Disturbed correlation between fungal biomass and
b
-glucuronidase
activity in infections of
Arabidopsis thaliana
with transgenic
Alternaria brassicicola
Bart P.H.J. Thomma
a,1, Yohannes S.H. Tadesse
b, Michel Jacobs
b,
Willem F. Broekaert
a,*
aF.A.Janssens Laboratory of Genetics,Katholieke Uni
6ersiteit Leu6en,K.Mercierlaan92,B-3001He6erlee-Leu6en,Belgium
bLaboratory of Plant Genetics,Vrije Uni
6ersiteit Brussel,Paardenstraat65,B-1640Sint-Genesius Rode,Belgium
Received 18 December 1998; received in revised form 7 June 1999; accepted 15 June 1999
Abstract
Several previous studies have described the use of the bacterial uidA(b-glucuronidase) gene to transform fungal pathogens of plants. In many casesb-glucuronidase activity has been considered as a quantitative parameter for the extent of development of
uidA-expressing transgenic fungi in infected plants. Here we present evidence that this method is not applicable in all plant-pathogen interactions. In Arabidopsis plants inoculated with an uidA-expressing transgenic Alternaria brassicicola fungus, there was a good correlation between lesion development and the level ofuidAmRNA whereasb-glucuronidase activity dropped as lesions extended. This indicates that plant factors accumulating or released during lesion development can interfere with
b-glucuronidase activity in the fungus. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Fungal Biomass;b-glucuronidase activity; Nucleic acid
www.elsevier.com/locate/plantsci
1. Introduction
When monitoring resistance of plants to fungal pathogens, detection of the pathogenin plantaand quantification of its biomass is a crucial step. Several methods have been used over the years to evaluate resistance of plants to fungi. For patho-gens causing either wilting or necrosis of entire plants resistance is usually expressed as the num-ber of wilted or dead plants over time. For patho-gens causing necrotic lesions or spots, quantitative data can be obtained by measuring the number and size of lesions over time or by measuring sporulation in or around the lesion site. Although these methods are relatively quick and easy to
perform, the disadvantage is that they are only suitable for those plant-fungus interactions that result in macroscopically scorable disease symp-toms. Moreover, the quantitative character of these methods can be questioned because they provide a measure for the extent of visible symp-toms rather than an accurate measure for the extent of fungal growth. Other techniques are based on the measurement of fungal constituents such as chitin, glucosamine or ergosterol, which are absent or nearly absent in uninfected plants [1 – 6]. However, these methods are time-consum-ing and not very sensitive. Moreover, the
correla-tion with fungal biomass is sometimes
questionable due to the inability of some tech-niques to discriminate between living and dead fungal tissue, to differences in content of these constituents in different fungal structures, or to interference with enzymatic activities in plants [7,8].
* Corresponding author. Fax: +32-16-32-19-66.
E-mail address: willem.broekaert@agr.kuleuven.ac.be (W.F. Broekaert)
1Research assistant of the ‘Fonds voor Wetenschappelijk
Onder-zoek-Vlaanderen’.
Relatively new methods for measuring fungal biomass include nucleic acid based techniques like quantitative PCR [9 – 11], RNA hybridization [12 – 15], or immunological methods like ELISA [16 – 19]. For all these methods, the main difficulty is interference of plant factors with the measured parameter as shown for nucleic acid based meth-ods [20] and immunological methmeth-ods [20,21].
Other researchers have developed methods
based on the measurement of b-glucuronidase
(GUS) activity in interactions with fungi
trans-formed with the bacterial gene uidA encoding
GUS [19,22 – 25]. Although the requirement of transgenic fungi is a disadvantage of this method, the advantage is that, once a transgenic fungus is available, quantification is technically easy and rapid. Moreover, such a transgenic fungus allows both quantification of its biomass and histochemi-cal visualization of its structuresin planta. GUS is normally very stable and uptill now no interfer-ence of plant factors with b-glucuronidase activity in plant-pathogen interactions have been reported. In this study we show, however, that also mea-surement of b-glucuronidase activity can suffer from interference with such factors. Quantification of a transgenic Alternaria brassicicola strain on Arabidopsis by measuring GUS activity did not correlate with the amount of GUS mRNA, proba-bly due to inactivation of the GUS enzyme in the lesion.
