APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1993, p. 1767-1773 Vol. 59, No.

6
0099-2240/93/061767-07$02.00/0
Copyright © 1993, American Society for Microbiology

The GUS Gene Fusion System (Escherichia coli ,3-D-Glucuronidase

Gene), a Useful Tool in Studies of Root Colonization by
Fusarium oxysporum
YVONNE COUTEAUDIER,lt* MARIE-JOSEE DABOUSSI,1 AGNES EPARVIER,2

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THIERRY LANGIN,1 AND JEAN ORCIVAL'
Institut de Ge'ne'tique et Microbiologie, Unite associee au Centre National de la Recherche Scientifique 1354,
Universite Paris-Sud, 91405 Orsay Cedex, 1 and Station de Recherches sur la Flore Pathogene dans le sol,
Institut National de la Recherche Agronomique, 21034 Dijon Cede ,2 France
Received 1 December 1992/Accepted 16 March 1993

The plant-pathogenic fungus Fusarium oxysporum was successfully transformed with the P-D-glucuronidase
gene from Escherichia coli (gusA) (GUS system) in combination with the gene for nitrate reductase (niaD) as
the selectable marker. The frequency of cotransformation, as determined by GUS expression on plates
containing medium supplemented with 5-bromo-4-chloro-3-indolyl glucuronide (GUS'), was very high (up to
75%). Southern hybridization analyses of GUS' transformants revealed that single or multiple copies of the
gusA gene were integrated into the genomes. High levels of GUS activity are expressed in some transformants,
but activity in F. oxysporum does not appear to be correlated with the copy number of the gusA gene. Since the
highest activity was found in a transformant with a single copy, it can be assumed that sequence elements of
F. oxysporum integrated upstream of the gene can act as a promoter or enhancer. Expression of the gusA gene
was also detected during growth of the fungus in plants, indicating that the GUS system can be used as a
sensitive and easy reporter gene assay in F. oxysporum.

The GUS gene fusion system (Escherichia coli P-D-glUCU-
ronidase gene) was described by Jefferson (14, 15) as a
powerful tool for the assessment of gene activity in trans-
genic plants and for the development of molecular genetic
analysis. The utility of this system has been shown for
bacteria, animals, and plants (15) and, more recently, for
yeasts and filamentous fungi (4, 20, 21, 23, 25). The first
report on the construction and testing of a plasmid express-
ing GUS in filamentous fungi (23) suggested that the system
could be developed for studies on disease spread and fungal
biomass quantification.
Our objective was to apply this tracking concept to the
important plant pathogen Fusarium oxysporum. Indeed,
difficulties are encountered in monitoring populations of a F.
oxysporum strain in association with roots because of the
presence of the F. oxysporum strain along with many other
fungal species and other strains of the same species. Isola-
tion on selective media of a marked strain by using a
pigmentation marker or resistance to benomyl has been used
for this purpose (2, 24), but this technique does not enable
visualization of hyphae at the root surface and quantification
of root colonization by pathogenic or nonpathogenic strains.
In this paper we describe the successful transformation of
strains of F. oxysporum with the gusA gene in combination
with the nitrate reductase structural gene of Aspergillus
nidulans (niaD) as the selectable marker (18). The usefulness
of the GUS gene fusion system for studies on root coloniza-
tion by F. oxysporum was then tested in preliminary exper-
iments by assaying GUS activity in vitro and in vivo in order
to localize and quantify the active biomass of the fungus in
roots. Moreover, our data on the molecular structure of
*
Corresponding author.
t Present address: Station de Recherches de Lutte Biologique,
Institut National de la Recherche Agronomique, La Miniere, 78285
Guyancourt, France.
1767
transformed strains should lead the way to further investi-
gations of gene expression in this fungus.
MATERIALS AND METHODS
Strains and media. The following wild-type strains of F.
oxysporum were used: a pathogen of melon (FOM150), a
pathogen of flax (FOLn3), and a nonpathogenic strain
(FO47) (obtained from C. Alabouvette, Institut National de
la Recherche Agronomique, Dijon, France). From the first
two strains, nitrate reductase-deficient mutants were se-
lected on the basis of resistance to chlorate. Mutant nia9
from FOM150 and mutant niaS2 from FOLn3, presumably
resulting from a mutation in the nitrate reductase structural
gene, as indicated by nutritional tests (5, 8), were used as
recipient strains in transformation experiments. The minimal
and complete media and culture conditions used were those
described previously (11).
Vectors. Plasmid pNOM102, constructed by Roberts et al.
(23), contains the E. coli 0-glucuronidase gene (gusA)
flanked by the glyceraldehyde 3-phosphate promoter (gpd)
fromAspergillus nidulans upstream and the A. nidulans trpC
transcription termination signal downstream (Fig. 1A). Plas-
mid pAN301 was obtained by insertion in pFB39 of an A.
nidulans DNA fragment carrying the nitrate reductase struc-
tural gene (niaD) (18).
Transformation procedure. F. oxysporum was transformed
as previously described by Langin et al. (16). Suspensions
containing 4 x 107 protoplasts were mixed with plasmids (5
,ug of pAN301 and 5 p,g of pNOM102). Transformants were
selected on medium containing nitrate as the sole nitrogen
source for the Nia+ phenotype conferred by pAN301. The
controls included protoplasts incubated with pFB39 to test
the stability of the recipient strains and protoplasts incu-
bated with pAN301 to test the efficiency of the transforma-
A

