a gal4-like protein is involved in the switch between
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The Plant Cell, Vol. 12, 1579–1589, September 2000, www.plantcell.org © 2000 American Society of Plant Physiologists
A GAL4-like Protein Is Involved in the Switch betweenBiotrophic and Necrotrophic Phases of the Infection Processof Colletotrichum lindemuthianum on Common Bean
Marie Dufresne,
a,1 Sarah Perfect,
b
Anne-Laure Pellier,
a
John A. Bailey,
b
and Thierry Langin
a
a
Laboratoire de Phytopathologie Moléculaire, Centre de Recherche sur les Plantes, Université Paris-Sud, 91405 Orsay
Cedex, France
b
Department of Agricultural Sciences, Institute of Arable Crops Research, Long Ashton Research Station, University of
Bristol, Bristol BS18 9AF, United Kingdom
Random insertional mutagenesis was conducted with the hemibiotrophic fungus Colletotrichum lindemuthianum
,
causal agent of common bean anthracnose. Nine mutants that were altered in their infection process on the host plant
were generated. One of these, H433 is a nonpathogenic mutant able to induce necrotic spots on infected leaves rapidly.
These spots are similar to those observed during the hypersensitive reaction. Cytological observations showed that the
development of the mutant H433 is stopped at the switch between the biotrophic and the necrotrophic phases. This
mutant carries two independent insertions of the transforming plasmid pAN7-1. Complementation studies using the
wild-type genomic regions corresponding to the two insertions showed that one is responsible for the H433 phenotype.
Sequencing analysis identified a single open reading frame that encoded a putative transcriptional activator belonging
to the fungal zinc cluster (Zn[II]
2
Cys
6
) family. The corresponding gene was designated CLTA1
(for C. lindemuthianum
transcriptional activator 1). Expression studies showed that CLTA1
is expressed in low amounts during in vitro culture.
Targeted disrupted strains were generated, and they exhibited the same phenotype as the original mutant H433. Com-
plementation of these disrupted strains by the CLTA1
gene led to full restoration of pathogenicity. This study demon-
strates that CLTA1
is both a pathogenicity gene and a regulatory gene involved in the switch between biotrophy and
necrotrophy of the infection process of a hemibiotrophic fungus.
INTRODUCTION
Fungi, one of the most damaging groups of microorganisms
to plants, are characterized by a wide variety of infection
strategies, from strict biotrophy to necrotrophy. The ability
to utilize multiple modes of infection makes it difficult to pro-
vide a comprehensive explanation of the mechanisms in-
volved in successful infection of a host plant.
During the past decade, different strategies have been
developed to identify the fungal genes specifically involved
in infection of the host plant, the pathogenicity genes (Oliver
and Osbourn, 1995; Hensel and Holden, 1996; Hamer and
Holden, 1997). Among these strategies, random insertional
mutagenesis has been used to study several plant patho-
genic fungi (Bölker et al., 1995; Dufresne et al., 1998;Sweigard et al., 1998; Balhadère et al., 1999). A major ad-
vantage of such a strategy is that there is no a priori postu-
late about the function or expression of the tagged genes.
Moreover, functions have been identified more readily than
when a direct candidate approach is used. In Magnaporthe
grisea
, random insertional mutagenesis made it possible to
identify many pathogenicity genes that potentially encode
various enzymes acyltransferase, neutral trehalase, and the
catalytic subunit of a cAMP-dependent kinase (Sweigard et
al., 1998). Studies of pathogenicity genes generally yield a
large number of path
mutants, thereby confirming that
pathogenicity genes constitute a major part of the genome
of plant pathogenic fungi.
Colletotrichum lindemuthianum
is the causal agent of an-
thracnose on common bean. This interaction has been as-
sumed to fit the gene-for-gene hypothesis (Flor, 1971), butgenetic studies have been performed only for the plant part-
ner (Young and Kelly, 1996; Geffroy et al., 1998). Phenotypi-
cally, the infection of a resistant cultivar of common bean by
a corresponding avirulent strain of C. lindemuthianum (in-
compatible interaction)
leads to the
appearance of localized
necrotic spots typical of a classical hypersensitive response.
The infection process has been well documented in previ-
ous cytological studies (O’Connell et al., 1985; Bailey et al.,
1992). Basic compatibility, that is, interaction between a
1
To whom correspondence should be addressed. E-mail marie.
[email protected]; fax 44-1603-250024.
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1580 The Plant Cell
susceptible cultivar of common bean and any virulent strain
of C. lindemuthianum
, begins with the adhesion of conidia
to aerial parts of the plant. Conidia then germinate and dif-
ferentiate to form an infection structure devoted to mechan-
ical penetration, the appressorium. After the penetration
step, the infection cycle is characterized by two successivephases. In the first phase, lasting 3 to 4 days, the fungus
grows biotrophically inside the infected epidermal cells. Dur-
ing this phase, referred to as the biotrophic phase, the fun-
gus differentiates infection vesicles and primary hyphae. The
second phase, which corresponds to the appearance of an-
thracnose symptoms, is completed 6 to 8 days after inocu-
lation. During this phase, the necrotrophic phase, the fungus
develops secondary hyphae that grow both intracellularly
and intercellularly and thus acts as a typical necrotrophic
pathogen. C. lindemuthianum
is classified as a hemi-
biotrophic fungus because of the succession of these two
phases during the infection cycle. Because the host plant is
not required for fungal growth, this pathogen can be manip-
ulated in vitro, in contrast to the case with strictly biotrophic(obligate) pathogens.
