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J Bacteriol, July 1998, p. 3592-3597, Vol. 180, No. 14
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of a Gene Product Induced by
Hard-Surface Contact of Colletotrichum gloeosporioides
Conidia as a Ubiquitin-Conjugating Enzyme by Yeast
Complementation
Zhi-Mei
Liu and
Pappachan E.
Kolattukudy*
Departments of Biochemistry and Medical
Biochemistry and Neurobiotechnology Center, The Ohio State
University, Columbus, Ohio 43210
Received 27 February 1998/Accepted 8 May 1998
 |
ABSTRACT |
The germinating conidia of many phytopathogenic fungi on hosts must
differentiate into an infection structure called the appressorium in
order to penetrate their hosts. Chemical signals, such as the host's
surface wax or fruit ripening hormone, ethylene, trigger germination
and appressorium formation of the avocado pathogen Colletotrichum
gloeosporioides only after the conidia are in contact with a hard
surface. What role this contact plays is unknown. Here, we describe
isolation of genes expressed during the early stage of hard-surface
treatment by a differential-display method and report characterization
of one of these cloned genes, chip1 (Colletotrichum hard-surface induced protein 1 gene), which
encodes a ubiquitin-conjugating enzyme. RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction. The predicted open reading frame
(ubc1Cg) would encode a 16.2-kDa
ubiquitin-conjugating enzyme, which shows 82% identity to the
Saccharomyces cerevisiae UBC4-UBC5 E2 enzyme, comprising a
major part of total ubiquitin-conjugating activity in stressed yeast
cells. UBC1Cg can complement the proteolysis deficiency of
the S. cerevisiae ubc4 ubc5 mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation.
 |
INTRODUCTION |
Many phytopathogenic fungi must
differentiate from the germ tube into an infection structure called the
appressorium in order to penetrate hosts (10, 33, 34).
Chemical and/or physical signals are known to trigger germination of
and appressorium formation by fungal conidia (7, 9, 16-18).
Some of the molecular events triggered by the physical signal in the
bean rust fungus Uromyces appendiculatus (4, 37,
38) and the rice rust fungus Magnaporthe grisea have
been studied (24). It has been known for a long time that
contact with a hard surface is necessary for many fungi to induce
appressorium formation (10). Conidia of Colletotrichum gloeosporioides are induced to germinate and differentiate to form
appressoria by chemical signals, including the host surface wax
(30) and a fruit ripening hormone, ethylene (11).
However, contact with a hard surface is necessary for the chemical
signals to induce appressorium formation. Conidia resting on either a hydrophilic hard surface (glass) or a hydrophobic hard surface responded to the chemical signals only between 2 and 4 h after the
initiation of contact with the hard surface (11, 12, 20). Recently, four genes expressed uniquely during appressorium formation induced by the host surface wax were cloned by differential screening of a library produced by a subtractive hybridization approach (19,
20). Disruption of one of these genes drastically decreased its
virulence for the host (19). However, the nature of the genes expressed during the 2 h of contact with the hard surface that primes the conidia to respond to the chemical signals is unknown.
To study molecular events triggered by hard-surface contact, genes
expressed in C. gloeosporioides conidia during hard-surface treatment were examined by an mRNA differential-display method (25, 26). Here, we report that one of the genes expressed during hard-surface treatment encodes a ubiquitin-conjugating enzyme,
which shows very high homology to the Saccharomyces
cerevisiae UBC4-UBC5 enzyme pair, comprising a major part of total
ubiquitin-conjugating activity in stressed yeast cells. We show that
the C. gloeosporioides gene expressed in S. cerevisiae can complement the proteolysis deficiency of an
S. cerevisiae ubc4 ubc5 mutant. These results suggest that
expression of this ubc gene triggered by hard-surface contact mediates ubiquitin-dependent protein degradation associated with germination and appressorium formation.
 |
MATERIALS AND METHODS |
Fungal and bacterial strains and materials.
