were required for saprophytic purposes. On the other hand, this does not exclude that they played some role during the active penetration of the fungus into the grain, since the fungi may have produced these enzymes at such low concentrations during the beginning of the infection that they could not be detected by the methods that we used. In addition, the fusaria may produce other, undetected, proteinases during the infection, or they may trigger the synthesis or activation of some of the barley proteinases that normally function during the germination process. This work indicates that Fusarium proteinases are undoubtedly present in barley during infection, but the question of exactly how or whether this facilitates the attack of the fungus on the grain is still unanswered. Acknowledgements The ®nancial support of the Raisio Group Research Foundation, the Tor-Magnus Enari Fund, the American Malting Barley Association and of Anheuser-Busch Inc. are greatly appreciated.
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5. Desjardins, A.E., Hohn, T.M. and McCormick, S.P. Trichothecene biosynthesis in Fusarium species: Chemistry, Genetics, and Signi®cance. Microbiological Reviews 57 (1993) 595±604. 6. Sunder, S. and Satyavir. Vegetative compatibility, biosynthesis of GA3 and virulence of Fusarium moniliforme isolates from bakanae disease of rice. Plant Pathology 47 (1998) 767±772. 7. Kolattukudy, P.E., Rogers, L.M., Li, D., Hwang, C.-S. and Flaishman, M.A. Surface signaling in pathogenesis. Proceedings of National Academy of Sciences USA 92 (1995) 4080±4087. 8. Stahl, D.J., Theuerkauf, A., Heitefuss, R. and SchaÈfer, W. Cutinase of Nectria haematococca (Fusarium solani f. sp. pisi) is not required for fungal virulence or organ speci®city on pea. Molecular Plant±Microbe Interactions 7 (1994) 713±725. 9. Urbanek, H. and Yirdaw, G. Hydrolytic ability of acid protease of Fusarium culmorum and its possible role in phytopathogenesis. Acta Microbiologica Polonica 33 (1984) 131±136. 10. Nightingale, M.J., Marchylo, B.A., Clear, R.M., Dexter, J.E. and Preston, K.R. Fusarium head blight: Effect of fungal proteases on wheat storage proteins. Cereal Chemistry 76 (1999) 150±158. 11. Di Pietro, A., Huertas-Gonz alez, M.D., GutierrezCorona, J.F., MartõÂnez-Cadena, G., Meglecz, E. and Roncero, M.I.G. Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum. Molecular Plant±Microbe Interactions 14 (2001) 653±662. 12. Schwarz, P.B., Jones, B.L. and Steffenson, B.J. Enzymes associated with Fusarium infection of barley. Journal of American Society of Brewing Chemists 60 (2002) 130±134. 13. Zalewska-Sobczak, J. Sequential secretion of cell wall degrading enzymes by Botrytis fabae and Fusarium avenaceum during growth on host and non-host plants. Biochemie und Physiologie der P¯anzen 180 (1985) 169±175. 14. Schwarz, P.B., Schwarz, J.G., Zhou, A., Prom, L.K. and Steffenson, B.J. Effect of Fusarium graminearum and F. poae infection on barley and malt quality. Monatsschrift fuÈr Brauwissenschaft 3/4 (2001) 55±63. 15. Schwarz, P.B. Impact of head blight on the malting and brewing quality of barley. In `Fusarium head blight of wheat and barley', (K.J. Leonard and W.R. Bushnell, eds), American Phytopathological Society Press, St Paul, MN, USA (2002) xxx±xxx In-press. 16. Pekkarinen, A.I., Jones, B.L. and Niku-Paavola, M.-L. Puri®cation and properties of an alkaline proteinase of Fusarium culmorum. European Journal of Biochemistry 269 (2002) 798±807. 17. Pekkarinen, A.I. and Jones, B.L. Trypsin-like proteinase produced by Fusarium culmorum grown on grain proteins. Journal of Agricultural and Food Chemistry 50 (2002) 3849± 3855. 18. Abildgren, M.P., Lund, F., Thrane, U. and Elmholt, S. Czapex-Dox agar containing iprodione and dichloran as a selective medium for isolation of Fusarium species. Letters in Applied Microbiology 5 (1987) 83±86.