A random insertional mutagenesis technique was devel-
oped in our laboratory to identify essential virulence deter-
minants of the fungus (Dufresne et al., 1998). Nine less-
pathogenetic mutants were recovered from 1200 transfor-
mants tested. The molecular analysis of mutant strain H290
allowed us to identify the first C. lindemuthianum
pathoge-
nicity gene, CLK1
, which encodes a putative serine/threo-
nine kinase and is involved in the regulation of appressorium
function (Dufresne et al., 1998).
We report here the cytological and molecular character-
ization of one nonpathogenic mutant, designated H433. We
first show that this path
mutant is blocked during the infec-
tion process at the switch between biotrophy and necrotro-
phy. Then, we demonstrate that the mutant phenotype is
related to the interruption of the CLTA1
gene (for C. linde-
muthianum
transcriptional activator 1), which encodes a pu-
tative GAL4-like transcriptional activator. These studies
allowed us to identify and characterize a regulatory gene in-
volved at the transition between biotrophic and necrotrophic
phases, a key step in the infection process of the hemi-
biotrophic fungus C. lindemuthianum
.
RESULTS
Macroscopic Characterization of the H433
Mutant Phenotype
The mutant strain H433 was assessed in vitro for radial
growth, sporulation, and spore morphology. No significant
differences were observed when compared with wild-type
strain UPS9 (data not shown), demonstrating that the H433
strain is specifically altered for pathogenicity. When inocu-
lated on the susceptible cultivar of common bean La Victoire
(excised leaves or plantlets; see Methods), the mutant strain
was unable to produce anthracnose symptoms even after a
long incubation (Figure 1). Moreover, more detailed obser-
vations revealed that infection by mutant H433 reproducibly
led to the appearance of small necrotic spots similar to
those observed in a hypersensitive response
3 days afterinoculation. These necrotic spots were also observed after
inoculating another susceptible cultivar of common bean,
P12S, with strain H433 (see Methods). Figure 2 illustrates
the similarity of symptoms induced on two near-isogenic
lines of common bean (see Methods), the susceptible line
P12S (Figure 2A) and the resistant line P12R (Figure 2B) af-
ter inoculation with mutant strain H433.
These results show that mutant strain H433 displays a
double phenotype: it is a nonpathogenic strain, and it is able
to induce on the two susceptible cultivars tested local ne-
crotic reactions similar to those observed during an incom-
patible interaction.
Figure 1. Pathogenicity Tests of Mutant Strain H433 on the Sus-
ceptible Cultivar La Victoire of Common Bean.
(A) Excised cotyledonary leaves were inoculated with wild-type
strain UPS9 and mutant strain H433. Leaves were photographed 7
days after inoculation.
(B) Eight-day-old plantlets were spray-inoculated with either wild-
type strain UPS9 or mutant strain H433. Photographs were taken 7
days after inoculation.
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Regulation of Biotrophy/Necrotrophy Transition 1581
H433 Is Blocked at the Switch between the Biotrophic
and Necrotrophic Phases
To determine the step altered in the mutant strain during the
infection process, we first assessed mutant strain H433 for
appressorium formation on the hypocotyl epidermis. For
wild-type strain UPS9 and mutant strain H433, the majority
of conidia germinated and produced appressoria (data not
shown), showing that H433 is not altered at this critical step
of the infection process.
To study the next steps of the infection cycle, we con-
ducted cytological observations with hypocotyl segments of
the cultivar La Victoire. Bright-field microscopic observa-
tions confirmed the induction of cell death, as visualized by
brown epidermal cells (Figure 3A). The brown color corre-
sponds to the accumulation of phenolic compounds after
cell death (O’Connell et al., 1985). These observations also
confirmed the formation of normal-looking appressoria (Fig-
ure 3A). Nomarski differential interference contrast observa-
tions revealed that appressorium function appears to be
normal because
infection vesicles and primary hyphae de-
velop and grow normally in infected epidermal cells beneath
appressoria (Figure 3B). However, even after a long incuba-
tion (14 days after inoculation; Figure 3B), no secondary hy-
phae were visible. After the same length of incubation, wild-
type strain UPS9 had invaded infected tissue with second-
ary hyphae (data not shown).
Such observations were confirmed by comparing the hy-
phal development of wild-type strain UPS9 and H433 ininfection time courses of hypocotyl segments of the suscep-
tible cultivar La Victoire. Mean values of hyphal length for
different points of the infection time course (Figure 3C)
clearly indicate that the hyphal growth of mutant strain H433
is inhibited at 3 to 4 days after inoculation, compared with
the extensive hyphal development of strain UPS9. This inhi-
bition kinetics is consistent with the previous observations
that only primary hyphae were differentiated by the mutant
strain H433.