C.
gloeosporioides, an isolate from avocado, was kindly provided by
Dov Prusky (Volcani Center, Bet-Dagan, Israel). Cultures were
maintained at 25°C on potato dextrose agar. Conidia were obtained by
gently scraping 5- to 7-day-old cultures in petri dishes flooded with
sterilized distilled water, as described previously (19,
20). Escherichia coli DH5
was used for propagating
all plasmids. Restriction and modification enzymes and Taq
DNA polymerase were from Life Technologies, Inc. (Bethesda Research
Laboratories [BRL]).
RNA preparation.
Conidia of C. gloeosporioides
(~5 × 106 conidia/dish) were spread into petri
dishes (150 by 15 mm) containing 30 ml of water and were incubated for
various periods of time. The conidia were harvested by scraping them
off the petri dishes with a rubber policeman (Fisher Scientific,
Cincinnati, Ohio) and were subjected to centrifugation at 12,000 × g for 15 min as described previously (19, 20).
For large-scale total-RNA isolation, the conidia from at least 50 petri
dishes were resuspended in a solution containing 4.5 M guanidinium
thiocyanate, 50 mM EDTA (pH 8.0), 100 mM
-mercaptoethanol, 25 mM
sodium citrate (pH 7.0), and 2% sodium N-lauroylsarcosine (3 to 5 ml) and disrupted for 5 min with 425- to 600-µm-diameter glass beads in a mini-bead beater (Biospec Products, Bartlesville, Okla.). The total RNA was isolated by density gradient centrifugation through CsCl (3). For small-scale total-RNA isolation, the conidia from ~10 petri dishes were suspended in 500 µl of
homogenization buffer (50 mM LiCl, 25 mM Tris-HCl [pH 8.0], 35 mM
EDTA, 35 mM EGTA, 0.5% sodium dodecyl sulfate [SDS]) and 500 µl of
phenol-chloroform (1:1) and disrupted for 5 min with 425- to
600-µm-diameter glass beads in a mini-bead beater. The aqueous phase
was then extracted with 500 µl of chloroform, and RNA was
precipitated with an equal volume of 4 M LiCl. The RNA pellet was
washed with 500 µl of 2 M LiCl and then with 70% ethanol.
Differential display of mRNA.
Total RNA was treated with
amplification-grade RNase-free DNase I (BRL) at 37°C for 30 min to
remove possible DNA contamination. The RNA concentration was calculated
from the absorbance at 260 nm. The differential-display procedure
recommended by the manufacturer (GenHunter Corporation, Brookline,
Mass.) was followed. For first-strand cDNA synthesis, a 19-µl mixture
containing 0.5 µg of total RNA, 4 pmol of oligo(dT) primer
5'-HT11M-3' (where M may be G, A, or C), 400 pmol of deoxynucleoside
triphosphate (dNTP), 25 mM Tris-HCl (pH 8.3), 37.6 mM KCl, 1.5 mM
MgCl2, and 5 mM dithiothreitol was heated at 65°C for 5 min. The temperature was then reduced to 37°C, and after 10 min, 1 µl of Moloney murine leukemia virus reverse transcriptase (200 U) was
added and incubation was continued at 37°C for another 50 min.
Finally, the 20-µl reaction mixture was heated at 75°C for 5 min
and then chilled to 4°C. For PCR, 2 µl of first-strand cDNA
solution was added to a mixture (18 µl) containing 1.5 U of
Taq DNA polymerase (BRL), 2.2 µM dNTP, 0.22 µM oligo(dT)
primer 5'-HT11M-3', 0.22 µM arbitrary decanucleotide primer, 11.1 µM Tris-Cl (pH 8.4), 55.6 mM KCl, 1.67 mM MgCl2, and
0.0011% gelatin. The reaction was carried out in a programmable thermal controller (MJ Research, Watertown, Mass.) as follows: 94°C
(30 s), 40°C (2 min), and 72°C (30 s) for 40 cycles. The additional
final extension step was performed at 72°C for 5 min. Each PCR
product (3.5 µl) was mixed with 2 µl of loading dye (95% formamide, 10 mM EDTA [pH 8.0], 0.09% xylene cyanole FF, and 0.09% bromophenol blue) and incubated at 80°C for 2 min immediately before
being loaded onto a 6% DNA sequencing gel. The gel was run at 60 W for
~3 h, placed on a piece of 3M paper, vacuum dried at 80°C for
1 h, and exposed to X-ray film. For reamplification of the cDNA
probe, gel segments representing DNA bands of interest were cut out
with razors, each gel slice along with the 3M paper was soaked in 100 µl of water for 10 min, and DNA was eluted by boiling for 15 min and
precipitated with ethanol in the presence of 50 µg of glycogen as a
carrier. To reamplify the DNA fragments, 4 µl of the total 10 µl of
eluted DNA was mixed with 36 µl of a reaction mixture containing 3 U
of Taq DNA polymerase (BRL), 2.2 µM dNTP, 0.22 µM
oligo(dT) primer 5'-T11M-3', 0.22 µM arbitrary decanucleotide primer,
11.1 µM Tris-Cl (pH 8.4), 55.6 mM KCl, 1.67 mM MgCl2, and
0.0011% gelatin. The PCR conditions were the same as those described
above. Finally, the amplified DNA fragments were cloned into a pCRII
vector (Invitrogen, Carlsbad, Calif.). Double-stranded plasmid DNAs
were prepared by the alkaline lysis-polyethylene glycol precipitation
method (31) and used directly for automated sequencing with
a model 373A sequencer from Applied Biosystems (Foster City, Calif.).