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probe used z A. nidulans gpd promoter sequence
- E. coli gusA coding sequence
r7 A. nidulans trpC terminator sequence
F. oxysporum DNA
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1768
VOL. 59, 1993
tion procedure. Transformants were subsequently purified
by uninucleated conidium isolation.
DNA isolation and Southern hybridization. Plasmids were
prepared from E. coli by the alkaline lysis method (19), and
total genomic DNAs from F. oxysporum transformants were
isolated as previously described (16). Restriction enzyme
digestions, DNA ligation, agarose gel electrophoresis, blot-
ting, and hybridizations were performed by using standard
procedures (19) and manufacturers' recommendations.
Probes were labeled with 32P by using a T7 random priming
kit (Pharmacia, Uppsala, Sweden). DNA sequencing was
performed by the dideoxy chain termination procedure (19),
using a T7 polymerase sequencing kit (Pharmacia). The
DNA was blotted onto a Hybond-N membrane (Amersham,
Les Ulis, France) by using a vacuum blotting system (Phar-
macia).
In situ colony color assay of 1-glucuronidase activity.
Young mycelial fragments from individual colonies of each
purified Nia+ transformant were transferred to enzyme-
linked immunosorbent assay plates containing 200 ,ul of
minimal medium per well. After 24 h of incubation, 4 ,ug of
the histochemical substrate 5-bromo-4-chloro-3-indolyl glu-
curonide (X-Gluc; Sigma, L'Isle d'Abeau, France) diluted in
phosphate buffer (pH 7.0) was added to each well. After 1 h
of incubation in the dark, the cotransformed colonies stained
blue (GUS'), while the controls, which were transformed
with plasmid pAN301 alone, were completely unstained and
remained so even after 24 h of incubation. An alternative
assay was performed by monitoring GUS activity qualita-
tively by fluorometry, using 4 methylumbelliferyl glucu-
ronide (MUG; Sigma) as the substrate. The fluorescence of
GUS' transformants was observed with a UV transillumi-
nator by using MUG at a final concentration of 1 mM. Both
tests were used to determine the frequency of cotransforma-
tion and to test the mitotic stability of GUS' transformants.
Mitotic stability test. A single spore of each GUS' trans-
formant was transferred to minimal medium. After five
subcultures, a spore suspension obtained from a single
colony was plated on minimal medium containing 0.6%
Triton X-100 (Prolabo, Paris, France). For each transfor-
mant, 96 colonies were picked and tested for the GUS'
phenotype on microtiter plates containing MUG as the
substrate, as described above. This test was performed in
triplicate.
Fluorometric 13-glucuronidase enzyme assay. Cell extracts
were made by grinding mycelium frozen in liquid nitrogen
and resuspending the resulting powder (1 g) in 3 ml of buffer
containing 100 mM Na2HPO4 (pH 6.8), 0.5 mM ,-mercap-
toethanol, 0.5 mM EDTA, 1% NaCl, and 0.5 mM phenyl-
methylsulfonyl fluoride. After centrifugation at 12,000 x g
for 30 min at 4°C, the supernatant was used as a crude
extract. The GUS specific activities of GUS' transformants
were assayed quantitatively by fluorimetry, using MUG as
the substrate as described by Jefferson (14). The fluorogenic
reaction mixture was incubated at 37°C, and 50-pul aliquots
were removed at time zero and at 10-min intervals for 40
min; the reaction was terminated by the addition of 0.95 ml
of 0.2 M Na2CO3. The fluorescence of each sample was read
by using excitation at 365 nm and emission at 455 nm with a
FIG. 1. (A) Partial restriction maps of plasmids pNOM102 and pGUS-T5. (B) Diagrammatic representation of different integration events