2. Materials and methods
2.1. Biolistic transformation of A. brassicicola
A. brassicicola (strain MUCL20297) was grown on half strength potato dextrose agar (PDA, Difco Laboratories, Detroit, USA) for 3 weeks until sufficient conidia were formed. Conidia were har-vested by rubbing the plates gently with a pipet tip while they were covered with sterile water. The spore suspension was filtered through glass wool
and subsequently centrifuged at 4000×g for 5
min. The spores were suspended in sterile water at a density of about 106 conidia per ml. A total of
150 ml spore suspension was dispersed over a
circular area with a diameter of about 20 mm located in the center of a Petri dish containing 10 ml half strength PDA containing 2% (w/v) agar.
Tungsten particles M5 (diameter 0.1 – 1.0 mm) (Sylvania, GTE Products Corp., Towanda, Pa., USA) were coated with plasmid DNA following the procedure of Sanford et al. [26]. The plasmid used to transform A.brassicicolawas pGUS5 con-taining a hygromycin-based selection marker gene and the E. coli uidA coding region driven by the
glyceraldehyde-3-phosphate dehydrogenase
(GPD1) promoter [27].
Per shot, 8ml of 100% ethanol containing 0.8mg of plasmid DNA associated with 500 mg of tung-sten particles, was spread on the center of a Kaplan flying disc. A helium driven device with flying discs was used for particle delivery (Biorad,
model PDS 1000/He). The distance between the
launch site and the target was 6 cm, and the helium pressure used to accelerate the flying disc under vacuum was 1300 psi. 24 h after delivery of the particles to the fungal spores the plates were overlaid with 10 ml of PDA amended with 200 mg/ml of hygromycin B. Most transformants grew through the layer of selective agar within 72 h. These transformants were transferred to new PDA plates containing 100 mg/ml hygromycin B. X-gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) was used to test whether the transformants exhibited b-glucuronidase activity [28].
2.2. Plant inoculations
Seeds of the jasmonate-insensitive Arabidopsis mutantcoi1-1 [29] were surface sterilized by wash-ing for 1 min in 70% ethanol followed by a 1 min wash in 20% commercial bleach. After three times washing with water, seeds were deposited on Mu-rashige and Skoog medium (Sigma Chemical Co.,
St. Louis, USA, M-5524) containing 100 mM
methyl jasmonate. After 7 days of germination, coi1-1 mutant seedlings are discriminated from segregating wild-type plants by the length of the roots [29]. These seedlings were transferred to soil. Inoculation of Arabidopsis plants withA.
brassici-cola was performed on 4-week-old soil-grown
plants by placing three 5 ml drops of a suspension of 5×105conidial spores/ml in water on each leaf [15]. Inoculated plants were incubated at 100% relative humidity. For each time point and each genotype, 30 discs (12.6 mm2) around an infection spot were punched out and stored at −80°C until
use in b-glucuronidase assays and RNA
2.3. b-Glucuronidase assays
To 30 leaf discs, 100 ml of GUS extraction
buffer [28] together with 300 mg quartz sand, 200 mg glass beads with an average particle diameter
of 425 – 600 mm and eight glass beads with an
average particle diameter of 3 mm was added in a screw-capped centrifuge tube. The extracts were prepared in a high-speed reciprocal shaker as de-scribed [30] and they were subsequently cleared by centrifugation (10 000×g for 5 min). GUS activity was measured fluorometrically as described [28] using 4-methylumbelliferyl-b-D-glucuronide as a
substrate. Protein contents of the extracts were determined by the method of Bradford [31] using bovine serum albumin as a standard.
2.4. RNA hybridisation assays
RNA was extracted from leaf discs cut from inoculated leaf material [30], quantified and hy-bridised with anuidA probe [30] after dot blotting of serial dilutions. Hybridisation signals were quantified using a luminescence CCD camera sys-tem (Night Owl, EG&G Berthold, Bad Wildbad, Germany).
3. Results and discussion
A. brassicicola transformants constitutively ex-pressing GUS were obtained by biolistic transfor-mation and screened for their virulence on Arabidopsis wild-type plants. A transformant with normal virulence was chosen for further testing. When inoculated with A. brassicicola, wild-type plants produced small brown necrotic lesions in which the fungus was contained. Microscopical examination showed no sporulation and only some weak colonization. This interaction clearly appeared to be of the incompatible type [15]. In contrast, when plants of the jasmonate-insensitive mutant coi1-1 were challenged with the same fun-gus clear signs of a compatible interaction were visible: spreading lesions with heavy colonisation by fungal hyphae as revealed by microscopic ex-amination [15].