We have thus demonstrated that mutant strain H433 is
blocked after primary hyphae differentiation, at the transition
between the biotrophic and necrotrophic phases, which cor-
relates with the nonpathogenic phenotype. Infection by this
mutant strain is further characterized by the appearance of
small necrotic spots, similar to hypersensitive lesions, on thetwo susceptible cultivars of common bean that were tested.
Molecular Analysis of the pAN7-1 Integration Pattern
and Recovery of Sequences Flanking the Insertion Loci
DNA gel blot experiments were conducted to determine the
integration pattern of the vector pAN7-1 (Punt et al., 1987),
Figure 2. Pathogenicity Tests of Mutant Strain H433 on Two Near-
Isogenic Cultivars of Common Bean.
For both cultivars, excised cotyledonary leaves were inoculated with
mutant strain H433. Photographs were taken 7 days after inoculation.
(A) Susceptible cultivar P12S.
(B) Resistant cultivar P12R.
Figure 3. Development of Fungal Infection Structures during Inter-
action of Susceptible Cultivar La Victoire with Mutant Strain H433.
(A) and (B) Leaves before and after, respectively, treatment with
chloral hydrate. AP, appressorium; HP, primary hyphae; VI, infection
vesicle. Bars in (A) and (B) 10 m.
(C) Time course of hyphal growth (see Methods). Square data points
are mutant strain H433; circles are wild-type strain UPS9.
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1582 The Plant Cell
which was used in transformating wild-type strain UPS9 to
generate the mutants. Two hybridizing bands were detected
after KpnI restriction digestion (KpnI does not cut within the
transforming plasmid) of genomic DNA from the H433 mu-
tant strain, indicating that the vector had inserted in at least
two different loci in the genome (data not shown). Theshorter KpnI hybridizing band (
12 kb) is referred to here as
the first locus and is designated 433.1. The longer hybridiz-
ing band (
12 kb) is the second locus and is designated
433.2. The number and lengths of hybridizing bands ob-
served with HindIII and EcoRI restriction digests confirmed
this result and are consistent with the insertion of a single
copy at the 443.1 locus and with tandem integration of at
least two copies at the 433.2 locus (data not shown).
A marker rescue strategy was used to recover the se-
quences flanking each integration site. For 433.1, the re-
striction enzyme KpnI was used to subclone a 2.0-kb
BamHI-HindIII fragment, designated SF433.1 (for sequences
flanking locus 433.1); for 433.2, because of the integration
pattern in tandem, we used the restriction enzyme HindIIIand recovered only one side of the locus, a 1.1-kb HindIII-
PstI fragment, designated SF433.2. Both fragments were
used as probes for restriction digests of UPS9 genomic
DNA. The simple hybridizing patterns were consistent with
the presence of a unique copy of each of these sequences
in the genome of C. lindemuthianum
(data not shown). The
probes SF433.1 and SF433.2 were then used to screen a
UPS9 genomic library to recover complete regions corre-
sponding to those of the wild-type strain. Long EcoRI frag-
ments of 11.0 and 8.0 kb, for the loci 433.1 and 433.2,
respectively, were subcloned. Restriction maps and location
of pAN7-1 insertions are shown in Figures 4A and 4B.
Complementation of the Insertion at Locus 433.2
Partially Restores Pathogenicity
To determine which integration event of vector pAN7-1—at
locus 433.1, locus 433.2, or both loci—was responsible for
the H433 mutant phenotype, we set up functional comple-
mentation experiments. Two functional complementation
plasmids, designated p433.1Phleo and p433.2Phleo, were
constructed by introducing the corresponding 11.0- and
8.0-kb EcoRI fragments into the unique EcoRI site of vec-
tor pAN8-1, which confers phleomycin resistance (Mattern
et al., 1988). H433 protoplasts were independently trans-
formed with the constructed plasmids p433.1Phleo andp433.2Phleo. For each transformation experiment, we re-
covered transformants carrying both the mutant allele from
the recipient strain H433 and the complete wild-type allele
from the transforming plasmid.
To establish whether complementation by either of the
two genomic regions could restore pathogenicity, we inocu-
lated excised primary leaves of the susceptible cultivar La
Victoire with spore suspensions of each type of transfor-
mant (at least four strains of each type), wild-type strain
UPS9 (Figure 5A), and the original mutant strain H433
(Figure 5B). All of the strains transformed with plasmid
p433.1Phleo (e.g., T433.1.2; Figure 5C) had the same mutant
phenotype as that of recipient strain H433, demonstrating
that the integration event at locus 433.1 is not responsible
for the mutant phenotype. In contrast, the strains trans-formed with plasmid p433.2Phleo (e.g., T433.2.19; Figure
5D) partly recovered pathogenicity on excised primary
leaves: reduced anthracnose symptoms were evident on
leaves, with a slight delay of 24 hr for symptoms to develop,
as compared with symptom development on leaves inocu-
lated with UPS9. All of the strains transformed with plasmid
p433.2Phleo also retained the ability to induce the appear-
ance of small necrotic spots, as did the original mutant
H433 (data not shown). These results demonstrate that the
integration event at locus 433.2 is responsible for the mutant
phenotype. The same results were obtained by using a 4-kb
HindIII subclone (see Figure 4). This fragment was therefore
selected for further analysis.
