CLNR1, the AREA/NIT2-like global nitrogen regulator of the plant fungal pathogen Colletotrichum lindemuthianum is required for the infection cycle

Authors


Summary

Nitrogen starvation is generally assumed to be encountered by biotrophic and hemibiotrophic plant fungal pathogens at the beginning of their infection cycle. We tested whether nitrogen starvation constitutes a cue regulating genes that are required for pathogenicity of Colletotrichum lindemuthianum, a fungal pathogen of common bean. The clnr1 (C. lindemuthianumnitrogen regulator 1) gene, the areA/nit-2 orthologue of C. lindemuthianum, was isolated. The predicted CLNR1 protein exhibits high amino acid sequence similarities with the AREA and NIT2 global fungal nitrogen regulators. Targeted clnr1 mutants are unable to use a wide array of nitrogen sources, indicating that clnr1 is the C. lindemuthianum major nitrogen regulatory gene. The clnr1 mutants are non-pathogenic, although few anthracnose lesions seldom occur on whole plantlets. Surprisingly, cytological analysis reveals that the clnr1 mutants are not disturbed from the penetration stage until the end of the biotrophic phase, but that they are impaired during the setting up of the necrotrophic phase. Thus, through CLNR1, nitrogen starvation constitutes a cue for the regulation of genes that are compulsory for this stage of the C. lindemuthianum infection process. Additionally, clnr1 mutants complemented with the Aspergillus nidulans areA gene are fully pathogenic, indicating that areA is able to activate the C. lindemuthianum suited genes, normally under the control of clnr1.

Introduction

Plant microbial pathogens must overcome large variations in environmental conditions during their infection cycle on the host. They need to respond and adapt to these changes in order to ensure the completion of their infection cycle. For complex pathogens such as most plant pathogenic fungi, the infection process requires the preparation and the development of the suited infection structures at the right time and the right place, which suggests a tight regulation of in planta fungal development. This regulation may be accomplished through specific fungal regulators acting according to a precise developmental programme. Furthermore, this regulation may be accomplished through global fungal regulators activated by environmental conditions recruited as precise cues for the expression of genes required for each step in the infection process. Nutrient availability is one of the most plausible environmental cue candidates during plant pathogen interactions. Several studies have suggested that lack of nutrients is one of the signals controlling the expression of genes involved in the pathogenicity of various plant microbial pathogens (Snoeijers et al., 2000).

Carbon and/or nitrogen starvation has been reported to induce or increase the expression of a number of in planta-induced genes of several fungal plant pathogens. For example, the mpg1 gene of the rice blast hemibiotrophic fungus Magnaporthe grisea encodes a hydrophobin-like protein involved in appressorium formation that is necessary for pathogenicity and has been shown to be expressed in vitro under carbon and nitrogen starvation conditions (Talbot et al., 1993). Similarly, the expression of the avirulence gene avr9 of the biotrophic fungal tomato pathogen Cladosporium fulvum is induced under nitrogen starvation but not under carbon starvation (Van den Ackerveken et al., 1994). Independently, nitrogen starvation has been widely used as a screen for the isolation of fungal genes that might be specifically induced in planta and/or involved in the pathogenicity of plant pathogenic fungi. Examples of such genes come from Colletotrichum gloeosporioides, which is a hemibiotrophic fungal pathogen of tropical legumes. The CgGS gene and the CgDN3 gene, which encode a glutamine synthetase and a product of unknown function, respectively, were obtained through a screen for genes that are specifically induced in vitro under nitrogen starvation (Stephenson et al., 1997; 2000). These genes have been shown to be expressed in planta during the early stages of the C. gloeosporioides infection process. Moreover, a CgDN3-targeted mutant is non-pathogenic, and data indicate that the CgDN3 gene is necessary for the early phases of the C. gloeosporioides infection process. Similarly, the pSI-9 and pSI-10 genes of the fungal tomato pathogen Cladosporium fulvum that encode an aldehyde dehydrogenase and an alcohol dehydrogenase, respectively, were identified through their specific expression under nitrogen-limiting conditions in vitro and were subsequently demonstrated to be expressed in planta (Coleman et al., 1997). Overall, it is frequently assumed that biotrophic and hemibiotrophic fungal pathogens encounter nitrogen-limiting conditions at the beginning of their infection process and that nitrogen starvation constitutes one of the signals involved in the regulation of genes that are induced in planta (Snoeijers et al., 2000).