19. Jones, B.L., Fontanini, D., Jarvinen, M. and Pekkarinen, A. Simpli®ed endoproteinase assays using gelatin or azogelatin. Analytical Biochemistry 263 (1998) 214±220. 20. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680±685. 21. Heisel, S.E., Peterson, D.M. and Jones, B.L. Identi®cation of United States barley cultivars by sodium dodecyl sulfate polyacrylamide gel electrophoresis of hordeins. Cereal Chemistry 63 (1986) 500±505. 22. Pekkarinen, A., Mannonen, L., Jones, B.L. and Niku-Paavola, M.-L. Production of proteases by Fusarium species grown on barley grains and in media containing cereal proteins. Journal of Cereal Science 31 (2000) 253±261. 23. Haikara, A., Laitila, A. and Kleemola, T. Effect of different Fusarium species and climatic conditions on the quality of barley and malt. In `Book of Abstracts, 6th European Fusarium Seminar & 3rd COST 835 Workshop, Agriculturally Important Toxigenic Fungi', Berlin (2000) 68±69. 24. Boisen, S. Protease inhibitors in cereals. Acta Agriculturae Scandinavica 33 (1983) 369±381. 25. Shewry, P.R. Barley seed proteins. In `Barley Chemistry and Technology', (A.W. MacGregor and R.S. Bhatty, eds), American Association of Cereal Chemists, St Paul, MN, USA (1993) pp 131±197. 26. Bechtel, D.B., Kaleikau, L.A., Gaines, R.L., Seitz, L.M. The effects of Fusarium graminearum infection on wheat kernels. Cereal Chemistry 62 (1985) 191±197. 27. Narziû, L., Back, W., Reicheneder, E., Simon, A. and Grandl, R. Untersuchungen zum gushing-problem. Monatsschrift fuÈr Brauwissenschaft 9 (1990) 296±305. 28. Oliver, R. and Osbourn, A. Molecular dissection of fungal phytopathogenicity. Microbiolology 141 (1995) 1±9. 29. Walton, J.D. Deconstructing the cell wall. Plant Physiology 104 (1994) 1113±1118. 30. Knogge, W. Fungal infection of plants. Plant Cell 8 (1996) 1711±1722. 31. Ball, A.M., Ashby, A.M., Daniels, M.J., Ingram, D.S. and Johnstone, K. Evidence for the requirement of extracellular protease in the pathogenic interaction of Pyrenopeziza brassicae with oilseed rape. Physiological and Molecular Plant Pathology 38 (1991) 147±161. 32. Movahedi, S. and Heale, J.B. The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by different isolates of Botrytis cinerea Pers ex. Pers. Physiological and Molecular Plant Pathology 36 (1990) 303±324. 33. Reddy, P.V., Lam, C.K. and Belanger, F.C. Mutualistic fungal endophytes express a proteinase that is homologous to proteases suspected to be important in fungal pathogenicity. Plant Physiology 111 (1996) 1209±1218. 34. Ryan, C.A. Protease inhibitors in plants: Genes for improving defences against insects and pathogens. Annual Review of Phytopathology 28 (1990) 425±449.