including integration of a single copy with a crossover point near the ends of the GUS construct (line 1) or within the gusA coding sequence
(line 2) and integration of tandemly repeated copies of pNOM102 involving the pUC19 part (line 3) or the GUS construct part (line 4) of the
plasmid.
GUS GENE FUSION SYSTEM 1769
Fluoroskan II fluorimeter (Labsystems, Les Ulis, France).
The GUS activity of each transformant was calculated from
the slope of the line showing the increase in fluorescence per
minute and was expressed in nanomoles of 4-methylumbel-
liferone (MU) produced per minute per milligram of protein
by extrapolating from an MU standard assay. Protein con-
centration was estimated by the Bradford method (3). Ex-
periments were performed at least in duplicate.
The GUS activities of inoculated plants were measured by
using extracts of young flax roots cultured as described
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below. Germinated seeds of flax (Linum usitatissimum cv.
Opaline) were aseptically transferred to Pyrex tubes (diam-
eter, 25 mm; length, 200 mm). Three seeds per tube were
placed on wire netting placed just above a 20-ml volume of
sterile nutrient solution. Suspensions of conidia of GUS'
transformant L-T6, alone (5 x 102 CFU/ml) or mixed with
conidia of nonpathogenic strain F047 (1 x 106 CFU/ml),
were transferred to the nutrient solution. Control treatments
consisted of noninoculated plants and plants inoculated with
either untransformed strain FOLn3 (5 x 102 CFU/ml) or
nonpathogenic strain F047 (1 x 106 CFU/ml). The bases of
the culturing tubes were wrapped in aluminum foil, and then
the tubes were incubated at 23°C. After a 7-day culture
period, three 2- to 3-cm roots were removed from each tube
and ground in 0.5 ml of extraction buffer. Suspensions of the
different extracts were then centrifuged at 12,000 x g for 10
min, and the supernatants were stored at -80°C. The activ-
ity of each sample extract was measured by using 50-,ul
samples and the fluorogenic method described above.
Histochemical localization of 1-glucuronidase activity in
roots infected with a GUS' transformant. Germinated flax (L.
usitatissimum cv. Opaline) seeds were cultivated in Carque-
fou soil (7) inoculated with conidia of a cotransformed strain
at a concentration of 1 x 103 conidia per ml. After 7 days the
histochemical localization of GUS activity was determined
by incubating flax plantlets for 2 h at 37°C in extraction
buffer containing the chromogenic substrate X-Gluc (0.44
mg/ml). The roots were then fixed in 4% glutaraldehyde-0.1
M sodium cacodylate (pH 7.2) overnight at room tempera-
ture.
Pathogenicity tests. Ten GUS' transformants and the
wild-type strains were tested for pathogenicity. For each
strain, 60 susceptible plantlets cultivated in rockwool plugs
(17) were inoculated with a suspension containing 5 x 102
conidia per ml. Observations of diseased plants were made
at the end of 6 and 7 weeks for muskmelon (Cucumis melo)
and flax experiments, respectively (17).
RESULTS
Two different strains of F. oxysporum were cotransformed
with plasmid pAN301 carrying the niaD selectable marker
and plasmid pNOM102 containing the gusA gene. A cotrans-
formation procedure was used because direct selection of
GUS' transformants failed. This can be explained by the
relatively low efficiency of transformation in filamentous
fungi (16), which implies that a large number of protoplasts
must be used. In the absence of selection pressure, the
growth of untransformed strains hides the GUS' phenotype,
1770 COUTEAUDIER ET AL. APPL. ENvIRON. MICROBIOL.