Fungi are able to use a wide array of nitrogen-containing compounds (Marzluf, 1997). In the saprophytic filamentous fungi Aspergillus nidulans and Neurospora crassa, nitrogen nutrition via the utilization of this variety of sources is mediated by the major positive control regulator genes areA (Caddick et al., 1986) and nit-2 (Stewart and Vollmer, 1986) respectively. Both genes display highly similar sequences and encode transcriptional factors of the GATA family (Scazzocchio, 2000). All members of this family carry a DNA-binding domain made of a single Cys-2/Cys-2 zinc finger followed by an adjacent basic region that recognizes the consensus GATA motif in promoter sequences of target genes. The global regulatory areA and nit-2 genes activate structural genes encoding a set of pathway-specific permeases and enzymes enabling uptake and catabolism of secondary nitrogen sources when the preferred nitrogen sources, ammonia and glutamine, are lacking (Marzluf, 1997). So far, for all filamentous ascomycete fungi in which the work has been done, it has been possible to identify one unique areA/nit-2 orthologous gene (Haas et al., 1995; Froeliger and Carpenter, 1996; Screen et al., 1998; Tudzynski et al., 1999; Pérez-Garcia et al., 2001). Among the saprophytic or plant pathogenic fungi in which functional analysis has been performed, these orthologues always show up to be global regulatory genes for the utilization of secondary nitrogen-containing compounds, indicating conserved pathways of nitrogen metabolism regulation among the different ascomycete fungi. As these AREA/NIT2-like global activators play a key role in the regulation of fungal nitrogen metabolism during nitrogen starvation/-limiting conditions, and if nitrogen starvation is indeed one of the signals required for the regulation of genes involved in the early phases of fungal infection cycle, then the inactivation of an areA/nit-2 orthologue in the genome of biotrophic or hemibiotrophic plant fungal pathogens should have a major effect on their capacity to cause disease on their hosts. The inactivation of the nut1 gene, the areA/nit-2 orthologue of M. grisea, did not lead to a significant decrease in pathogenicity (Froeliger and Carpenter, 1996); however, non-orthologous additional nitrogen regulatory gene(s) may exist in the genome of this fungus (Lau and Hamer, 1996). Likewise, the inactivation of the nrf1 gene, the areA/nit-2 orthologue of C. fulvum, did not result in a reduction in pathogenicity (Pérez-Garcia et al., 2001). Thus, the hypothesis of nitrogen starvation as an environmental cue for the induction of genes necessary for the infection cycle is still questioned.

Colletotrichum lindemuthianum, a hemibiotrophic plant fungal pathogen, is the causal agent of anthracnose on common bean, Phaseolus vulgaris. The infection process has been well documented in previous cytological studies (O’Connell et al., 1985; Bailey et al., 1992). The infection cycle begins with the adhesion of conidia to aerial parts of the plant (leaf, stem, fruit). Then, the conidium germinates and develops a spherical structure, the appressorium, which is necessary for the penetration of host epidermal cells. After the penetration stage, the infection cycle is characterized by the succession of two phases. In the first phase, the biotrophic phase, which lasts 3–4 days, the fungus differentiates infection vesicles and primary hyphae inside a few of the host's cells. During the biotrophic phase, the fungus grows between the wall and the plasma membrane of host cells without causing their death. The second phase, which corresponds to the necrotrophic phase and to the onset of anthracnose symptoms, is completed within 6–7 days after the beginning of the infection cycle. During the necrotrophic phase, the fungus differentiates secondary hyphae, which are thinner than primary hyphae and grow extensively, leading to the disorganization and death of the infected host cells. Major nutritional changes are believed to occur during: (i) the first early phases from outside the plant tissue until penetration, during which the fungus might live on its stock nutrients; (ii) the onset of biotrophy, during which the fungus might organize nutrient uptake from adjacent living host cells; and (iii) the switch between the biotrophic phase and the necrotrophic phase. Few molecular determinants possibly (Centis et al., 1997; Perfect et al., 1998) or unequivocally (Dufresne et al., 1998; 2000) involved in the pathogenicity of C. lindemuthianum have been identified. Whether the variable environmental conditions encountered in planta by C. lindemuthianum along its infection cycle could be cues for the regulation of genes encoding molecular determinants necessary to its infection process has not been investigated. We hypothesize that nitrogen starvation occurs during the biotrophic phase of C. lindemuthianum and that it could act as a signal for the regulation of genes necessary to this first phase of the infection cycle.

We isolated the clnr1 (C.lindemuthianumnitrogen regulator 1) gene, the areA/nit-2 orthologue of C. lindemuthianum, and demonstrated by a gene replacement strategy that the clnr1 gene is the major nitrogen-regulatory gene required for the regulation of nitrogen metabolism in C. lindemuthianum. We have also assessed the role of the clnr1 gene in the infection cycle of this fungus by testing the pathogenicity of clnr1-replaced strains.

Results

Molecular characterization of the clnr1 gene, the areA/nit-2 orthologue of C. lindemuthianum