Mutant H433 Is Affected in a Gene Encoding a Putative
Zn(II)
2
Cys
6
Transcriptional Activator
The 4130-bp HindIII fragment was sequenced on both
strands. The SF433.2 fragment was used as a probe to
screen a cDNA library generated from wild-type strain UPS9
grown on rich medium (see Methods). Two independent
cDNAs were also recovered and sequenced on both
strands. Nucleotide sequence analysis revealed the exist-
ence of an open reading frame of 2526 bp (including introns)
Figure 4. Location of pAN7-1 Insertions in the Wild-Type GenomicRegions Corresponding to Loci 433.1 and 433.2.
(A) Restriction map of the 433.1 locus.
(B) Restriction map of the 433.2 locus. The two boldface H’s indi-
cate the location of the 4-kb HindIII fragment used in the comple-
mentation experiments.
B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SacI; X, XhoI; Xb,
XbaI. For each locus, the location of the insertion event is indicated
by the arrowhead. The locations of the recovered flanking se-
quences SF433.1 and SF433.2 are indicated by bars.
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Regulation of Biotrophy/Necrotrophy Transition 1583
that potentially could encode a 746–amino acid polypeptide
having substantial homology with fungal transcriptional acti-
vators of the GAL4 family. This gene was designated
CLTA1
. Its nucleotide and deduced amino acid sequences
are presented in Figure 6.
The gene contains five introns, all of which exhibit the ex-
pected splice sites (Ballance, 1986). The putative transcrip-
tion start point is located 722 bp upstream of the initiation
codon, as determined by identical 5
ends for the two
cDNAs. The insertion event of vector pAN7-1 in the mutant
strain H433 occurred 303 bp upstream of the initiation
codon of the CLTA1 gene. This site is between the putative
transcription start point and the initiation codon of the
CLTA1
gene.Within the putative 746–amino acid polypeptide, three
major domains that are relatively conserved among the
many fungal transcriptional activators belonging to the zinc
cluster family can be identified. As shown in Figure 7, the first
one is a typical zinc binuclear cluster domain (Zn[II]
2
Cys
6
do-
main) located at the N terminus of the protein between resi-
dues 21 and 50 and may be a DNA binding domain. The
second, named MHR (for middle homology region), is less
conserved but is mostly specific for proteins belonging to the
zinc cluster family (Schjerling and Holmberg, 1996). The MHR
is found between amino acids 279 and 318 of the CLTA1
protein. The third domain, located at the C terminus of the
protein, is a possible activation domain that covers at least
95 amino acids from residue positions 605 to 700. In most
proteins of the zinc cluster family, the activation domain ispredominantly acidic. In the CLTA1 protein, this region is
glutamine, histidine, and proline rich (accounting for 39, 23,
and 25% of the amino acid content, respectively). Further
sequence analysis indicated that a putative dimerization ele-
ment (Suarez et al., 1995) is present between amino acids
67 and 80. By considering the first two protein domains,
CLTA1 seems to be more closely related to the Aspergillus
nidulans transcriptional activator UaY (Suarez et al., 1995),
having 56.7% identical residues within the zinc cluster do-
main (Figure 7) and 39.5% identity within the MHR. These
sequence features indicated that the CLTA1 protein is a po-
tential transcriptional activator belonging to the fungal zinc
cluster family.
Insertion of pAN7-1 at the 433.2 Locus Is Responsible
for the H433 Mutant Phenotype
Functional complementation experiments demonstrated
that the integration event at locus 433.2 is responsible for
blocking the H433 mutant at the switch between biotrophic
and necrotrophic phases. Final disease symptoms induced
by complemented strains on the susceptible cultivar La Vic-
toire were not as severe as those that occurred with the
wild-type strain (Figure 5). Therefore, we cannot exclude
that another mutation exists. If so, it would occur indepen-
dently of the insertion of vector pAN7-1 and would lead to
this additional mutant phenotype, which is masked by the
blockage between biotrophy and necrotrophy in the H433
mutant. To demonstrate that the insertion event in the
CLTA1
gene alone is responsible for the mutant phenotype,
we conducted gene disruption experiments. Two gene dis-
ruption vectors, p433.2A:hygro and p433.2B:hygro, contain-
ing the hygromycin resistance gene cassette flanked on
both sides by homologous sequences, were constructed as
shown in Figures 8A and 8C, respectively. The location of
the hygromycin resistance cassette in p433.2A:hygro (Fig-
ure 8A) was chosen to reproduce the insertion event at lo-
cus 433.2 in mutant strain H433. In p433.2B:hygro, an
internal fragment of the CLTA1
coding sequence was de-
leted and replaced by the hygromycin resistance cassette togenerate a null mutant allele (Figure 8C). These disruption
vectors were used independently to transform protoplasts of
wild-type strain UPS9. For each transformation experiment,
60 hygromycin-resistant transformants were arbitrarily se-
lected for DNA gel blot analysis. After transformation with
p433.2A:hygro, three of the 60 hygromycin-resistant trans-
formants analyzed were found to carry the mutant allele
introduced by transformation, as determined by the switch
of the 4.0-kb fragment present in the genomic DNA of
Figure 5. Pathogenicity of Complemented Transformants on the
Susceptible Cultivar La Victoire of Common Bean.
