- © 2000 American Society of Plant Physiologists
Abstract
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]2Cys6) 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. Complementation of these disrupted strains by the CLTA1 gene led to full restoration of pathogenicity. This study demonstrates 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 provide a comprehensive explanation of the mechanisms involved 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 pathogenic fungi (Bölker et al., 1995; Dufresne et al., 1998; Sweigard et al., 1998; Balhadère et al., 1999). A major advantage of such a strategy is that there is no a priori postulate 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 anthracnose on common bean. This interaction has been assumed to fit the gene-for-gene hypothesis (Flor, 1971), but genetic studies have been performed only for the plant partner (Young and Kelly, 1996; Geffroy et al., 1998). Phenotypically, the infection of a resistant cultivar of common bean by a corresponding avirulent strain of C. lindemuthianum (incompatible interaction) leads to the appearance of localized necrotic spots typical of a classical hypersensitive response. The infection process has been well documented in previous cytological studies (O’Connell et al., 1985; Bailey et al., 1992). Basic compatibility, that is, interaction between a 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 differentiate to form an infection structure devoted to mechanical penetration, the appressorium. After the penetration step, the infection cycle is characterized by two successive phases. In the first phase, lasting 3 to 4 days, the fungus grows biotrophically inside the infected epidermal cells. During this phase, referred to as the biotrophic phase, the fungus differentiates infection vesicles and primary hyphae. The second phase, which corresponds to the appearance of anthracnose symptoms, is completed 6 to 8 days after inoculation. 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 hemibiotrophic 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 manipulated in vitro, in contrast to the case with strictly biotrophic (obligate) pathogens.
A random insertional mutagenesis technique was developed in our laboratory to identify essential virulence determinants of the fungus (Dufresne et al., 1998). Nine less-pathogenetic mutants were recovered from 1200 transformants tested. The molecular analysis of mutant strain H290 allowed us to identify the first C. lindemuthianum pathogenicity gene, CLK1, which encodes a putative serine/threonine kinase and is involved in the regulation of appressorium function (Dufresne et al., 1998).
We report here the cytological and molecular characterization of one nonpathogenic mutant, designated H433. We first show that this path– mutant is blocked during the infection process at the switch between biotrophy and necrotrophy. Then, we demonstrate that the mutant phenotype is related to the interruption of the CLTA1 gene (for C. lindemuthianum transcriptional activator 1), which encodes a putative GAL4-like transcriptional activator. These studies allowed us to identify and characterize a regulatory gene involved at the transition between biotrophic and necrotrophic phases, a key step in the infection process of the hemibiotrophic 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 inoculated 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 observations 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 after inoculation. 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) after 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 necrotic reactions similar to those observed during an incompatible interaction.
Pathogenicity Tests of Mutant Strain H433 on the Susceptible 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.
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.
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 conducted cytological observations with hypocotyl segments of the cultivar La Victoire. Bright-field microscopic observations confirmed the induction of cell death, as visualized by brown epidermal cells (Figure 3A). The brown color corresponds to the accumulation of phenolic compounds after cell death (O’Connell et al., 1985). These observations also confirmed the formation of normal-looking appressoria (Figure 3A). Nomarski differential interference contrast observations revealed that appressorium function appears to be normal because infection vesicles and primary hyphae develop and grow normally in infected epidermal cells beneath appressoria (Figure 3B). However, even after a long incubation (14 days after inoculation; Figure 3B), no secondary hyphae were visible. After the same length of incubation, wild-type strain UPS9 had invaded infected tissue with secondary hyphae (data not shown).
Such observations were confirmed by comparing the hyphal development of wild-type strain UPS9 and H433 in infection time courses of hypocotyl segments of the susceptible 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 inhibition 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 correlates with the nonpathogenic phenotype. Infection by this mutant strain is further characterized by the appearance of small necrotic spots, similar to hypersensitive lesions, on the two 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), 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 mutant strain, indicating that the vector had inserted in at least two different loci in the genome (data not shown). The shorter KpnI hybridizing band (∼12 kb) is referred to here as the first locus and is designated 433.1. The longer hybridizing band (>12 kb) is the second locus and is designated 433.2. The number and lengths of hybridizing bands observed 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).