The proteins encoded by the areA/nit-2 orthologous genes of filamentous fungi exhibit a strict conservation of their DNA-binding domain, which consists of a single Cys-2/Cys-2 zinc finger followed by an adjacent basic region (Scazzocchio, 2000). Polymerase chain reaction (PCR) strategies with two degenerated oligonucleotide primers derived from the highly conserved zinc finger domain of the A. nidulans areA gene and the N. crassa nit-2 gene (Haas et al., 1995) have been developed successfully in order to isolate the areA/nit-2 orthologues of the plant fungal pathogens Gibberella fujikuroi (Tudzynski et al., 1999) and C. fulvum (Pérez-Garcia et al., 2001). We followed a similar PCR approach in order to isolate the areA/nit-2 orthologue of the plant fungal pathogen C. lindemuthianum. A resulting C. lindemuthianum 141 bp PCR fragment was cloned and sequenced. The deduced amino acid sequence of this PCR product showed 100% and 98% identity to the DNA-binding domain of the corresponding AREA and NIT2 amino acid sequences respectively. Subsequently, this fragment was used as a probe to screen a genomic library of the C. lindemuthianum UPS9 strain. Seven hybridizing plaques were identified as putative clones containing part of the putative areA/nit-2 orthologue of C. lindemuthianum. All clones exhibited similar restriction patterns as well as similar hybridization patterns when the original PCR fragment was used as a probe (data not shown). A 7.1 kb XhoI–XhoI genomic fragment was subcloned, resulting in the plasmid pclnr1. This plasmid was used to generate a physical map of the 7.1 kb genomic insert as well as extensive sequencing of DNA surrounding the original PCR fragment (data not shown). The genomic insert contains the putative areA/nit-2 orthologue of C. lindemuthianum, the clnr1 (C. lindemuthianumnitrogen regulator 1) gene. Restriction patterns derived from this map are consistent with the hybridization bands that were observed on the Southern blot of genomic DNA of C. lindemuthianum UPS9 strain (data not shown), which indicates that the clnr1 gene is present as a single-copy gene in the genome of this fungus.

DNA sequence analysis of the clnr1 gene revealed the existence of a 3263 bp open reading frame (ORF) that encodes a putative AREA/NIT2-like 971-amino-acid protein. Based on amino acid sequence alignment of the predicted CLNR1 protein with the AREA and NIT2 protein sequences and on the conservation of consensus splicing sites of introns, the coding region of the clnr1 gene is probably interrupted by two introns of 120 bp and 227 bp, which show typical 5′ and 3′ consensus splicing sites for genes of filamentous fungi (Ballance, 1986). The positions of the two introns on the nucleotide sequence are conserved between the clnr1 gene and the areA and nit-2 genes, which exhibit one single intron and two introns respectively. The first intron of the clnr1 gene corresponds to the single intron present within the A. nidulans areA gene and to the first intron present within the N. crassa nit-2 gene. The second intron of the clnr1 gene corresponds to the second intron present within the N. crassa nit-2 gene. The existence of these two introns was confirmed by reverse transcription (RT)-PCR analysis (data not shown). Figure 1 provides an alignment of the predicted CLNR1 protein with the amino acid sequences of the global nitrogen regulators AREA and NIT2 from the fungal saprophytes A. nidulans and N. crassa, and with the amino acid sequences of the global nitrogen regulators AREA-GF, NUT1 and NRF1 from the fungal plant pathogens G. fujikuroi, M. grisea and C. fulvum, respectively. Comparison of the predicted CLNR1 protein with the five global nitrogen regulators reveals significant similarities at the amino acid level throughout most of the protein (Fig. 1). The higher percentage of amino acid identity reaches 54% between the CLNR1 and AREA-GF proteins (Table 1). Moreover, the CLNR1 protein is most closely related to NIT2 from N. crassa (51% amino acid identity) than to AREA from A. nidulans (35% amino acid identity) (Table 1). Amino acid identity scores are in agreement with the phylogenetic position of C. lindemuthianum, which belongs to the Pyrenomycete subclass as do N. crassa, G. fujikuroi and M. grisea, whereas A. nidulans and C. fulvum belong to the Plectomycete subclass. The best conserved region between CLNR1 and the five fungal global nitrogen regulators corresponds to the 53-residue sequence (from 695 to 747 in amino acid sequence) that constitutes the DNA-binding domain: amino acid identity is extremely high in this region as 52 out of 53 amino acids are identical in all six proteins. Moreover, the next 19 residues downstream from the DNA-binding domain are identical between the CLNR1 and NIT2 proteins. A high percentage of amino acid identity between CLNR1 and NIT2 is also observed for the residues at the N-terminus extremity, a sequence required for nitrogen repression in N. crassa: 25 out of the first 33 residues of the amino extremity are conserved (from 1 to 33 in amino acid sequence). The last 11 C-terminal amino acids of CLNR1 are also highly conserved with those of the five global nitrogen regulators. In N. crassa, an α-helix within the DNA-binding domain and the 12 C-terminal residues have been demonstrated to be involved in interaction with the NMR protein, a nitrogen metabolic repressor (Xiao et al., 1995; Pan et al., 1997) that also exists in A. nidulans (Andrianopoulos et al., 1998). This suggests the probable existence of an NMR-like protein in C. lindemuthianum. Given the results of multiple alignment between the CLNR1 protein and the various fungal global nitrogen regulators, the clnr1 gene appears to be the areA/nit-2 orthologue of C. lindemuthianum.

Figure 1.