TABLE 1. Morphological, biochemical, and molecular

characteristics of transformants and wild-type strains
Straina
Strain' GUS Mitotic MU Copy
phenotype" stability' activityd no.e
FOLn3 GUS- <0.005
L-T89 GUS- <0.005 1
L-Tl GUS+ u 12 1
L-T3 GUS+ u 266 >1
L-T4 GUS+ s 347 >1
L-T6 GUS+ s 571 >1 <-

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7. 2 kb
FOM150 GUS- <0.005
M-T6 GUS- <0.005 0
M-T1 GUS+ s 683 >1
M-T2 GUS+ s 61 >1
M-T5 GUS+ s 951 1
M-T7 GUS+ s 302 >1
a FOLn3 and FOM150 are wild-type strains; transformants from these
strains were designated by the prefixes L and M, respectively.
b GUS phenotype was determined on X-Gluc medium
plates. GUS+,
colonies stained blue; GUS-, unstained colonies.
c Mitotic stability was determined with a sample of 96 colonies in a
microtiter assay in which MUG was the substrate. s, stable (all of the colonies
had the GUS+ phenotype; u, unstable (colonies had GUS- and GUS+
phenotypes).
d Expressed as nanomoles of MU produced per minute per milligram of
protein.
e The number of copies of pNOM102 was determined by Southern blotting.