Fusarium Species Synthesize Alkaline Proteinases in Infested Barley Anja I. Pekkarinen ²³, Tuija H. Sarlin³, Arja T. Laitila³, Auli I. Haikara³ and Berne L. Jones²§ ²Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706, USA; ³VTT Biotechnology, P.O. Box 1500, 02044 VTT, Finland and §Cereal Crops Research Unit, Agricultural Research Service, US Department of Agriculture, Madison, Wisconsin 53726, USA Received 5 June 2002 ABSTRACT Barley (Hordeum vulgare L.) that is infested with Fusarium head blight (FHB, `scab') is unsuitable for malting and brewing because it may contain mycotoxins and has unacceptable malting quality. Fungal proteinases are apparently often involved in plant-microbe interactions, where they degrade storage proteins, but very little is known about the enzymes that the fungi produce in the infected grain. We have shown previously that one plant pathogenic fungus, Fusarium culmorum, produced subtilisin- and trypsinlike enzymes when grown in a cereal protein medium. To establish whether these proteinases were also synthesized in FHB-infested barley in vivo, ®eld-grown barley was infested as the heads emerged. Extracts were prepared from the grain as it developed and matured and their proteolytic activities were measured with N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and N-benzoyl-Val-Gly-Arg p-nitroanilide. The heavily infested barleys contained both subtilisin- and trypsin-like activities. These enzymes reacted with antibodies prepared against each of the two F. culmorum proteinases, indicating that those produced in the laboratory cultures and in the ®eld-infested barley were the same. The presence of these proteinases correlated with the degradation of speci®c buffer-soluble proteins in the infested grains. These enzymes readily hydrolyzed barley grain storage proteins (C- and D-hordeins) in vitro. The presence of these Fusarium proteinases in the barley indicates that they probably play an important role in the infestation, but exactly how and when they function is not clear. # 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Fusarium, scab, proteinase, barley.
INTRODUCTION Fusarium head blight (FHB, scab) is a fungal disease of wheat and barley that is caused predominantly by Fusarium graminearum, F. culmorum and F. avenaceum1. Severe FHB infestations have occurred in humid ABBREVIATIONS USED: BVGRpNA N-benzoyl-Val-GlyArg p-nitroanilide; CBBR-250 Coomassie Brilliant Blue R-250; FAN free amino nitrogen; MH mechanically harvested; SAAPFpNA N-succinyl-AlaAla-Pro-Phe p-nitroanilide; SL subtilisin-like; TL trypsin-like.
Corresponding Author. Anja Pekkarinen, VTT Biotechnology, P.O. Box 1500, 02044 VTT, Espoo, Finland. Tel: 358±9-456-4461; Fax: 358±9-455-2103; E-mail: anja.pekkarinen@vtt.®
0733±5210/03/030349 08 $35.00/0
regions in the Americas, Asia and Europe and have often disastrously affected both producers and consumers2±4. Heavily infected cereals are unsuitable for use as either food or feed due to their poor processing properties and possible mycotoxin contamination. Infection by fusaria may lead to the production of toxins5 and hormone-like compounds6, as well as of various hydrolytic enzymes such as cutinase7,8, proteinases9±12, xylanases and cellulases12,13. These may be associated with pathogenesis but, so far, none of these have been considered as a clearly determining factor in virulence. The roles of proteinases that form during the FHB infection of cereals is of interest because protein degradation can strongly # 2003 Elsevier Science Ltd. All rights reserved.
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affect the baking, malting or brewing quality of the diseased grain10,14,15. Nightingale et al.10 showed that an extraneous alkaline proteinase occurred in wheat that was infested with F. graminearum and Schwarz et al.12 showed that there was an increase in the nonspeci®c proteinase activity of diseased barley grain. However, no one had previously identi®ed Fusarium proteinases in cereal grains. Recently, we puri®ed and described the characteristics of a subtilisin- and a trypsin-like proteinase that were produced by F. culmorum that was grown in a medium that contained wheat gluten16,17. For this study, arti®cially inoculated ®eld grown barley grain was used to study whether these enzymes were produced in vivo during Fusarium infection. Their presence in extracts prepared from the inoculated grain was established by using speci®c synthetic substrates to measure their activities and by using antibodies raised against these enzymes.