Alignment of the amino acid sequence of the predicted CLNR1 (accession no. AY168017) protein of C. lindemuthianum with AREA/NIT-2-like proteins of other filamentous fungi. The AREA/NIT-2-like proteins AREA (accession no. X52491NID), NIT2 (accession no. M33956NID), AREA-GF (accession no. Y11006NID), NUT1 (accession no. U60290NID) and NRF1 (accession no. AF312694) are from the saprophytic fungi Aspergillus nidulans and Neurospora crassa, and from the plant fungal pathogens Gibberella fujikuroi, Magnaporthe grisea and Cladosporium fulvum respectively. The black box, the dark grey box and the light grey box represent 100%, 80% and 60% of amino acid conservation respectively. The plain line indicates the position of the zinc finger domain. Dashes represent a gap position. Amino acid sequences were compared with clustal X (1.8) (Thompson et al., 1997).

Table 1. . Percentages of amino acid identity and amino acid similarity between the predicted CLNR1 protein of Colletotrichum lindemuthianum and AREA/NIT2-like proteins of other filamentous fungi.
% identity
% similarity
CLNR1AREANIT2AREA-GFNUT1
  1. The AREA/NIT-2-like proteins AREA, NIT2, AREA-GF, NUT1 and NRF1 are from the saprophytic fungi Aspergillus nidulans and Neurospora crassa, and from the fungal plant pathogens Gibberella fujikuroi, Magnaporthe grisea and Cladosporium fulvum respectively. Percentages of amino acid identity and amino acid similarity are indicated on the upper line and on the lower line of each comparison respectively.

AREA35    
50    
NIT25132   
6446   
AREA-GF543148  
674461  
NUT147314444 
60464958 
NRF13838313333
5151444645

The clnr1 gene encodes the major nitrogen regulator of C. lindemuthianum

To ascertain that the clnr1 gene controls the regulation of nitrogen metabolism in C. lindemuthianum, we aimed to inactivate the clnr1 gene by a gene replacement strategy. A gene replacement vector, pR-clnr1, was constructed through the replacement of the 4.3 kb EcoRI–EcoRI fragment that carries most of the clnr1 ORF (from 960 bp downstream ATG until the stop codon) as well as 2.0 kb downstream sequence by a hygromycin B resistance cassette consisting of an A. nidulans trpC promoter followed by the hph gene conferring hygromycin resistance from the pCB1003 vector (Fig. 2A). Subsequently, the 4.2 kb XhoI–XhoI linear fragment carrying the hph cassette flanked on the left and right sides by 1.3 kb and 1.5 kb, respectively, of homologous sequences to the clnr1 locus was used to transform the wild-type strain UPS9. Fifty-two independent transformants chosen randomly were screened for the replacement of the clnr1 gene by the hph cassette. The screening was performed by Southern blot analysis of XhoI-digested genomic DNAs probed with the 1.5 kb EcoRI–XhoI fragment, as indicated in Fig. 2A. For clnr1-replaced strains, the switch of the 7.1 kb fragment present in the wild-type genomic DNA to a 4.2 kb fragment is observed as illustrated in Fig. 2B with the R8 and R54 clnr1-replaced strains. Among the 52 transformants, 18 transformants were found to be replaced for the clnr1 gene. For all these transformants, the gene replacement experiment led to a unique integration of the linearized replacement cassette at the clnr1 locus. Two clnr1-replaced strains, R8 and R54, and one ectopic transformant, E42, were chosen for further study.

Figure 2.

clnr1 mutant construction by gene replacement.A. Construction of the clnr1 gene replacement vector pR-clnr1. The 4.3 kb EcoRI–EcoRI region carrying most of the clnr1 gene (from 960 bp downstream of ATG until the stop codon) and a 2.0 kb downstream sequence was replaced by the hygromycin resistance gene cassette (a 1.4 kb fragment from the pCB1003 plasmid containing the A. nidulans trpC promoter followed by the hph hygromycin resistance gene). The hygromycin resistance gene cassette is flanked with border sequences of the wild-type clnr1 genomic locus. The left and right flanking regions are 1.3 kb and 1.5 kb respectively. The dotted lines represent the sequence of the cloning vectors. The bold line represents the 1.5 kb EcoRI–XhoI fragment used as a probe for Southern analysis of the resulting transformants in (B). B, BglII; E, EcoRI; X, XhoI.
B. Southern blot analysis of the resulting transformants recovered after transformation of the wild-type strain UPS9 with the 4.2 kb XhoI–XhoI fragment from the pR-clnr1 vector. Total genomic DNAs were digested with XhoI and probed with the 1.5 kb EcoRI–XhoI fragment, underlined by the bold line in (A). The size of the wild-type clnr1 locus, 7.1 kb, and the size of the clnr1-replaced locus after double homologous recombination, 4.2 kb, are indicated on the left.