Development of Fungal Infection Structures during Interaction 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 μm.
(C) Time course of hyphal growth (see Methods). Square data points are mutant strain H433; circles are wild-type strain UPS9.
A marker rescue strategy was used to recover the sequences flanking each integration site. For 433.1, the restriction 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 HindIII and 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 corresponding to those of the wild-type strain. Long EcoRI fragments 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 complementation 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 vector pAN8-1, which confers phleomycin resistance (Mattern et al., 1988). H433 protoplasts were independently transformed with the constructed plasmids p433.1Phleo and p433.2Phleo. For each transformation experiment, we recovered 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 inoculated excised primary leaves of the susceptible cultivar La Victoire with spore suspensions of each type of transformant (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 transformed 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 inoculated with UPS9. All of the strains transformed with plasmid p433.2Phleo also retained the ability to induce the appearance 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)2Cys6 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 existence of an open reading frame of 2526 bp (including introns) that potentially could encode a 746–amino acid polypeptide having substantial homology with fungal transcriptional activators of the GAL4 family. This gene was designated CLTA1. Its nucleotide and deduced amino acid sequences are presented in Figure 6.
Location of pAN7-1 Insertions in the Wild-Type Genomic Regions 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 indicate the location of the 4-kb HindIII fragment used in the complementation 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 sequences SF433.1 and SF433.2 are indicated by bars.
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.
The gene contains five introns, all of which exhibit the expected splice sites (Ballance, 1986). The putative transcription 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]2Cys6 domain) located at the N terminus of the protein between residues 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 is predominantly 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 element (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 domain (Figure 7) and 39.5% identity within the MHR. These sequence features indicated that the CLTA1 protein is a potential 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 Victoire 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 independently 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 disruption vectors, p433.2A:hygro and p433.2B:hygro, containing 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 (Figure 8A) was chosen to reproduce the insertion event at locus 433.2 in mutant strain H433. In p433.2B:hygro, an internal fragment of the CLTA1 coding sequence was deleted and replaced by the hygromycin resistance cassette to generate 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 selected for DNA gel blot analysis. After transformation with p433.2A:hygro, three of the 60 hygromycin-resistant transformants 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 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 transformants analyzed were disrupted (Figure 8D).
Nucleotide and Predicted Amino Acid Sequences of the C. lindemuthianum CLTA1 Gene.
Nucleotides are numbered with reference to the putative transcription 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 sequence of the C. lindemuthianum CLTA1 gene has GenBank accession number AF190427.
Excised primary leaves of the susceptible cultivar La Victoire were inoculated with spore suspensions of the disrupted strains (e.g., R433.2.16, a p433.2A:hygro transformant; 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 ectopic transformants retained their ability to induce anthracnose symptoms (data not shown). Identical results were observed with disruptants resulting from transformation with the p433.2B:hygro plasmid (data not shown). Therefore, disruption 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 pathogenicity, suggesting that the mutant possessed a virulence defect independent of the phenotype of the clta1 mutant. Consequently, similar functional complementation experiments using the 4-kb HindIII fragment (see Figure 4) were performed with a disrupted strain (R433.2.41, a p433.2A:hygro transformant) 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 the appearance of anthracnose symptoms as rapidly and extensively as did the wild-type strain (data not shown). The difference between complementation of H433 and R433.2.41 is discussed later.
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). Sequence alignment was determined by using the CLUSTALV algorithm (Higgins et al., 1992).
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 by Punt 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.
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 complete 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 demonstrated in several cases in which pathogenicity or avirulence genes were shown to be induced under nitrogen or carbon starvation conditions (Talbot et al., 1993; Van den Ackerveken et al., 1994). RNA gel blots prepared with either 20 μg of total RNA or 2 μg 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 expression 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.