Isolation of C. gloeosporioides full-length cDNA by
5' rapid amplification of cDNA ends (RACE) and sequence analysis.
To obtain the upstream nucleotide sequence, an internal specific primer
(5'-GTG CTC CTA ACT CTG ATC GGT C-3') and Lambda ZAP vector primers (T7
and T3) were used for PCR, with a Lambda ZAP cDNA library from
hard-surface-treated conidia as a template. A Lambda ZAP cDNA library
was prepared according to the manufacturer's instructions
(Stratagene). The PCR was initiated by denaturation at 94°C for 2.5 min and then carried out for 40 cycles as follows: 94°C (25 s),
54°C (35 s), and 72°C (1.5 min). The additional final extension
step was performed at 72°C for 8 min. The ~1-kb PCR product was
purified from the 1% agarose gel with a Geneclean kit (Bio 101, Vista,
Calif.), cloned into a pCRII vector (TA cloning kit; Invitrogen), and
sequenced as indicated above. The DNA sequence from both strands was
analyzed with DNA Stride 1.2. Amino acid homology searches were
conducted with the BLAST program from the National Center for
Biotechnology Information (1). Homology comparison was
performed with the SeqApp program.
RNA blot analysis.
Total RNA isolated from conidia or
germinating conidia of C. gloeosporioides was dissolved in a
solution containing 50% formamide, 16% formaldehyde, 20 mM MOPS
[3-(N-morpholino)propanesulfonic acid], 5 mM sodium
acetate, and 1 mM EDTA (pH 7.0), incubated for 15 min at 65°C, and
chilled on ice. Denatured samples were subjected to electrophoresis on
1% agarose gels containing 2.2 M formaldehyde and were blotted onto
Nytran membranes. The blots were prehybridized for ~4 h at 65°C in
a solution containing 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M
sodium citrate [pH 7.6]), 2× Denhardt's solution, 0.1% SDS, and
100 µg of sheared salmon DNA/µl and hybridized for ~16 h in the
same solution with 106 cpm of a 32P-labeled
cDNA probe/ml prepared by randomly primed labeling. The membranes were
washed twice for 10 min at room temperature in 2× SSC plus 0.1% SDS,
briefly washed at 65°C with 0.2× SSC plus 0.1% SDS, and exposed to
X-ray film at
80°C in the presence of an intensifying screen.
32P was quantitated with a PhosphorImager (Molecular
Dynamics, Sunnyvale, Calif.).
Southern blot analysis.
Genomic DNA was isolated from
mycelium grown in mineral medium (15) containing 1% yeast
extract and 1% glucose with shaking (200 rpm) for 36 h. The
genomic DNA was digested to completion with restriction enzymes,
subjected to electrophoresis on 1% agarose gels, and transferred to
Nytran membranes. The conditions for prehybridization, hybridization,
and washing were the same as those described above for RNA blots. The
ubcCg genomic DNA was amplified by PCR with a 5'
noncoding region primer (5'-GAC TCT CAC AAT CCA AAT CAA AAG-3') and the
internal specific primer (5'-GTG CTC CTA ACT CTG ATC GGT C-3'). The PCR
was initiated by denaturation at 94°C for 2.5 min and was then
carried out for 38 cycles as follows: 94°C (25 s), 54°C (35 s), and
72°C (1.5 min). The additional final extension step was performed at
72°C for 8 min.