preventing direct selection of such transformants. The intro-
duction of the gusA gene by cotransformation was very
efficient for both strains, as indicated by the proportion of
GUS+ phenotypes on plates containing X-Gluc medium (50
and 75% GUS+ transformants in Fusanum oxysporum f. sp.
melonis and Fusarium f. sp. lini, respectively). Transfor-
mants were designated with a letter prefix according to
whether they were obtained by transformation of Fusarium
oxysporum f. sp. melonis (M) or Fusarium f. sp. lini (L)
strains. Eight cotransformants exhibiting a GUS' phenotype
on X-Gluc plates were tested for mitotic stability, assayed
fluorometrically for GUS activity, and analyzed by Southern
blotting. The results are summarized in Table 1.
The mitotic stabilities of different individual transformants
tested in the microtiter assay with the MUG substrate
indicated that most of the transformants analyzed retained
the GUS activity of the original transformant after five
transfers on nonselective medium.
Fluorometric measurements of GUS activity in different
strains showed that no significant GUS activity was detected
in strains exhibiting a GUS- phenotype on X-Gluc medium
plates (the two wild-type strains and transformants L-T89
and M-T6). In GUS' transformants, the levels of GUS
activity varied greatly; a maximum 100-fold difference was
observed, and in general the levels were similar to those
expressed in other fungi with the same vector (23).
Southern hybridizations were performed to determine the
fate of plasmid DNA in transformants. Total genomic DNAs
were isolated from 10 Nia+ transformants (8 GUS+ and 2
GUS- transformants), digested to completion with EcoRI,
which cuts plasmid pNOM102 once, and probed with the
4.5-kb HindIII-EcoRI pNOM102 fragment containing the
entire GUS construct (Fig. 1A). Sequences homologous to
pNOM102 were detected in the eight GUS' transformants
and in one of the two GUS- transformants tested (Fig. 2).
The hybridization pattern found in transformants M-T5 and
L-T89 consists of two hybridizing bands, neither of which is
the size of the original plasmid (Fig. 2, lanes 2 and 6). This is
FIG. 2. Southern blot analysis of F. oxysporum transformants.
Genomic DNAs from individual transformants were digested with
EcoRI, electrophoresed on 0.6% agarose, transferred to a nylon
membrane, and probed with the 4.5-kb HindIII-EcoRl pNOM1O2
fragment, which contains the entire GUS construct (Fig. 1). Lane 1
contained the control (transformant M-T6; lanes 2 through 10
contained transformants M-T5, M-Tl, M-T2, M-T7, L-T89, L-Tl,
L-T3, L-T4, and L-T6, respectively.
consistent with integration of a single copy into the chromo-
some via recombination within the GUS construct se-
quences. Transformant L-Tl (Fig. 2, lane 7) produced one
hybridizing band larger than plasmid pNOM102. Further
analysis of this transformant with another restriction enzyme
revealed two distinct bands after hybridization with the same
probe (data not shown), indicating that a single copy of
pNOM1O2 was integrated. On the basis of the relative
intensities of the two bands, transformants L-Tl and M-T5
probably arose from integration via a crossover point located
near the ends of the GUS construct to give two hybridizing
bands, only one of which (the larger) hybridized strongly to
the probe (Fig. iB, line 1). The pattern of hybridization
observed in five GUS' transformants (M-T1, M-T2, L-T3,
L-T4, and L-T6) consists of a major hybridizing band at a
size corresponding to linearized copies of pNOM1a2 associ-
ated with additional bands at larger and smaller sizes (Fig. 2,
lanes 3 and 4, and 8 through 10). This pattern can be
explained by the integration of unmodified, tandemly re-
peated copies of the plasmid at the same site, leading to one
or two flanking fragments, depending on the position of the
recombination event1B, in the plasmid (Fig. lines 3 and 4).
With transformant M-T7 (Fig. 2, lane 5), more than three
bands were discerned, indicating that integration had oc-
curred at different sites in the recipient strain genome or that
rearrangements had occurred.
From this analysis, it should be noted that the integration
of one copy of the plasmid can lead to no GUS expression or
low or high levels of GUS expression. To explain the
VOL. 59, 1993 GUS GENE FUSION SYSTEM 1771

30 T b

25 +
z
20 t
IL
E. 15
0 +
+

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10
a
5 a
a

0 l ---
I-.
0 2 0
I-li V
0
0 U.
IL
U
I-
FIG. 3. GUS activities in flax roots inoculated with suspensions of conidia of transformed and untransformed strains. The following
treatments were used: noncontaminated plants (CONTROL); plants contaminated with wild-type strain FOLn3 (5 x 102 conidia per ml) (FO
LINI); plants contaminated with nonpathogenic strain F047 (1 x 106 conidia per ml) (FO47); plants contaminated with GUS transformed
strain L-T6 (5 x 102 conidia per ml) (LT-6); plants contaminated with both nonpathogenic strain F047 (1 x 106 conidia per ml) and GUS
transformed strain L-T6 (5 x 102 conidia per ml) (LT-6 + FO 47). Values are averages of three replicates and are expressed as nanomoles
of MU produced per milligram of protein after a 40-min reaction. Bars labelled with the same letter are not significantly different (P = 0.05;
Newmann-Keuls test).