EXPERIMENTAL Field experiments Two-rowed malting barley (Hordeum vulgare L.) cultivars `A' and `B' were grown during the summer of 1998 at two experimental farms that were located at Hauho and Jokioinen in southern Finland. The plants were sprayed with conidia suspensions from Fusarium graminearum (VTT D-95470), F. culmorum (VTT D-80148) or F. poae (VTT D-82182) when the incipient kernels were emerging from the boot. Triplicate plots (each 10 m2) were treated with each of the three Fusarium species and three plots were left untreated to serve as controls. Thirty randomly chosen spikes were collected manually from each plot 18, 40 and 53 days (Hauho) or 21, 36, 49 and 63 days (Jokioinen) after inoculation and the samples from the triplicate plots were pooled. Fifteen spikes from each pool were freeze-dried and stored at ÿ20 C for the proteinase studies. The barleys that are called `mechanically harvested' (MH) were machine harvested 54 or 63 days after inoculation at Hauho and Jokioinen, respectively. Those samples were dried in warm air to moisture contents of approximately 12% and stored at 12±14 C. A replicate experiment with F. culmorum was carried out during the summer of 2000 with cv. A at Hauho. In this case, spike samples were collected manually 33, 46 and 55 days after inoculation and grain was machine harvested 55 days after inoculation.
Measuring infection rates The percentage of the grain that was infected by Fusarium was estimated by plating non-disinfected kernels on a selective CZID-agar plate18. In addition, samples of the 1998 MH grain and of the year 2000 grain were homogenized and analyzed for yeast and mold counts. Ten grams of grain were soaked in 90 mL of 09% NaCl for 30 min at 8 C and homogenized for 10 min with Stomacher LabBlender 400 BA.6021 (Seward Medical, London, UK). Dilutions of the homogenates of year 1998 and 2000 samples were plated on YM nutrient or potato dextrose (PD) agar, respectively. Both nutrient media were purchased from Difco (Detroit, MI, USA.) and supplemented with 001% chloramphenicol, 001% chlorotetracycline and 002% Triton X. The colonies formed by Fusarium species were counted after 7 days of incubation at room temperature. The results were expressed as a number of colony forming units (cfu) per gram of homogenized grain. Extracting protein from the barley grain Freeze-dried kernels and the MH grain were ground with a BuÈhler Universal Laboratory disc mill, type DLFU (BuÈhler-Miag GmbH, Braunschweig, Germany) that was set to `30'. Their proteins were extracted by incubating 20 g of ground grain with 50 mL of 50 mM Na acetate, pH 55, buffer at 22 C for 15 h. The mixtures were centrifuged at 1700 g for 5 min, after which the supernatants were transferred to microcentrifuge tubes and centrifuged at 11 600 g for 5 min. The supernatants were ®ltered with 13 mm wide, 045 mm Millex-HV ®lters (Millipore, Bedford, MA, USA) and stored at ÿ20 C until analyzed. Proteinase activity assays The proteinase activities of the grain extracts were measured using speci®c synthetic substrates as described previously16. The subtilisinlike (SL) activities were measured at 25 C with 50 mM N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA, Sigma, St Louis, MO, USA) in 180 mM Tris-HCl, pH 90, and the trypsin-like (TL) activities at 28 C with 05 mM N-benzoyl-Val-GlyArg pNA (BVGRpNA) in 4% dimethyl sulfoxide (DMSO) and 175 mM Tris-HCl, pH 90. The nonspeci®c proteinase activities were measured at 40 C
Fusarium proteinases in infested barley
and pH 89 using an azogelatin method19, as modi®ed by Pekkarinen et al.16. The activities were expressed as nkat (synthetic substrates) or change in absorbance 100/min (azogelatin) per mL of grain extract. All of the analyses were duplicated. SDS-PAGE analysis of grain extracts Grain extracts (80 mL) prepared from the year 2000 samples were mixed with 5 SDS-sample buffer (20 mL) that contained 15% b-mercaptoethanol and held in a boiling water bath for 2 min. The proteins from 5 mL of the boiled samples were separated with a 12% SDS-PAGE gel20 and stained with 01% Coomassie Brilliant Blue R-250 (CBBR-250) that was dissolved in a 40% methanol±1% acetic acid solution. Antibody detection of F. culmorum proteinases The presence of the TL and SL enzymes in the year 2000 samples was examined using the western blotting method. Sixteen mL-samples of each grain extract and of the puri®ed TL proteinase17 (03 mg) were separated with SDS-PAGE as above and blotted, for 16 h, onto a nitrocellulose membrane