We assessed the role of the clnr1 gene in the regulation of C. lindemuthianum nitrogen metabolism. In the fungal model organisms A. nidulans and N. crassa, an areA mutant strain and a nit-2 mutant strain are able to grow on ammonia and glutamine that are the preferred nitrogen sources used by fungi. However, they are unable to use a variety of alternative nitrogen sources, including amino acids, amides, purines, nitrate and nitrite that can be assimilated by wild-type strains (Marzluf, 1997). The growth properties of the two clnr1-replaced strains R8 and R54 on minimal medium supplemented with diverse nitrogen-containing compounds as the sole nitrogen source were compared with those displayed by the wild-type strain UPS9. As shown in Table 2, the C. lindemuthianum wild-type strain is able to grow extensively on a diverse array of nitrogen-containing compounds as sole nitrogen sources in the same way as it does on complete medium. In contrast, R8 and R54 are unable to grow on the majority of the nitrogen compounds tested, except for aspartate, asparagine and alanine. The ectopic strain E42 displays identical growth properties to those displayed by the wild-type strain UPS9 on the various nitrogen sources. The growth properties of R8 and R54 on the various nitrogen sources are quite similar to those displayed by the A. nidulans areA mutant strain, except for aspartate, asparagine and alanine and also, to a lesser extent, for ammonia and glutamine. Surprisingly, these two nitrogen sources allow only reduced growth of R8 and R54, but similar results have already been observed for Aspergillus oryzae (Christensen et al., 1998), Aspergillus niger (Lenouvel et al., 2001) and C. fulvum (Pérez-Garcia et al., 2001).

Table 2. . Growth properties of Colletotrichum lindemuthianum and Aspergillus nidulans wild-type and mutant strains on minimal medium supplemented with various nitrogen sources.
Nitrogen source A. nidulans strains C. lindemuthianum strains 
Wild type areA mutantUPS9R8aE422.13b
  • a

    .C. lindemuthianum clnr1-replaced strains R8 and R54 display the same phenotype.

  • b

    . The four C. lindemuthianum clnr1-replaced strain R8 complemented with the clnr1 gene, 2.13, 2.17, 2.118 and 2.119, display the same phenotype.

  • +++, growth similar to that displayed by the wild-type strain on rich medium; ++, reduced growth compared with that displayed by the wild-type strain on rich medium; +, delayed and greatly reduced growth compared with that displayed by the wild-type strain on rich medium; –, no growth; ND, not determined.

Ammonia++++++++++++++++
Glutamine+++++++++++++++
Nitrate++++++++++++
Nitrite++++++++++++
Adenine++++++
Guanine++++++++++++
Hypoxanthine++++++++++++
Xanthine++++++++++++
Urea++++++++++++
Formamide++++++
Acetamide++++++
Histidine+++++++++++
Glutamate+++++++++++++
Aspartate++++++++++++++
Asparagine++++++++++++++
Alanine++++++++++++++
Proline++++++++++++
Glycine+++++++++++++
Leucine+++++++++++
Isoleucine+++++++++
Valine++++++++++
Serine+++++++++++++
Threonine+++++++++++++
Tryptophan++++++++++++
Tyrosine++++++++++++
Phenylalanine++++++++++++
Arginine++++++++++++
Ornithine+++++++++
CytosineNDND+++

In addition to the deletion of most of the clnr1 ORF, the gene replacement event with the pR-clnr1 vector involved the deletion of a 2.0 kb genomic fragment downstream the clnr1 gene. Although sequence analysis of this region did not reveal any putative ORF (data not shown), we could not exclude the possibility that a gene involved in nitrogen metabolism regulation of C. lindemuthianum is present in this region. Thus, to confirm that the phenotype of the clnr1-replaced strain on the various nitrogen sources results from the inactivation of the clnr1 gene only, the clnr1-replaced strain R8, which displays exactly the same phenotype as the clnr1-replaced strain R54, was complemented with only the clnr1 gene (the region corresponding to the 4.5 kb XhoI–BglII fragment of the 7.1 kb genomic insert including 360 bp upstream from the transcription start and 570 bp downstream from the stop codon). Four resulting complemented strains, 2.13, 2.17, 2.118 and 2.119, have been tested for their growth properties on the various alternative nitrogen sources tested previously. The growth properties of the four complemented strains are similar to that displayed by UPS9, as indicated for strain 2.13 in Table 2, confirming that the phenotype displayed by the clnr1-replaced strains on the various nitrogen sources results from the replacement of the clnr1 gene. Thus, the overall results of growth properties confirm that the clnr1 gene encodes a major positive-acting regulator involved in the control of the assimilation of a wide array of nitrogen sources in C. lindemuthianum.

The C. lindemuthianum clnr1 gene and the A. nidulans areA gene are symmetrical functional homologues