absence of GUS activity in transformant L-T89, the restric-
tion map of the GUS construct was determined more pre-
cisely for this strain. A hybridization analysis (data not
shown) indicated that the integration event involved the
1.9-kb NcoI fragment of pNOM102 (Fig. 1B, line 2), leading
to the disruption of the gusA coding sequence. For the other
two single-copy transformants (M-T5 and L-T1), the great
difference in the GUS activity levels suggests that the site of
integration may influence the expression of the gusA gene. A
molecular analysis of the integration event in transformant
M-T5, which exhibited the highest GUS activity level, was
performed. A hybridization analysis showed that the site of
the crossover leading to the integration of pNOM102 was
located within the EcoRI-NcoI fragment which contains the
gpd promoter sequences. To analyze more precisely the
effect of both the truncation of this region and the fusion with
Fusanum sequences, a plasmid, designated pGUS-T5, was
rescued from transformant M-T5. This plasmid carried the
gusA coding sequence, the trpC transcriptional terminator,
and the recombinant EcoRI-NcoI fragment (Fig. 1A). Partial
sequencing of the 5' region of the new GUS construct (400
bp upstream from the ATG) showed that the sequence is
identical to that present in pNOM102. Thus, the elements
responsible for the strong GUS activity should be located
upstream from the promoter region. In an additional exper-
iment, the glucuronidase activities of the three cotransfor-
mants obtained via introduction of pGUS-T5 in F. oxyspo-
rum were similar to or higher than the activity of
transformant M-T5. A Southern blot analysis of these
cotransformants did not permit us to determine the inte-
grated copy number. Nevertheless, these results argue for
the presence of total sequences in pGUS-T5 that are respon-
sible for the level of strong activity observed in transformant
M-T5.
In pathogenicity assays, all transformants gave wilt symp-
toms indistinguishable from those caused by the two wild-
type strains. Extracts of flax roots infected by wild-type
strains or GUS + transformant L-T6, alone or mixed with
antagonistic strain F047, were assayed fluorometrically for
GUS activity. A significant level of GUS activity was found
when strain L-T6 was inoculated alone, indicating that the
transformant still expressed GUS during growth in plants
(Fig. 3). When this strain was associated with nonpathogenic
strain F047, the GUS activity detected did not differ signif-
icantly from the background activity in the noncontaminated
control roots. In this combination, the reduction in GUS
activity should reflect a reduction in the level of infection,
revealing the antagonistic role of the nonpathogenic strain.
Expression of GUS activity was detected by the observa-
tion of blue mycelium on young flax roots contaminated with
transformant L-T6 and stained with the X-Gluc substrate. In
contrast, stained hyphae were not observed on roots inocu-
lated with both pathogenic and nonpathogenic strains (Fig. 4
and data not shown).
DISCUSSION
Our results show that the introduction of nonselectable
genes into Fusanium sp. is very efficient since cotransforma-
tion frequencies of more than 50% were obtained by using
equimolar amounts of selected and unselected DNAs. The
levels of efficiency observed are similar to those reported in
other filamentous fungi (9, 22, 23) and allow the introduction
of desired genes without the need for complex plasmid
construction. Identification of GUS' transformants can be
performed quickly by testing a large number of strains in a
simple microtiter assay. The maintenance of GUS activity
through conidiation correlates well with the stable integra-
tion of the reporter gene in the genomes of cotransformants.
Various levels of GUS activity are found in transformants,
and these levels are higher than those observed in Clados-
porium fulvum but lower than those observed in A. nidulans
(23) or Pseudocercosporella herpotrichoides (4). The varia-
tion observed in the expression of GUS activity does not
1772 COUTEAUDIER ET AL.
FIG. 4. Histochemical localization of GUS activity in the mycelium of GUS' transformant L-T6 at the root surface of 7-day-old flax roots.
A blue precipitate (arrows) indicated the location of GUS activity in the mycelium. Magnification, x600.
reflect differences in plasmid copy number between trans-