at 8 C and in 25 mM Tris and 192 mM glycine, pH 83, with a voltage of 60 V. On a duplicate gel, 8 mL-samples of each grain extract and of the puri®ed SL proteinase16 (07 mg) were separated. The proteins were transferred onto the membrane for 24 h in pH 83-transfer buffer that contained 0035% SDS. Rabbit polyclonal antibodies that were raised against each of the puri®ed F. culmorum proteinases were used as primary antibodies (Animal Care Unit Polyclonal Antibody Service, University of Wisconsin Medical School, Madison, WI, USA) and the secondary antibody was a goat anti-rabbit horseradish peroxidase conjugate (Bio-Rad, Hercules, CA, USA). Hordein digestion with puri®ed enzyme The hordein hydrolyzing activity of the puri®ed F. culmorum SL proteinase16 was studied at pH 60 and 40 C using a previously described procedure17. The reaction suspension contained 31 mM Na citrate, pH 60, 4 mg/mL of the enzyme, 25% DMSO, and 25% (v/v) hordein extract that was prepared with 55% isopropanol and 2%
351
b-mercaptoethanol21. The reactions were carried out in the presence or absence of a class speci®c inhibitor, chymostatin (40 mM, Sigma). A reaction mixture that contained no enzyme was prepared as a control for detecting any unspeci®c hordein degradation. Samples were removed from the suspensions after 0, 30 and 120 min of incubation and analyzed with a 12% SDS-PAGE gel. The gel was stained with CBBR-250 as above.
Hydrolysis of buffer-soluble proteins with puri®ed proteinases A sample of cv. A (grown in Finland in 1995) was ground with a U/D Cyclone Sample Mill (UDY, Ft. Collins, CO, USA) to pass a 05 mm screen and an extract was prepared in Na acetate, pH 55, buffer as described above. Seventy mL of extract was mixed with 3 mg of puri®ed F. culmorum SL or TL enzyme in a total volume of 350 mL of 130 mM Na citrate, pH 6, and incubated at 40 C for 0, 15, 30, 60 and 120 min. The reactions were stopped by mixing 50 mL of each sample with 13 mL of 5 SDS-sample buffer and heating in boiling water bath for 3 min. The hydrolysis products were analyzed with SDSPAGE on 12% acrylamide gels.
RESULTS AND DISCUSSION Infection rates Both the 1998 and 2000 summers were rainy, which provided excellent growth conditions for fusaria. These conditions caused a proliferation of the fungi, which resulted in the control barley also being contaminated with Fusarium. About 3 weeks after the treatments were applied in 1998, the noninoculated samples had a lower proportion of infected kernels than the Fusarium-inoculated ones, but the treated vs. control differences became smaller as the growing period progressed (Tables I and II). The data obtained by counting the colony forming units of the harvested samples indicated that the inoculated kernels generally contained more fungus than the controls. This implies that despite the high contamination level of all of the samples, the disease had proceeded further in the F. culmorum and F. graminearum-inoculated heads than in the nontreated or F. poae-inoculated ones.
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Table I Fusarium-infection rates of barley kernel samples from ®eld trials grown during the summer of 1998 in southern Finland
Location Hauho
Jokioinen
Control
Days after inoculation
A
18 40±53 54d 54d 21 36±63 63d 63d
4%c 80±96% 74% 10 103 cfu/ge 14% 86±100% 98% 550 cfu/g
a
F. culmorum b
F. graminearum
F. poae
B
A
B
A
B
A
B
8 58±66 94 550 18 76±100 100 63 103
76 96±100 100 40 104 46 96±100 100 40 104
24 100 100 20 104 64 100 100 16 105
96 100 100 40 105 96 100 100 20 106
74 100 100 63 105 98 100 99 40 106
30 68±98 86 32 103 32 92±100 100 550
32 82±90 93 40 103 44 66±100 96 10 105
a
Cultivar `A'. Cultivar `B'. c The percentage (%) of kernels that showed Fusarium contamination when whole kernels were incubated on CZID agar. d Mechanically harvested. Other samples were collected by hand. e The number of colony forming units per gram of homogenized grain that was analyzed using the YM agar plating method. b
Table II Fusarium-infection rates of control and F. culmorum-inoculated cv. `A' ®eld-grown barley kernels grown at Hauho during 2000 Days after inoculation 33 46 55 55c
Control
F. culmorum
93%a 550 cfu/gb 100% 55 103 cfu/g 97% 50 104 cfu/g 97% 50 102 cfu/g
100 22 105 100 17 106 100 24 106 100 41 105
a
The percentage (%) of kernels that showed Fusarium contamination when whole kernels were incubated on CZID agar. b The number of colony forming units per gram of homogenized grain that was analyzed using PD agar. c Mechanically harvested.