Excised cotyledonary leaves were inoculated using the following
strains. Photographs were taken 7 days after inoculation.
(A) Wild-type strain UPS9.
(B) Mutant strain H433.
(C) Complemented strain T433.1.2.
(D) Complemented strain T433.2.19.
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1584 The Plant Cell
wild-type strain UPS9 to a 6.5-kb fragment in the disrupted
strains (Figure 8B). For the second disruption plasmid,
p433.2B:hygro, two of the 60 hygromycin-resistant transfor-
mants analyzed were disrupted (Figure 8D).
Excised primary leaves of the susceptible cultivar La
Victoire were inoculated with spore suspensions of the dis-rupted strains (e.g., R433.2.16, a p433.2A:hygro trans-
formant; Figure 9C), corresponding ectopic transformants,
wild-type strain UPS9 (Figure 9A), and mutant strain H433
(Figure 9B). The three disrupted strains obtained with the
p433.2A:hygro plasmid had exactly the same phenotype as
mutant strain H433 (R433.2.16; Figure 9C), whereas the ec-
topic transformants retained their ability to induce anthrac-
nose symptoms (data not shown). Identical results were
observed with disruptants resulting from transformation with
the p433.2B:hygro plasmid (data not shown). Therefore, dis-
ruption of the CLTA1
with either of the two mutant alleles is
sufficient to reproduce the H433 mutant phenotype.
Complementation of CLTA1
-Disrupted Strains Leads to
Complete Restoration of Pathogenicity
Complementation of H433 did not fully restore pathogenic-
ity, suggesting that the mutant possessed a virulence defect
independent of the phenotype of the clta1
mutant. Conse-
quently, similar functional complementation experiments us-
ing the 4-kb HindIII fragment (see Figure 4) were performed
with a disrupted strain (R433.2.41, a p433.2A:hygro trans-
formant) as a recipient strain. Complemented transformants
were recovered and assessed for pathogenicity on excised
cotyledonary leaves of the susceptible cultivar La Victoire.
Complemented transformants were able to induce theappearance of anthracnose symptoms as rapidly and exten-
sively as did the wild-type strain (data not shown). The dif-
ference between complementation of H433 and R433.2.41
is discussed later.
Figure 6. Nucleotide and Predicted Amino Acid Sequences of the
C. lindemuthianum CLTA1 Gene.
Nucleotides are numbered with reference to the putative transcrip-
tion start point (indicated by the asterisk). The location of the pAN7-1
insertion is shown by the arrow. The locations of the forward primer(1821 to 1847) and the reverse primer (2267 to 2244) are underlined.
Introns are presented in lowercase letters. The putative Zn(II)2Cys6
DNA binding domain is indicated in boldface. The nucleotide se-
quence of the C. lindemuthianum CLTA1 gene has GenBank acces-
sion number AF190427.
Figure 7. Sequence Alignment of Zn(II)2Cys6 Domains from CLTA1
and from Other Fungal Zinc Cluster Transcriptional Activators.
Identical residues are indicated by asterisks and similar residues by
dots. The UaY and FACB genes were isolated from A. nidulans
(Suarez et al., 1995; Todd et al., 1997). The AMDR gene was isolated
from A. oryzae (Wang et al., 1992). The GAL4 gene was isolated
from Saccharomyces cerevisiae (Laughon and Gesteland, 1984). Se-
quence alignment was determined by using the CLUSTALV algo-
rithm (Higgins et al., 1992).
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Regulation of Biotrophy/Necrotrophy Transition 1585
Expression Analysis of the CLTA1 Gene in Fungal
Cultures from the Wild-Type and Mutant Strains
The expression of the CLTA1 gene was first examined in
pure cultures of wild-type strain UPS9 grown in either com-
plete or nitrogen-depleted medium. The latter condition was
tested because it reflects leaf conditions at the beginning of
a pathogen’s infection cycle. Expression has been demon-
strated in several cases in which pathogenicity or avirulence
genes were shown to be induced under nitrogen or car-
bon starvation conditions (Talbot et al., 1993; Van den
Ackerveken et al., 1994). RNA gel blots prepared with either
Figure 8. Gene Disruptions of the 433.2 Locus.
(A) p433.2A:hygro contains the hygromycin resistance gene cassette (a 3638-bp XhoI-SpeI fragment from the pAN7-1 plasmid demonstrated byPunt et al. [1990] to be sufficient to confer hygromycin resistance) flanked with border sequences of the wild-type genomic locus. The left and
right flanking regions are 260 bp (HindIII-SnaBI fragment) and 3.4 kb (XhoI-HindIII fragment), respectively.
(B) DNA gel blot analysis of the disrupted transformants recovered after transformation with p433.2A:hygro. Total genomic DNAs of the wild-
type strain (lane 1), one ectopic transformant (lane 2), and the two disrupted transformants obtained (lanes 3 and 4) were digested with HindII
and probed with a 363-bp KpnI-SacI fragment, indicated as probe in (A).