Functional complementation has already been shown between the areA gene and the nit-2 gene of the fungal saprophytes A. nidulans and N. crassa. Similarly, the nut1 gene and the nrf1 gene of the fungal plant pathogens M. grisea and C. fulvum, respectively, have been demonstrated to be functional homologues of the A. nidulans areA gene, and the areA-GF gene of the fungal plant pathogen G. fujikuroi is the functional homologue of the N. crassa nit-2 gene. We tested whether the C. lindemuthianum clnr1 gene is a functional homologue of the areA gene in A. nidulans. For this purpose, the A. nidulans areA loss-of-function mutant was transformed with the vector pclnr1, which carries the clnr1 ORF and its own promoter sequence. Ten A. nidulans transformants were obtained and analysed by Southern hybridization using the clnr1 gene as a probe. The clnr1 gene was present in one or two copies in the genome of the A. nidulans transformants (data not shown). Five of these A. nidulans transformants (1.1, 2.1, 3.2, 4.1, 5.1) were tested for their growth properties on minimal medium supplemented with the various nitrogen sources (Table 3). The A. nidulans areA mutants complemented with the clnr1 gene exhibit growth properties that are similar to those of the A. nidulans wild-type strain except surprisingly for ammonia and glutamine, for which the growth was not as extensive as the growth of the A. nidulans wild-type strain. From these results, we can conclude that the C. lindemuthianum clnr1 gene is a functional homologue of the A. nidulans areA gene, as the clnr1 gene restores the wild-type growth properties of the A. nidulans areA mutant on alternative nitrogen sources.

Table 3. . Growth properties of Colletotrichum lindemuthianum and Aspergillus nidulans wild-type strains, mutant strains and symmetrically complemented strains on minimal medium supplemented with various nitrogen sources.
Nitrogen source A. nidulans strains C. lindemuthianum strains
Wild type areA mutant5.1aUPS9R84.2b
  • a

    . The five A. nidulans areA mutants complemented with the C. lindemuthianum clnr1 gene, 1.1, 2.1, 3.2, 4.1 and 5.1, display the same phenotype.

  • b

    . The three C. lindemuthianum clnr1-replaced strain R8 complemented with the A. nidulans areA gene, 4.2, 4.16 and 4.19, display the same phenotype.

  • +++, growth similar to that displayed by the wild-type strain on rich medium; ++, reduced growth compared with that displayed by the wild-type strain on rich medium; +, delayed and greatly reduced growth compared with that displayed by the wild-type strain on rich medium; –, no growth; ND, not determined.

Ammonia++++++++++++++
Glutamine+++++++++++++
Nitrate++++++++++++
Nitrite++++++++++++
Adenine++++++++
Guanine++++++++++++
Hypoxanthine++++++++++++
Xanthine++++++++++++
Urea+++++++++++
Formamide++++++++
Acetamide++++++++
Histidine+++++++++
Glutamate+++++++++++++
Aspartate++++++++++++++
Asparagine++++++++++++++
Alanine++++++++++++++
Proline++++++++++++
Glycine+++++++++++++
Leucine++++++++++
Isoleucine++++++++++
Valine+++++++++++
Serine+++++++++++++
Threonine++++++++++++
Tryptophan++++++++++++
Tyrosine++++++++++++
Phenylalanine++++++++++++
Arginine++++++++++++
Ornithine++++++++++
CytosineNDNDND++

We tested reciprocally whether the areA gene of A. nidulans is a functional homologue of the clnr1 gene in C. lindemuthianum. The clnr1-replaced strain R8 was complemented with the areA gene, and the resulting C. lindemuthianum transformants 4.2, 4.16 and 4.19 carrying the areA gene in their genome were tested for their growth properties on minimal medium supplemented with various nitrogen sources (Table 3). The capacity to grow on secondary nitrogen compounds was restored for the clnr1-replaced strain complemented with the areA gene in the same way as when complementation is achieved with the clnr1 gene. Thus, the two functional complementation experiments clearly demonstrate that the clnr1 gene and the areA gene are symmetrical functional homologues in both fungal organisms.

The major nitrogen regulatory gene clnr1 plays a crucial role in the infection cycle of C. lindemuthianum

We assessed the importance of the major nitrogen regulator CLNR1 of C. lindemuthianum in its pathogenicity. When inoculated on a detached leaf of the common bean cultivar La Victoire, the wild-type strain UPS9 of C. lindemuthianum induces anthracnose symptoms that first appear as brown lesions on the main vein between 4 and 5 days after inoculation, then extend further to secondary veins and, finally, leads to complete maceration of the whole leaf within 6–7 days after inoculation (Fig. 3A). Whereas the ectopic strain E42 retains its ability to induce anthracnose symptoms similar to those of the wild-type strain UPS9 (Fig. 3D), the clnr1-replaced strains R8 and R54 are unable to produce anthracnose symptoms (Fig. 3B and C), even after an increased incubation period of 5 days. Despite the non-pathogenic phenotype of R8 and R54 on a detached leaf, rare small anthracnose lesions can occur on the main vein, but with a delay of 1–2 days compared with those produced by UPS9 and E42. Moreover, these rare lesions never extend. The four independent complemented strains of C. lindemuthianum, 2.17, 2.118, 2.119 and 2.13, obtained through complementation of the clnr1-replaced strain R8 with the clnr1 gene, show fully restored pathogenicity (Fig. 3G–J). Overall, these results demonstrate that the non-pathogenic phenotype of the clnr1-replaced strains R8 and R54 on a detached leaf results from the inactivation of the clnr1 gene.

Figure 3.