Yeast complementation.
The S. cerevisiae ubc4
ubc5 double mutant [Y0096; his3-
200 leu2-3,2-112
lys2-801 trp1-1(Am) ura3-52
ubc4::HIS3 ubc5::LEU2] was kindly provided by Stefan Jentsch, Friedrich Miescher
Laboratory, Heidelberg, Germany. The
ubc1Cg cDNA was cloned into the EcoRI site in both orientations of a low-copy-number yeast expression vector, pBM272 (kindly provided by Douglas Johnson, University of
Vermont, Burlington), under the control of a GAL10 promoter with a URA3 selectable marker. Plasmids with inserts in both
orientations with regard to the GAL10 promoter, as well as
the plasmid without any insert, were used to transform the Y0096
strain. Yeast transformation was carried out according to standard
protocols (3). Ura+ transformants were
obtained in synthetic complete medium lacking uracil with 2%
glucose (SC
U) at 30°C. They were streaked onto SC
U plates
and SC(Gal)
U plates (plates with synthetic complete medium
lacking uracil with 2% galactose) and incubated at either 30 or
37°C.
Nucleotide sequence accession number.
The nucleotide
sequence for the ubc1Cg cDNA is in the GenBank
database under accession no. AF030296.
 |
RESULTS |
Differential display of RNA from C. gloeosporioides
during hard-surface contact.
Total RNAs from hard-surface-treated
(2 h) or control (untreated) conidia were reverse transcribed with
primers as indicated in Materials and Methods. Products were amplified
by using combinations of eight arbitrary 5' decamers and three
oligo(dT) HT11M primers. Figure 1 shows
the area of a differential-display gel including the amplified products
obtained with primer combination HT11A and H-AP2 or HT11A and H-AP3. A
band representing an enhanced level of expression of a gene during the
hard-surface treatment is present at ~190 bp. The same pattern was
observed when PCR and electrophoresis were repeated. When the ~190-bp
DNA band recovered from the gel was amplified by PCR and used as a
probe for Northern blot analysis, two transcripts were found: a
strongly hybridizing band at ~1 kb and a much less strongly
hybridizing band at ~2.4 kb. Both transcripts were induced by 2 h of hard-surface treatment (data not shown). The reamplified PCR
product was used directly as the substrate for automated sequencing
with the 5' decamer as the primer. The sequence is shown in Fig.
2. When the PCR product was cloned and
independent clones were sequenced, four different sequences were found;
one of them was identical to that underlined in Fig. 2. Since the
direct sequencing of the PCR product gave this sequence, further
studies were focused on this clone, which we designated
chip1 (for Colletotrichum hard-surface-induced
protein 1).

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FIG. 1.
Area of a differential-display gel showing the amplified
products obtained with primer combinations of oligo(dT) primer HT11A
and arbitrary 5' decamer H-AP2 (A2) or H-AP3 (A3) by using as templates
cDNAs derived from conidia treated on a hard surface for 2 h (H)
and an untreated control (C). The amplified product of interest is
indicated by an arrow at ~190 bp. Experimental details are provided
in the text.
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FIG. 2.
Nucleotide sequence and deduced amino acid sequence of
the cloned cDNA fragment showing the ORF coding for UBC1Cg.
The sequence of the differential-display product is underlined, and the
specific internal primer used for 5' RACE is in boldface and is
underlined, as is the active site, Cys, required for
ubiquitin-thioester formation.
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Isolation of full-length cDNA for CHIP1 by 5' RACE and sequence
analysis.