formants. Similar observations were reported in C. fulvum
by Roberts et al. (23), but these observations appear to be
different from those observed by Bunkers (4) for P. herpo-
trichoides. The differences in GUS activity levels could be
mediated by different integration positions. It was interesting
to note that the highest level of GUS activity was obtained in
a transformant carrying only one copy of pNOM102. Such
strong activity does not result from structural modifications
of the GUS construct produced by the integration event. The
analysis of the structure of rescued plasmid pGUS-T5 indi-
cated that F. oxysporum sequences located near the inte-
grated plasmid promoted or enhanced GUS gene activity.
Such a plasmid could be an alternative to pNOM102 in
experiments involving F. oxysporum.
Fluorogenic measurement of GUS activity by using MUG
as a substrate provides a useful tool for quantifying the
active root-colonizing biomass of a strain containing the
gusA gene. Previously, the basic methods used to evaluate
the colonization of roots by F. oxysporum have consisted of
the identification of colonies arising from root fragments
plated on selective medium. This kind of bioassay was not
suitable for investigations focused on interactions between
strains. Our results show that the GUS marker system can
allow rapid quantification of fungal growth in roots. This
system is also a new, accurate method for the study of
competition between pathogenic and nonpathogenic F. oxy-
sporum strains at the root level. The reduction in the GUS
activity level in flax roots exposed to the GUS' pathogenic
strain in association with nonpathogenic strain F047 sug-
gests that the nonpathogenic strain inhibited the colonization
of living root tissues by the pathogenic strain. These results
are in agreement with earlier observations showing that
APPL. ENVIRON. MICROBIOL.
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strain F047 inhibited the germination of wild-type strain
FOLn3 in the rhizosphere of flax roots (6). Additional
studies (12) showed that strain F047 drastically reduced the
GUS activity of the pathogen in the root tissues, while other
strains did not modify the activity of the pathogen, and
reduction was related to the population ratio of FOLn3 GUS
to F047.
X-Gluc-stained hyphae of the transformed strain were
clearly visible on root tissues and could be easily observed
after different contact times between roots and pathogenic
strains. Direct examination of the location of hyphae by
histochemical localization of glucuronidase activity should
permit workers to study the root colonization pattern of F.
oxysporum in various environments; indeed, little is known
about what happens between germination in root exudates
and the detection of the pathogen in the vascular system
(13). Moreover, a combination of histochemical techniques
and the GUS marker technique may be feasible for the
development of a staining procedure capable of locating
hyphae of both nonpathogenic and pathogenic strains in host
tissues. The resulting information would be of considerable
value in elucidating the mechanism by which a nonpatho-
genic F. oxysporum strain reduces the incidence of Fusar-
ium wilt disease (1, 6). Our results illustrate the suitability of
GUS genetic marking of F. oxysporum for identifying and
monitoring a specific population throughout studies of plant-
fungus interactions. Another application of the expression of
the GUS gene in F. oxysporum is in the study of the basic
mechanism of transposition recently reported in this fungus
(10). In this case, the 3-glucuronidase marker gene can be
used as a reporter gene to develop a sensitive assay for the
excision of transposable elements introduced into various F.
oxysporum strains.
VOL. 59, 1993
ACKNOWLEDGMENTS
We thank R. Oliver for kindly providing the pNOM102 plasmid,
C. Gerlinger for excellent technical assistance, and M. F. Turlier for
the photography.
REFERENCES
1. Alabouvette, C., and Y. Couteaudier. 1992. Biological control of
Fusarium wilts with nonpathogenic Fusana, p. 415-426. In
E. C. Tjamos, G. C. Papavizas, and R. J. Cook (ed.), Biological
control of plant diseases: progress and challenges for the future.
Plenum Press, New York.
2. Alabouvette, C., Y. Couteaudier, and J. Louvet. 1984. Recher-
ches sur la r6sistance des sols aux maladies. IX. Dynamique des
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