Pathogenicity of
C. lindemuthianum strains on detached leaves of the common bean cultivar La Victoire. Detached cotyledonary leaves were inoculated with the wild-type strain UPS9 (A, E and K), the clnr1-replaced strains R8 (B, F and L) and R54 (C), the ectopic strain E42 (D and M), the strains 2.17, 2.118, 2.119, 2.13 (G–J) obtained through complementation of the R8 strain with the clnr1 gene, and the strains 4.2, 4.16, 4.19 (N–P) obtained through complementation of the R8 strain with the
A. nidulans areA gene. Leaves were photographed 7 days after inoculation.

Variation in pathogenicity has been observed for fungal plant pathogens according to the plant organ used for the pathogenicity test (Dufresne and Osbourn, 2001). As C. lindemuthianum is a natural pathogen of all aerial bean parts, we also tested the pathogenicity of the clnr1-replaced strains R8 and R54 on a whole plantlet. When inoculated on a whole plantlet of the common bean cultivar La Victoire, the wild-type strain UPS9 induces anthracnose symptoms starting with brown lesions on leaf, petiole and stem between 4 and 5 days after inoculation that extend as large brown water-soaked lesions within the next 2–3 days (Fig. 4A). Whereas the E42 ectopic strain produces disease in a similar way to UPS9 (Fig. 4D), the R8 and R54 strains are non-pathogenic on whole plantlets (Fig. 4B and C). Nevertheless, these mutants are able to induce rare genuine anthracnose lesions on whole plantlets. Moreover, these lesions are reduced in size and appear with a delay of 1–2 days compared with both the wild-type and the ectopic strains. Interestingly, in contrast to the symptoms caused by either the wild-type strain or the ectopic strain, the lesions caused by the clnr1 mutants vary according to the organ. On the leaf of a whole plantlet, R8 and R54 display rare lesions, which are consistent with the results observed in the detached leaf assay. Furthermore, lesions on the main veins of leaves of whole plantlets never extend further like the rare existing lesions on leaves in the detached leaf assay. Unlike the lesions on the leaves of whole plantlets, the few lesions on the petiole and stem caused by R8 and R54 could spread infrequently to become brown water-soaked lesions. Additionally, in these brown water-soaked lesions, conidia occasionally could be found that germinated efficiently in vitro (data not shown). Thus, the clnr1-replaced strains R8 and R54 are non-pathogenic on detached leaf and whole plantlets of common bean, although a few rare anthracnose symptoms that are mostly abortive can be observed on plantlets. These results indicate that the major nitrogen regulatory gene clnr1 of C. lindemuthianum is crucial for the infection cycle of this fungus on common bean.

Figure 4.

Pathogenicity of
C. lindemuthianum wild-type, clnr1-replaced and ectopic strains on whole plantlets of the common bean cultivar La Victoire. Whole plantlets were inoculated with the wild-type strain UPS9
(A), the clnr1-replaced strains R8
(B) and R54 (C) and the ectopic strain E42
(D). The few anthracnose lesions caused by both R8 and R54 strains are indicated by the white arrows on (B) and (C). Plantlets were photographed 7 days after inoculation.

To characterize further the phenotype displayed by the clnr1-replaced strains R8 and R54 during the infection process, cytological analyses were done on lesions appearing on the stem from inoculated whole plantlets of common bean. Both R8 and R54 strains display the same phenotype, and both UPS9 and ectopic E42 strains display the same phenotype. Photographs of the R8 strain in comparison with the wild-type strain UPS9 are presented in Fig. 5. R8 conidia germinate and form appressoria similarly to the wild-type strain UPS9 (Fig. 5A and B). Cytological observations on subsequent steps in the infection process reveal that R8 develops normal primary hyphae in a period of 3–4 days after inoculation in the same way as the wild-type strain UPS9 (Fig. 5C and D). Therefore, the clnr1-replaced strains develop a penetration stage as well as a biotrophic phase, both similar to that developed by the wild-type strain UPS9. In contrast, the clnr1-replaced strain R8 differentiates rare secondary hyphae and, moreover, the differentiation of these structures is delayed 1–2 days compared with the wild-type strain UPS9 (Fig. 5E and F). Thus, cytological analyses indicate that the clnr1-replaced strains R8 and R54 are not disturbed during the penetration stage and the biotrophic phase of the infection cycle. In contrast, the clnr1 mutants are impaired during the development of secondary hyphae and thus at the necrotrophic phase. The difficulties displayed by the clnr1 mutants in developing secondary hyphae must result in the macroscopic abortive symptoms observed on common bean.

Figure 5.

Infection structures developed by the
C. lindemuthianum UPS9a wild-type strain and the R8bclnr1-replaced strain during the infection process on the stem of whole plantlets of the common bean cultivar La Victoire. Whole plantlets were inoculated with the wild-type strain UPS9 (A, C and E) and the R8 clnr1-replaced strain (B, D and F). Few secondary hyphae can be observed for the clnr1-replaced strains as displayed in the subframe in
(F). Ap, appressorium; C, conidia; S, stomatal guard cells; EC, epidermal cell; PH, primary hyphae; SH, secondary hyphae; DAI, days after inoculation. The 50 µm scale is indicated by the white line at the bottom of each photograph.
aBoth the UPS9 wild-type strain and the E42 ectopic strain display the same phenotype.
bBoth the R8 and R54 clnr1-replaced strains display the same phenotype.