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.
Because of its location within the promoter region, the insertion event within the CLTA1 gene (Figure 6) could have led either to a null mutation or to a deregulation of expression. 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 transcription–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 observed when total RNA of wild-type strain UPS9 (Figure 10B) was amplified. These results confirm that the insertion event of 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 mutant derived from C. lindemuthianum, designated H433. This nonpathogenic strain is able to induce local necrotic reactions in susceptible cultivars of common bean. These reactions 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 local 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 hypotheses 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 observed 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 induction during infection. In mutant strain H433, the insertion 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 results show that most likely another mutation in mutant strain H433 (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 putative 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 binding, an MHR, and a possible transcriptional activation domain. In Fusarium solani f sp pisi, another Zn(II)2Cys6 protein, CTF1, involved in transcriptional activation of a cutinase-encoding gene, was identified (Li and Kolattukudy, 1997). However, gene replacement experiments still need to be performed to demonstrate its role in fungal pathogenicity.
CLTA1 Expression in the Wild Type, Mutant Strain H433, and Corresponding Complemented or Replaced Strains.
(A) RNA gel blot analysis. Samples (2 μg per lane) were fractionated on a formaldehyde–agarose gel and transferred to a nylon membrane (see Methods). The blot was hybridized with the 363-bp KpnI-SacI fragment. Lanes 1 and 2, wild-type strain UPS9 grown in complete or minimal medium, respectively. Lanes 3 to 6, samples extracted 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 kilobases.
(B) Ethidium bromide–stained RT-PCR products corresponding to CLTA1-specific transcripts. These experiments were conducted with 4 μg 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 control); lanes 2 and 3, wild-type strain UPS9 grown in complete or minimal 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.
Identifying the CLTA1 gene will lead to identification of the regulatory pathway and of the genes controlling the transition 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 phase involves major nutritional changes, in particular the ability of the pathogen to utilize plant constituents by activating catabolic pathways. One hypothesis is that the putative CLTA1 transcriptional regulator controls genes that allow necrotrophic 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 putative 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 powerful 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 and media 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, 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 pathogenicity 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 assays with excised hypocotyl segments were used as described in O’Connell et al. (1985). Hypocotyl segments of the susceptible cultivar La Victoire were inoculated with a spore suspension of the mutant strain H433 at a concentration of 5 × 103 spores mL–1. Strips of epidermal tissue were sampled from beneath the site at which inoculation droplets were applied and were directly mounted in distilled water for bright-field microscopy.
For microscopic observations using Nomarski differential interference contrast, strips were cleared in a solution of chloral hydrate (2.5 g mL–1) before being mounted in distilled water. Hyphal development was measured as follows. For each strain, wild-type strain UPS9 and mutant strain H433, nine hypocotyl segments of La Victoire plantlets were inoculated with a spore suspension at a concentration of 5 × 105 spores mL–1. Subsequently, for each hypocotyl, one infection site was arbitrarily selected for microscopic examination. 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 were separated by formaldehyde–agarose gel electrophoresis and transferred 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 manufacturer’s instructions.
Reverse transcription–polymerase chain reaction (RT-PCR) experiments were conducted with the Ready-To-Go RT-PCR beads kit (Pharmacia Biotech S.A., Saint-Quentin en Yvelines, France). Each reaction used 4 μg 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 designed for specific amplification of CLTA1 transcripts and were used at an annealing temperature of 60°C.
Construction and Screening of the cDNA Library
A C. lindemuthianum cDNA library was constructed in vector λZAP Express by using the ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA) and mRNA of wild-type strain UPS9 grown in liquid potato dextrose 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-forming units plated for the screening was approximately three to four times the initial number of recombinants. Screening was performed according 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 Terminator 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 sequences 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.
- Published September 1, 2000.
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