To obtain the upstream nucleotide sequence, an internal
specific primer (5'-GTG CTC CTA ACT CTG ATC GGT C-3') and Lambda ZAP vector primers (T7 and T3) were used for PCR, with a Lambda ZAP cDNA
library of hard-surface-treated conidia as a template. An ~1-kb PCR
product was obtained with the internal specific primer and T7 vector
primer. Cloning and sequencing of this product revealed an open reading
frame (ORF) that would encode a 147-amino-acid protein with a deduced
molecular mass of 16.2 kDa (Fig. 2). The DNA sequence surrounding the
ATG translation start site (underlined) (GCCAAAATGGC)
has a conserved Kozak sequence found in filamentous fungi
(CA[C/A][A/C]ATGNC) and closely resembles the
Kozak sequence from mammals (GCC[A/G]CCATGG)
(14, 23).
The protein that is predicted to be encoded by this ORF shows very high
homology to ubiquitin-conjugating enzymes from various
organisms (Fig.
3): 91.2% to UBC4
Sp of
Saccharomyces pombe (
5),
85.7% to
UBC1
Dm of
Drosophila (
32), 8.4%
to UBC2
Ce of
Caenorhabditis elegans
(
38), 83.0% to human UBC5
Hs
(
21), 82% to UBC4
Sc of
S. cerevisiae
(
32), and 42.2% to UBC
Wh of wheat
(
13). Therefore,
we designate CHIP1
C. gloeosporioides ubiquitin-conjugating enzyme
1, or
UBC1
Cg.

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FIG. 3.
Homology comparison of UBC1Cg with
UBC4Sp of S. pombe, UBC2Ce of
C. elegans, UBC1Dm of Drosophila,
human UBC5Hs, and UBCWh of wheat. The
homologous residues are shaded.
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ubc1Cg transcript levels induced by
hard-surface and ethylene treatment.
When the ~1-kb PCR product
was used as a probe for Northern blot analysis, a single transcript of
~1 kb was found, indicating that the cloned cDNA represents a nearly
full-length transcript. Analysis of the time course of induction by
hard-surface treatment showed that induction of
ubc1Cg was readily detectable in 2 h, increased until about 6 h of hard-surface treatment, and
subsequently decreased (Fig. 4A).

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FIG. 4.
(A) Northern blots showing time course of induction of
ubc1Cg transcription by hard-surface contact in
C. gloeosporioides conidia. The equal loading amounts of
total RNAs (20 µg/lane) were reflected by ethidium bromide staining
of 28S and 18S rRNA. (B) Northern blots showing time course of
induction of ubc1Cg transcription by 10 µM
ethephon on a hard surface in C. gloeosporioides conidia.
The amount of total RNAs used was 20 µg/lane. (C) Northern blots
showing induction of ubc1Cg by a hard surface
with or without ethylene treatment. Total RNAs (10 µg/lane) isolated
from C. gloeosporioides conidia that had been on a hard
surface for 4 h (H4), on a hard surface with 10 µM ethephon
(HE4) for 4 h, or left in the tube at room temperature for 4 h (C4) were subjected to electrophoresis and blotted onto Nytran
membranes. 32P-labeled ~1.0-kb cDNA containing the coding
sequence of ubc1Cg was used as a probe.
Estimation of RNA sizes was based on the 0.24- to 9.5-kb RNA ladder
(BRL). Experimental details are provided in the text.
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Since ethylene is known to induce germination and appressorium
formation by
C. gloeosporioides conidia on a hard surface
(
11),
the effect of ethylene on induction of
ubc1Cg in conidia resting
on a hard surface was
tested. The time course of induction was
quite similar to that observed
on a hard surface in the absence
of ethylene, with maximal induction
occurring at ~4 h (Fig.
4B).
The degree of induction on a hard
surface with ethylene was higher
than that observed on a hard surface
without ethylene. A direct
comparison of the RNA blots shown in Fig.
4C
demonstrates that
the
ubc1Cg transcript level
was higher on a hard surface with
ethylene than that reached on a hard
surface without ethylene.
Quantitation showed that the hard-surface
treatment alone caused
a twofold increase in transcript level, whereas
hard-surface and
ethylene treatment caused a sixfold increase in
transcript level.
Southern blot analysis of ubc1Cg.
The
genomic DNA isolated from C. gloeosporioides was digested
with BamHI, EcoRI, HindIII,
SstI, XbaI, or XhoI, and Southern blots of the digests were hybridized with the cDNA clone. The results
showed only one band in the case of all digests except the
HindIII digest, which showed two bands (Fig.