Measurement of GUS activity was chosen as a method to quantify the biomass ofA. brassicicola in both Arabidopsis genotypes. Plants were inocu-lated with the transgenicA. brassicicolastrain and
leaf discs containing infection spots were collected every 24 h till 6 days after inoculation. Proteins were extracted from the leaf discs, incubated with methylumbelliferyl glucuronide (MUG) and the GUS enzyme activity was measured by determin-ing methylumbelliferone (MU) levels. For wild-type plants, MU-levels did not rise during the first 48 h after inoculation. Between 48 and 72 h the MU level rose, and slightly declined after this time point (Fig. 1A). For coi1-1 plants inoculated with A. brassicicola, MU levels were essentially the same as for wild-type plants during the first 48 h after inoculation. Between 48 and 72 h MU levels in inoculated coi1-1 plants markedly increased to reach a higher level relative to wild-type plants. However, after 72 h the MU level dropped and fell back to initial levels (Fig. 1A). Although the rise in MU levels in A. brassicicola-inoculated wild-type plants correlated more or less with macro-scopic lesion development, the evolution of MU levels in inoculatedcoi1-1 plants was clearly not in line with that of lesion development in this geno-type (Fig. 1B). We therefore set out to determine transcript levels of the uidA gene in Alternaria-in-fected plants. In Fig. 1C it is shown that transcript levels of the uidAgene slowly increase between 48 and 72 h after inoculation for wild-type plants inoculated with transgenicA.brassicicola, which is in line with the determination of MU-levels as well as with lesion development. For the inoculated coi1-1 plants, the increase inuidA transcript levels was much stronger compared to wild-type plants (Fig. 1C). After 6 days, the amount of uidA tran-script in coi1-1 plants was roughly seven times higher than in wild-type plants. The levels of uidA transcript in A. brassicicola-infected coi1-1 plants therefore appears to correlate with lesion develop-ment. In contrast, the drop in GUS-activity ob-served between 72 and 96 h after inoculation did not correlate with either uidA-transcript levels or lesion development.
enzyme due to components released in the lesion. We therefore assessed whether compounds pro-duced during lesion development would affect en-zyme activity of purified GUS. For this purpose extracts were prepared from 25 discs cut out from healthycoi1-1 leaves and from the same number of discs taken around lesions on coi1-1 leaves 120 h
after inoculation with A. brassicicola. Addition of purified GUS (200 ng/ml) to extracts from the inoculated leaves caused a reduction in activity after 24 h of about 30% relative to the GUS enzyme incubated in presence of extracts from healthy leaves. This reduction was consistently observed and indicates the presence of GUS-inacti-vating compounds in leaves showing lesions. It is unclear, however, whether the reduction of GUS activity observed in the experiment described in Fig. 1A can be fully accounted for by the interac-tion of GUS with lesion-derived GUS-inactivating compounds.
Irreversible inactivation of the GUS enzyme has previously been reported to occur in cells from transgenic GUS-expressing plants when such cells underwent cell death and collapse [32]. The inacti-vation was demonstrated to be due to oxidizing agents produced by such cells, which interfere with proper folding of the enzyme during biosynthesis [32]. It is known that cells in a lesion caused by pathogen infection produce high amounts of reac-tive oxygen species [33,34], which could conceiv-ably disturb the intracellular redox state of fungi growing in such lesions.
Most papers reporting the use of the GUS enzyme to quantify fungal infection in plant tissues have found good correlations between GUS activ-ity and the level of infection [19,23 – 25]. One paper, however, briefly mentioned that the GUS enzyme was not a good marker for colonization of tomato leaf tissue with Botrytis cinerea [14]. To-gether with the findings described in this paper, this indicates that the GUS enzyme is not always an adequate marker for plant-pathogen
interac-tions. Alternaria and Botrytis are both
necro-trophic fungi, whereas most of the previous studies were performed on biotrophic fungi. Hence, it appears that the infection strategy of the fungus and the extent of lesion development associated with the infections, determine whether or not GUS can be used as a true marker for infections.
Acknowledgements
The Arabidopsis mutant coi1-1 was a kind gift of Dr J. Turner, University of East Anglia, Nor-wich, UK. The plasmid pGUS5 was a kind gift of Dr W. Scha¨fer.
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