Fusarium proteinases were present in inoculated grains The SL activities of extracts prepared from barley samples that were grown in the ®eld during 1998 are presented in Table III. The activities of the inoculated and control extracts from both cultivars and both locations were quite similar after 18 days of inoculation. However, more pronounced differences were observed in the samples that were collected between 36 and 63 days after treatment. The activities of the extracts that were prepared from the F. culmorum and F. graminearum-inoculated
samples were generally 10±20 times higher than those of the corresponding controls. The differences between the inoculated and non-treated cv. A activities from the Jokioinen samples were, at most, eight-fold. This was partly due to the relatively low activities of the inoculated samples and partly because the non-treated samples of cv. A from Jokioinen contained slightly higher activities than the other controls. The activities of the F. poae inoculated barleys remained, in all cases, at the same level as those of the controls. Thus, the production of the alkaline proteinases was associated with higher pathogenicity, since both F. culmorum and F. graminearum are generally more pathogenic than F. poae1. However, whether the proteinase synthesis is important for cell wall penetration or whether it occurs only after penetration via other mechanisms, is not clear. When these three fungi were grown in a gluten medium, F. poae synthesized mostly acid proteinase(s) while the other two produced alkaline proteinase(s), implying that the synthesis of their proteinases were regulated differently22. Therefore, they may act in different ways in living organisms. However, when grown saprophytically on autoclaved barley, all three Fusarium species produced mainly alkaline proteinases. This implies that F. poae might produce alkaline proteinases in developing kernels if it could overcome the grain's defenses. The MH samples typically contained less SL activity than the ®nal manually picked samples, which were collected at nearly the same time. This difference may be due to the collection methods used since, during the mechanical harvesting, the lightest
Fusarium proteinases in infested barley
353
Table III The subtilisin-like activities (nkat/mL) of extracts of control and Fusarium-inoculated barleys that were grown during the summer of 1998 Control Location Hauho
Jokioinen
F. culmorum
F. graminearum
F. poae
Days after inoculation
Aa
Bb
A
B
A
B
A
B
18 40 53 54d 21 36 49 63 63d
077 020 009 013 036 029 062 090 078
054 013 009 008 025 029 026 024 016
182 295c 202 119 051 105 120 241 116
046 161 175 021 052 300 349 221 302
303 292 154 117 059 225 169 181 109
094 348 418 060 090 304 209 310 243
106 026 023 015 031 018 040 054 046
041 022 014 007 018 047 027 048 026
a
Cultivar `A'. Cultivar `B'. c The activities that are between 5 and 10 times higher than those of the corresponding controls are shaded and those that are more than 10 times higher are bolded and shaded. d Mechanically harvested. b
8 Activity, AU or nkat/mL
(most shrivelled) kernels were separated away from the grain pool by the harvesting machine. During hand harvesting, all of the grains were retained. When malts were produced from the 1998 MH grains and their malting qualities were analyzed, those from the F. culmorum- or F. graminearuminoculated cv. B barleys contained more wort soluble nitrogen and/or free amino nitrogen (FAN) than those made from the non-treated and F. poaeinoculated samples (A. Haikara, personal communication). With these two particularly virulent species, the highest SL activities were detected in the corresponding barley samples, especially at Jokioinen. This implies that the proteinase was probably at least partially responsible for the elevated protein degradation. The soluble nitrogen and FAN levels were also elevated in all of the inoculated cv. A malts23. These data do not indicate whether the increased protein hydrolysis occurred in the ®eld, during malting, or both. Similar results, i.e. increased levels of wort FAN and of neutral and alkaline proteinase activities in Fusarium-inoculated barley, have also been obtained in other studies12,14. In addition, the concentrations of the fungal toxins deoxynivalenol and zearalenone in the cv. B samples correlated strongly with their SL proteinase activities23. The non-speci®c (measured using the azogelatin substrate) and the SL and TL proteinase activities of the cv. A samples of the summer 2000 experiment showed the same trend as those from 1998 (Fig. 1). The differences between the inoculated and control samples were consistent for all three proteinase
6 4 2 0 33
C, non-sp.