5). However, the restriction map of the
cDNA clone showed that there is no HindIII site within
the cDNA. To test whether there is a HindIII site in
the genomic DNA, PCR-amplified ~1.5-kb genomic DNA was digested with HindIII. This digestion yielded ~0.9-
and ~0.6-kb fragments, indicating that there is a
HindIII site in this genomic DNA (data not
shown). Apparently, there is an intron containing a
HindIII site in this genomic DNA. Thus, the Southern
blot analysis indicates that the genome of C. gloeosporioides contains one copy of the
ubc1Cg gene.

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FIG. 5.
Southern hybridization of restriction enzyme-digested
genomic DNA (10 µg/lane) from C. gloeosporioides with a
32P-labeled ~1.0-kb cDNA containing the coding sequence
of UBC1Cg as the probe. Molecular sizes (determined with a
DNA HindIII size marker) are shown on the left.
Experimental details are provided in the text.
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Complementation of ubc yeast mutant with
ubc1Cg.
To test whether the sequence
similarity of UBC1Cg to yeast UBC4 is also reflected in its
function, we tried to complement the S. cerevisiae ubc4 ubc5
mutant by expression of ubc1Cg. The yeast
ubc4 ubc5 mutant is heat sensitive; it cannot grow at 37°C and can grow only very slowly at 30°C (32). The
ubc1Cg cDNA was cloned into the EcoRI
site in both orientations in a low-copy-number yeast expression vector,
pBM272, under the control of the GAL10 promoter with a URA3
selectable marker. Plasmids with inserts in both orientations, as well
as pBM272 without any insert, were used to transform the yeast
ubc4 ubc5 mutant strain. Transformants were streaked onto
SC
U plates or SC(Gal)
U plates and incubated at either 30 or 37°C.
When yeast ubc4 ubc5 mutant cells were transformed with
plasmids with ubc1Cg inserted in the proper
orientation, they grew relatively quickly at 37°C on inducible medium
(containing galactose) but not on noninducible medium (containing
glucose) (data not shown). Plasmids alone or with a
ubc1Cg insert in the opposite orientation did
not grow at 37°C on either inducible medium (containing galactose) or
noninducible medium (containing glucose). Therefore, UBC1Cg
complemented the growth deficiency and heat sensitivity of the
ubc4 ubc5 mutant on inducible medium but not on noninducible
medium. Thus, UBC1Cg is not only structurally but also
functionally similar to yeast UBC4.
 |
DISCUSSION |
The formation of appressoria is essential for penetration of the
avocado pathogen C. gloeosporioides into its host. Contact with a hard surface is necessary for the chemical signals ethylene and
avocado wax to induce appressorium formation in C. gloeosporioides. C. gloeosporioides conidia can form appressoria
on both a hydrophilic cover glass and a hydrophobic polystyrene petri
dish when exposed to the chemical signals. On the other hand, on soft
hydrophilic or hydrophobic substrates, such as 2% agar or petrolatum,
respectively, only germination occurs (27). The
hydrophilicity or hydrophobicity of the surface does not play an
important role in appressorium formation by C. gloeosporioides conidia. The molecular mechanism by which
hard-surface treatment assists appressorium formation remains unknown.
Elucidation of the nature of genes uniquely expressed during
hard-surface treatment could help in understanding the molecular basis
of the early events in plant-fungus interaction. Chemical signals, such
as ethylene or avocado wax, showed no effect on appressorium formation
in C. gloeosporioides conidia during the first 2 h.
Treatment for the next 2 to 3 h with chemical signals induced
appressorium formation, but subsequent treatment had no effect
(11, 12, 20). These observations suggest that a chain of
molecular events that ultimately leads to differentiation of the germ
tubes into appressoria is initiated upon contact with a hard surface.
Breaking the chain of events at any critical stage should interfere
with appressorium formation. The early contact with a hard surface
presumably initiates molecular changes that prime the conidia to
respond to chemical signals, such as the host wax or ethylene. Although
some of the genes induced by the chemical signals have been cloned,
nothing is known about genes expressed in the early phase. Therefore,
we chose to concentrate on transcripts induced during 2 h of
hard-surface treatment.