(C) p433.2B:hygro was constructed by replacing a 759-bp SacI-HindIII fragment located at the 3 end of the CLTA1 open reading frame by the
hygromycin resistance gene cassette (a 2704-bp SacI-HindIII fragment from the pAN7-1 vector).
(D) DNA gel blot analysis of the disrupted transformants recovered after transformation with p433.2B:hygro. Total genomic DNAs of the wild-
type strain (lane 1), two ectopic transformants (lanes 2 and 3), and the two disrupted transformants obtained (lanes 4 and 5) were digested with
HindIII and probed with a 363-bp KpnI-SacI fragment, indicated as the probe in (C).
As expected in both disruption experiments, the native 4.0-kb band of strain UPS9 remained in the ectopic transformants but was no longer
present in the disrupted strains. B, BamHI; H, HindIII; K, KpnI; P, PstI; S, SacI; Sn, SnaBI; X, XhoI. Homologous recombination through a double
crossover event (denoted by crossed lines) results in the replacement of a part of the wild-type region by the hygromycin resistance gene ( hph ).
The curved lines represent the genome. The small arrowheads indicate the location of the pAN7-1 insertion sites. Asterisks in (A) and (C) denote
borders of the restriction fragments deleted in the disruption vectors. Numbers at left in (B) and (D) indicate lengths in kilobases.
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1586 The Plant Cell
20
g of total RNA or 2 g of mRNA were probed with the
363-bp KpnI-SacI internal fragment. No hybridization signal
was observed in total RNA, even after long exposure, in any
sample (data not shown). Thus, we concluded that expres-
sion of the CLTA1 gene was too weak to allow detection with
total RNA. A weak hybridization signal corresponding to the
expected size, 3.0 kb, was observed in mRNA (Figure 10A).
Signal intensity was not markedly different under the two
growth conditions previously described (Figure 10A, lanes 1
and 2), indicating that CLTA1 is expressed constitutively at a
very low level in pure cultures of wild-type strain UPS9 and is
probably not induced under nitrogen starvation.
Because of its location within the promoter region, the in-
sertion event within the CLTA1 gene (Figure 6) could have
led either to a null mutation or to a deregulation of expres-
sion. We thus investigated the consequences of such an
event on the expression of the CLTA1 gene in mutant strain
H433. RNA gel blot analysis was conducted with poly(A)
RNA of mutant strain H433 grown on complete medium. No
hybridization signal was detected, indicating that the gene
does not appear to be expressed in the mutant strain (Figure
10A). Using the more sensitive method of reverse transcrip-
tion–polymerase chain reaction (RT-PCR), no amplification
product specific for the CLTA1 gene was obtained from total
RNA of the mutant strain, whereas product was clearly ob-
served when total RNA of wild-type strain UPS9 (Figure 10B)
was amplified. These results confirm that the insertion eventof vector pAN7-1 in H433 resulted in nonexpression of the
CLTA1 gene in pure fungal cultures.
DISCUSSION
In this study, we have characterized a nonpathogenic mu-
tant derived from C. lindemuthianum, designated H433. This
nonpathogenic strain is able to induce local necrotic reac-
tions in susceptible cultivars of common bean. These reac-
tions are similar to those resulting from an incompatible
interaction between an avirulent strain of C. lindemuthianum
and a corresponding resistant genotype of common bean.
Cytological studies have shown that this mutant strain is
unable to differentiate secondary hyphae, which allow
necrotrophic growth in a basic compatible interaction
(O’Connell et al., 1985; Bailey et al., 1992). H433 is therefore
a mutant blocked at the transition between biotrophy and
necrotrophy.
An interesting aspect to the phenotype is the ability
of mutant strain H433 to induce on susceptible cultivars lo-
cal necrotic spots very similar to hypersensitive lesions
(O’Connell et al., 1985). The ability of biotrophic pathogens
not to induce host cell death in a compatible interaction has
been discussed by Morel and Dangl (1997). Three hypothe-
ses can be used to explain the mutant phenotype of strain
H433. The ability to induce local necrotic spots could result
from (1) the deregulated production of an elicitor molecule
(which is nonspecific because the same reaction was ob-
served with different susceptible genotypes of common
bean); (2) a defect in the suppression of defense responses
leading to cell death; or (3) the inability of the fungus to
achieve its developmental program within the plant, allowing
the completion of nonspecific host defense responses.
These hypotheses will be tested in future work.Molecular analyses allowed us to identify the CLTA1
gene, which encodes a potential 746-residue polypeptide
and is responsible for the phenotype observed in H433.
Gene disruption experiments demonstrated that inactivation
of CLTA1 results in the exact same phenotype as that of
mutant strain H433, proving that CLTA1 is a pathogenicity
gene. Expression of CLTA1 in pure cultures of the fungus is
very low. Preliminary experiments did not detect any induc-
tion during infection. In mutant strain H433, the insertion
Figure 9. Pathogenicity of the CLTA1 Disruptants on the Susceptible Cultivar La Victoire of Common Bean.
Excised cotyledonary leaves were inoculated with the following strains. Photographs were taken 7 days after inoculation.