46 55 Days after inoculation C, SL
C, TL
Fc, non-sp.
MH, 55 Fc, SL
Fc, TL
Figure 1 The proteinase activities of extracts that were prepared from control (C) and F. culmorum-inoculated (Fc) barley (cv. `A') in Hauho, Finland, in 2000. The activities were measured at pH 9 using the substrates N-succinyl-Ala-Ala-ProPhe pNA (subtilisin-like, SL), N-benzoyl-Val-Gly-Arg pNA (trypsin-like, TL) or azogelatin (non-speci®c proteinase).
analyses. The activities of the inoculated grains increased with time and 55 days after inoculation they were about 30 (non-speci®c), 70 (SL) or 20 (TL) times higher than those of the controls. The activities in the MH grain were once again very low compared to those of the manually collected samples, but they were still about 10 times higher in the inoculated than in the control samples, and were roughly the same as those of the corresponding cv. B samples from Jokioinen in 1998. Both the SL and TL proteinases were detected in extracts of inoculated grain, using antibodies that were raised against the puri®ed enzymes (Fig. 2). The TL enzyme antibodies did not cross react with
354
A. I. Pekkarinen et al.
A
kD -100 - 50 - 37 - 25
- 15
C Fc 33 d
C
Fc 46 d
C Fc C Fc_ TL 55 d MH 55d
C Fc 33 d
C Fc C Fc M C Fc_ 46 d 55 d MH, 55d
Figure 3 SDS-PAGE separations of buffer soluble proteins from barley (cv. `A') grown at Hauho during 2000. C, controls; Fc, F. culmorum inoculated; M, molecular weight standard. The samples were as in Figure 2. The protein bands that had disappeared or clearly diminished in the inoculated sample are indicated with arrows.
B
C Fc 33 d
C
Fc 46 d
C Fc 55 d
C Fc_ SL MH 55d
Figure 2 Western blots of control (C) and F. culmoruminoculated (Fc) barley (cv. `A') extracts. The barley was grown at Hauho, Finland, in 2000. Antibodies against A) the TL or B) the SL enzymes were used to detect the proteinases. The samples were collected by hand 33, 46, or 55 days after inoculation, or mechanically (MH) after 55 days. The right hand lanes were loaded with puri®ed TL (03 mg), or SL (07 mg) enzymes. Sixteen and 8 mL extract samples were loaded onto gels A and B, respectively.
the SL ones, and vice versa. The responses with both of the antibodies were strongest in the manually collected 55-d sample. Both the activity (Fig. 1) and antibody reactions (Fig. 2) of the inoculated MH samples were subtle, indicating that their low activity levels were not the result of enzyme inactivation, but that the lightest, most heavily infected, grains were apparently removed by the mechanical harvester.