By using a differential-display method, we found eight genes,
designated chip genes, expressed during the hard-surface
treatment of conidia of C. gloeosporioides. chip1 encodes a
ubiquitin-conjugating enzyme, which shows very high homology to the
yeast UBC4-UBC5 enzyme pair. To test whether this clone, obtained from
RNA from conidia subjected to hard-surface treatment, represents the
transcript induced during hard-surface treatment, Northern blot
analyses were performed. The results showed that the transcript reached its maximum level after 4 to 6 h of treatment with a hard surface and then decreased. ubc1Cg was induced to a
higher level by exposure to an ethylene-generating compound, ethephon,
on a hard surface. The increase ceased by 6 h, just before
appressorium formation began to be detectable, and the transcript level
decreased quite rapidly during the next few hours. The genes discovered
by the present approach are probably involved in the induction of
appressorium formation, although there is no direct proof that the
cloned transcripts induced by hard-surface treatment are actually
involved in the chain of events that lead to appressorium formation.
Since C. gloeosporioides ubc complemented the ubc4
ubc5 yeast mutant, it is clear that ubc1Cg
is functionally equivalent to yeast ubc4 ubc5. In
eukaryotes, the ubiquitin-proteasome system is involved in degradation
of various proteins. The ubiquitination of target proteins is catalyzed
by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes
(E2) and in some cases also requires auxiliary substrate recognition
proteins (E3). The targets of this degradation pathway include
calmodulin and subunits of trimeric G protein (28, 29). A
calmodulin gene was cloned from Colletotrichum trifolii
(8). When an antisense strategy was used to reduce the
expression of this calmodulin gene, appressoria were formed at a
reduced frequency (6). Calmodulin was recently found to be
involved in appressorium formation in C. gloeosporioides (22), and G protein was found to be essential for
appressorium formation in M. grisea (6).
Selective protein degradation by the ubiquitin-proteasome system has
been found to play a critical role in many situations, such as the
cellular stress response and differentiation, that involve
reprogramming of protein synthesis (36). In yeast, at least
12 different ubc genes encode ubiquitin-conjugating enzymes, which mediate strikingly diverse functions. One of the
best-characterized yeast E2 enzymes is the UBC4-UBC5 pair. ubc4
ubc5 mutants are sensitive to heat shock, canavanine (an arginine
analog), and cadmium, suggesting that the UBC4-UBC5 enzyme pair
mediates selective degradation of short-lived and abnormal proteins
(32). The UBC4-UBC5 enzyme pair comprises a major part of
total ubiquitin-conjugating activity in stressed yeast cells
(2). UBC4-UBC5 homologs have been found in several
organisms. In C. elegans, UBC2 is developmentally regulated
by becoming specific to the nervous system in L4 larvae and adults
(40), and unlike the yeast UBC4-UBC5 enzyme pair, it is not
induced by heat shock (39). UBC1Dm in
Drosophila is also involved in selective protein degradation
(35). Our finding that UBC1Cg can complement the
proteolysis deficiency of the yeast ubc4 ubc5 mutant
indicates that it may also mediate selective proteolysis pathways. In
the present case, hard-surface contact probably signals a chain of
molecular events that involve reprogramming of protein synthesis needed
for conidial germination and differentiation into appressoria. The
signal transduction processes involved in transmitting the hard-surface
contact to the cellular machinery remain to be elucidated. It is
possible that the physical signals and the chemical signals share some
signal transduction pathways involved in the differentiation process
that are essential for infection by many fungi. Such pathways could
serve as targets of antifungal strategies to protect plants.
 |
ACKNOWLEDGMENTS |
We thank Daoxin Li and Yeon-ki Kim for many helpful discussions.
This work was supported by National Science Foundation grant
IBN-9318554.
 |
FOOTNOTES |
*
Corresponding author. Mailing address:
Neurobiotechnology Center, The Ohio State University, 206 Rightmire
Hall, 1060 Carmack Rd., Columbus, OH 43210. Phone: (614) 292-5682. Fax:
(614) 292-5379. E-mail: kolattukudy.2{at}osu.edu.
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J Bacteriol, July 1998, p. 3592-3597, Vol. 180, No. 14
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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