(A) Wild-type strain UPS9.
(B) Mutant strain H433.
(C) Disrupted strain R433.2.16.
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Regulation of Biotrophy/Necrotrophy Transition 1587
event of vector pAN7-1 led to nonexpression of CLTA1 in
pure cultures. Complementation of the mutant strain by the
corresponding region led to weak anthracnose symptoms,
whereas full restoration of pathogenicity was observed
when a CLTA1-disrupted strain was the recipient. These re-
sults show that most likely another mutation in mutant strainH433 (possibly the second pAN7-1 insertion), which does
not itself affect the infection process, does not allow the full
recovery of wild-type pathogenicity.
These results indicate that the CLTA1 gene encodes a pu-
tative GAL4-like transcriptional activator. The deduced
amino acid sequence of the CLTA1 protein suggests that it
contains the three functional domains characteristic of the
GAL4-like family of transcriptional activators (Schjerling and
Holmberg, 1996): the C6 zinc cluster involved in DNA bind-
ing, an MHR, and a possible transcriptional activation do-
main. In Fusarium solani f sp pisi, another Zn(II)2Cys6protein, CTF1, involved in transcriptional activation of a cuti-
nase-encoding gene, was identified (Li and Kolattukudy,
1997). However, gene replacement experiments still need to
be performed to demonstrate its role in fungal pathogenicity.
Identifying the CLTA1 gene will lead to identification of the
regulatory pathway and of the genes controlling the transi-
tion between biotrophy and necrotrophy. Most of the fungal
transcriptional activators belonging to the zinc cluster family
control catabolic pathways: FACB in A. nidulans controls
the expression of acetate utilization genes (Todd et al.,
1997), NIT4 in Neurospora crassa mediates nitrate induction
(Yuan et al., 1991), and UaY in A. nidulans regulates purine
utilization (Suarez et al., 1995). During the infection process
of hemibiotrophic fungi, entry into the necrotrophic phaseinvolves major nutritional changes, in particular the ability of
the pathogen to utilize plant constituents by activating cata-
bolic pathways. One hypothesis is that the putative CLTA1
transcriptional regulator controls genes that allow necro-
trophic growth on infected plant tissues.
In C. lindemuthianum, two mutants obtained by random
insertional mutagenesis have been characterized. Their
genes encode a serine/threonine protein kinase and a puta-
tive GAL4-like transcriptional activator (Dufresne et al., 1998;
this study). Each mutant was demonstrated to be affected at
a key step of the infection process: the first, H290, is affected
at the penetration step, whereas H433 is blocked at the
switch between biotrophy and necrotrophy. Both studies
demonstrate that random insertional mutagenesis is a pow-
erful tool to isolate pathogenicity determinants of plant
pathogenic fungi. A comparative analysis with results from
the other pathosystems should lead to a better knowledge of
the infection processes of aerial fungal pathogens.
METHODS
Strains, Media, and Growth of Colletotrichum lindemuthianum
Colletotrichum lindemuthianum UPS9 (Fabre et al., 1995) was used
as the wild-type recipient strain in this study. Growth conditions andmedia are as described in Dufresne et al. (1998).
Bean Cultivars and Near-Isogenic Lines
The common bean ( Phaseolus vulgaris ) cultivar La Victoire (Tezier,
Valence-sur-Rhone, France) was used as the susceptible cultivar in
all pathogenicity assays. A pair of near-isogenic common bean lines,
P12S and P12R, differing only by the resistant gene present in P12R,
Figure 10. CLTA1 Expression in the Wild Type, Mutant Strain H433,
and Corresponding Complemented or Replaced Strains.
(A) RNA gel blot analysis. Samples (2 g per lane) were fractionatedon a formaldehyde–agarose gel and transferred to a nylon mem-
brane (see Methods). The blot was hybridized with the 363-bp KpnI-
SacI fragment. Lanes 1 and 2, wild-type strain UPS9 grown in com-
plete or minimal medium, respectively. Lanes 3 to 6, samples ex-
tracted from cultures grown in complete medium: lane 3, mutant
strain H433; lane 4, complemented strain T433.2.19; lane 5, another
H433-complemented strain; and lane 6, replaced strain R433.2.16.
The 3.3 at left indicates the length of the hybridizing band in kilo-
bases.
(B) Ethidium bromide–stained RT-PCR products corresponding to
CLTA1-specific transcripts. These experiments were conducted
with 4 g of total cellular RNA. For fungal samples, total cellular
RNA was extracted from culture grown in complete medium, except
for those in lanes 3 and 5, which correspond to the wild-type strain
UPS9 and the mutant strain H433, respectively, grown in nitrogen-
starved medium. Two amplification products were observed, one
corresponding to the genomic DNA amplification product (447 bp)
and the other corresponding to the mRNA amplification product
(398 bp). Lane 1, susceptible cultivar La Victoire (plant negative con-
trol); lanes 2 and 3, wild-type strain UPS9 grown in complete or min-
imal medium, respectively; lanes 4 and 5, mutant strain H433 grown
in complete or minimal medium, respectively; lane 6, replaced strain
R433.2.16; and lane 7, genomic DNA of the wild-type strain UPS9
(DNA control). Numbers at left denote the lengths of the different
amplification products in base pairs.