The effect of inoculation on buffer soluble grain proteins When extracts of the various year 2000 grain samples were separated by SDS-PAGE, their
protein compositions differed (Fig. 3). The approximately 62, 32, 30 and 25 kD proteins that were present in the 55-d control samples had disappeared from the inoculated grain and a new protein of about 44 kD had formed. These changes were most apparent in the 55-d samples, that contained the highest alkaline proteinase activities, but some differences were also visible in the earlier samples. On the other hand, when an extract of these soluble proteins was prepared and treated with each of the puri®ed proteinases in vitro under conditions similar to those used in the hordein hydrolysis experiment, no hydrolysis was observed (results not shown). This may have been due to the added enzyme being bound to proteinase inhibitors that occur in barley grains24. The apparent protein decomposition shown in Figure 3 may have occurred either during the fungal invasion or during the protein extraction process (15 h at 22 C), due to the `excess' proteinase that was not bound by the endogenous inhibitors. The soluble protein patterns of the control and experimental MH barleys were identical (Fig. 3), probably because they contained little proteinase activity (Fig. 1). Fusarium proteinases hydrolyze hordeins in vitro The F. culmorum TL proteinase hydrolyzed the D hordein and all but one of the C hordeins of Morex barley in vitro17 and the SL proteinase hydrolyzed all of the C and D hordeins (Fig. 4). None of
Fusarium proteinases in infested barley
kD 10075-
-D
50-
C
25B 151
2
3
4
5
6
7
8
Figure 4 The in vitro hydrolysis of a hordein extract by the puri®ed F. culmorum subtilisin-like enzyme. Lanes 2±4: Enzyme, no inhibitor reactions were stopped at 0, 30 and 120 min, respectively; lanes 5±6: Enzyme plus chymostatin, 0 and 120 min; lanes 7±8: hordein, no enzyme, no chymostatin. A molecular weight standard sample was loaded in lane 1. The letters B, C and D indicate the positions of the different classes of hordeins as in reference 25.
the hordeins were degraded in the absence of the puri®ed enzyme, and chymostatin inhibited the hydrolysis, indicating that no nonspeci®c protein degradation occurred in the reaction mixtures. The C and D hordeins comprise 10±20% and 2±4% of the barley prolamins, respectively25. No degradation of the major, B, hordein fraction was detected under these conditions. Whether or not these enzymes contribute to the protein matrix degradation that occurs in vivo10,15,26,27, remains to be determined with immunomicroscopy. What roles do the Fusarium proteinases play in grain invasion? The synthesis of one particular enzyme is normally not crucial for the invasion of a fungus; the pathogens generally produce a multitude of enzymes that work together to decompose the host plant structures28±30. The roles of proteinases in different plantmicrobe interactions may differ: in some cases, the expression of proteinases has been suggested as being a primary pathogenesis factor31,32, but a fungal proteinase has also been detected in a symbiotic fungal-plant interaction33. A subtilisin-like proteinase was produced by F. oxysporum f. sp. lycopersici in tomato roots and stems during infection, but disruption of its gene did not affect the pathogenicity of the fungus11. Regardless of how essential the proteinases may be for pathogenicity, it has been hypothesized that certain proteinase inhibitors
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protect the host plant from pathogen invasion34. Both the SL and TL proteinases were produced by Fusarium in the grain, indicating that the interaction between the fungus and barley involves fungal extracellular proteinases at some stage(s) of disease development. The highest activities were detected in grains that were inoculated with the most pathogenic species (Table III), implying that the proteinase production is associated with pathogenicity. However, because the activities were found only in heavily infected grain, it seems that they were produced (or at least detected) only after a successful invasion had occurred and were required for saprophytic purposes. On the other hand, this does not exclude that they played some role during the active penetration of the fungus into the grain, since the fungi may have produced these enzymes at such low concentrations during the beginning of the infection that they could not be detected by the methods that we used. In addition, the fusaria may produce other, undetected, proteinases during the infection, or they may trigger the synthesis or activation of some of the barley proteinases that normally function during the germination process. This work indicates that Fusarium proteinases are undoubtedly present in barley during infection, but the question of exactly how or whether this facilitates the attack of the fungus on the grain is still unanswered. Acknowledgements The ®nancial support of the Raisio Group Research Foundation, the Tor-Magnus Enari Fund, the American Malting Barley Association and of Anheuser-Busch Inc. are greatly appreciated.
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