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1588 The Plant Cell
were used to demonstrate the similarity of the symptoms induced by
mutant strain H433 on susceptible and resistant cultivars (Geffroy et
al., 1998).
Infection Assays, Light Microscopy, and Induction of
Appressorium Formation
The screening procedure for identification of the altered pathogenic-
ity mutants, the pathogenicity tests, and the assays for induction of
appressorium formation were performed as previously described
(Dufresne et al., 1998). For cytological observations, infection as-
says with excised hypocotyl segments were used as described in
O’Connell et al. (1985). Hypocotyl segments of the susceptible culti-
var La Victoire were inoculated with a spore suspension of the mu-
tant strain H433 at a concentration of 5 103 spores mL1. Strips of
epidermal tissue were sampled from beneath the site at which inoc-
ulation droplets were applied and were directly mounted in distilled
water for bright-field microscopy.
For microscopic observations using Nomarski differential interfer-
ence contrast, strips were cleared in a solution of chloral hydrate
(2.5 g mL1 ) before being mounted in distilled water. Hyphal devel-opment was measured as follows. For each strain, wild-type strain
UPS9 and mutant strain H433, nine hypocotyl segments of La Vic-
toire plantlets were inoculated with a spore suspension at a concen-
tration of 5 105 spores mL1. Subsequently, for each hypocotyl,
one infection site was arbitrarily selected for microscopic examina-
tion. At each infection site, the length of hyphae produced from 270
appressoria was measured 2, 3, 4, 6, and 8 days after inoculation.
Nucleic Acid Manipulation and Fungal Transformation
Genomic DNA extractions were performed according to Ross (1995).
DNA gel blot analysis was conducted as described in Dufresne et al.
(1998). Total cellular RNA was extracted according to the procedure
described in Vallélian-Bindschedler et al. (1998). RNA samples wereseparated by formaldehyde–agarose gel electrophoresis and trans-
ferred to nylon membranes N (Amersham France SA, Les Ulis,
France) by capillary elution (Sambrook et al., 1989). Hybridizations
were conducted as previously described (Dufresne et al., 1998).
mRNA was isolated from total RNA by using magnetic Dynabeads
oligo(dT)25 (Dynal, Compiègne, France) according to the manufac-
turer’s instructions.
Reverse transcription–polymerase chain reaction (RT-PCR) exper-
iments were conducted with the Ready-To-Go RT-PCR beads kit
(Pharmacia Biotech S.A., Saint-Quentin en Yvelines, France). Each
reaction used 4 g of total RNA and was conducted according to the
manufacturer’s instructions with 2 mM MgCl2 (final concentration).
The two primers—forward, 5-cgcagtacgccatgttggaccccgtcgc-3;
and reverse, 5-GCCCAATAAAGGACGCTGCGTCAGG-3 —were de-
signed for specific amplification of CLTA1 transcripts and were usedat an annealing temperature of 60C.
Construction and Screening of the cDNA Library
A C. lindemuthianum cDNA library was constructed in vector ZAP
Express by using the ZAP-cDNA Synthesis Kit (Stratagene, La Jolla,
CA) and mRNA of wild-type strain UPS9 grown in liquid potato dex-
trose medium. cDNA synthesis was conducted according to the
manufacturer’s instructions. After in vitro packaging with the Giga-
packII Gold Packaging Extract Kit (Stratagene), recombinant phages
were infected in Escherichia coli XL1Blue-MRF (Sambrook et al.,
1989). The complexity of the library before amplification was 1.2
106 recombinants. For amplification steps and screening, E. coli
Q358 (Sambrook et al., 1989) was used. The number of plaque-form-
ing units plated for the screening was approximately three to four
times the initial number of recombinants. Screening was performedaccording to standard procedures (Sambrook et al., 1989).
DNA Sequencing and Sequence Analysis
Nucleotide sequences of the genomic region and cDNA of the
CLTA1 gene were determined by using ABI PRISM Big Dye Termina-
tor cycle sequencing (Perkin-Elmer Corp., Norwalk, CT). Sequencing
reactions were separated on an Applied Biosystems (Foster City, CA)
AB377 autosequencer. To search for homologies with known se-
quences in the data banks, we used the BLAST program (Altschul et
al., 1990). Multiple sequence alignments were determined using the
CLUSTAL V software (Higgins et al., 1992).
ACKNOWLEDGMENTS
We thank John P. Morrissey and Richard Laugé for critical reading of
the manuscript and the anonymous reviewers for their helpful advice.
This work was supported by the Centre National pour la Recherche
Scientifique and the Université Paris-Sud.
Received January 10, 2000; accepted June 14, 2000.
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DOI 10.1105/tpc.12.9.15792000;12;1579-1590Plant Cell
Marie Dufresne, Sarah Perfect, Anne-Laure Pellier, John A. Bailey and Thierry Langinon Common BeanColletotrichum lindemuthianumInfection Process of
A GAL4-like Protein Is Involved in the Switch between Biotrophic and Necrotrophic Phases of the
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