The major nitrogen regulator AREA of A. nidulans restores the full pathogenicity of a clnr1 mutant strain

We demonstrated above that the A. nidulans areA gene is a functional homologue for the function of a major nitrogen regulator of the clnr1 gene in the fungal pathogen C. lindemuthianum. We tested whether functional homology also occurs during the natural in planta life cycle of C. lindemuthianum. For this purpose, we tested the pathogenicity of the three independent complemented strains, 4.2, 4.16, and 4.19, obtained through complementation of the clnr1-replaced strain R8 with the areA gene of A. nidulans. These three strains are able to produce anthracnose symptoms on a detached leaf with the same timing and as severe as those caused by the wild-type strain UPS9 (Fig. 3K and N–P). Thus, the clnr1-replaced strain R8 recovers its full pathogenicity upon complementation with the areA gene of the fungal saprophyte A. nidulans. This result indicates that the major nitrogen regulator AREA of A. nidulans is able to regulate not only C. lindemuthianum genes that are important for nitrogen nutrition in axenic culture, but also C. lindemuthianum genes important for nitrogen nutrition in planta as well as putative genes involved in other important processes for C. lindemuthianum pathogenicity on bean; all these genes are under the control of the major nitrogen regulator CLNR1 in the wild-type strain of C. lindemuthianum.

Discussion

Nitrogen starvation is a condition that is widely believed to be encountered by biotrophic or hemibiotrophic fungal plant pathogens early in infection of the host. Assuming that this is the case for the hemibiotrophic bean fungal pathogen C. lindemuthianum, we tested whether this condition acts as an environmental cue for the regulation of genes required for the early phases of its complex infection cycle. The cloning of clnr1, the areA/nit-2 orthologous gene of C. lindemuthianum, and the construction of clnr1 mutants by a gene replacement strategy allowed us to test this hypothesis.

As expected, sequence comparisons of the C. lindemuthianum CLNR1 protein with the global nitrogen regulatory proteins AREA and NIT2 from the fungal saprophytes A. nidulans and N. crassa and with the global nitrogen regulatory proteins AREA-GF, NUT1 and NRF1 from the plant fungal pathogens G. fujikuroi, M. grisea and C. fulvum, respectively, reveal high levels of amino acid sequence similarities throughout the protein. The highest overall degrees of amino acid identity are observed between the CLNR1 protein and the global nitrogen regulators AREA-GF, NIT2 and NUT1 from G. fujikuroi, N. crassa and M. grisea respectively. This is consistent with the phylogenetic relationship of these four ascomycete species that belong to the Pyrenomycete subclass, whereas A. nidulans and C. fulvum belong to the Plectomycete subclass and, accordingly, CLNR1 exhibits lower amino acid identity with AREA and NRF1, the respective major nitrogen regulators of these two fungi.

The comparison of growth between the C. lindemuthianum wild-type strain and the clnr1 mutants on various nitrogen sources confirms that the clnr1 gene acts as a global nitrogen regulatory gene involved in the control of nitrogen metabolism in C. lindemuthianum. Moreover, complementation experiments demonstrate that the clnr1 gene and the areA gene are symmetrical functional homologues. Unlike the areA mutant of A. nidulans and the nit2 mutant of N. crassa, the clnr1 mutants display difficulties in growing on ammonia and glutamine that are the preferred nitrogen sources for fungi. This suggests that inactivation of the clnr1 gene could act on ammonia and glutamine uptake in C. lindemuthianum as observed previously with the inactivation of the areA/nit-2 orthologues from A. oryzae (Christensen et al., 1998), A. niger (Lenouvel et al., 2001) and C. fulvum (Pérez-Garcia et al., 2001), whereas data are missing for M. grisea (Froeliger and Carpenter, 1996) and G. fujikuroi (Tudzynski et al., 1999). Interestingly, a family of ammonia transporters in which most members are induced during nitrogen starvation has recently been defined for plants and yeast (Van Dommelen et al., 2001).

Thus, the occurrence of the clnr1 gene in the genome of C. lindemuthianum and the availability of the corresponding clnr1 mutants allowed us to test the hypothesis that nitrogen limitation could act as a cue for the regulation of genes that are required for the development and the functionality of specific structures of the biotrophic phase: the infection vesicle and the primary hyphae. In this work, we demonstrate that the global nitrogen regulator CLNR1 is required for C. lindemuthianum to accomplish a complete infection cycle on common bean. However, surprisingly, the inactivation of the clnr1 gene has no disturbing effect on the early phases of the infection process. The clnr1 mutants are able to differentiate the infection structures required for the penetration stage (the appressorium), the biotrophic phase (the infection vesicle and primary hyphae) and progress without delay in these first two steps of the infection cycle compared with the wild-type strain. In contrast, the inactivation of the clnr1 gene impairs the setting up of the necrotrophic phase of the fungus. The clnr1 mutants are able to enter the necrotrophic phase, but they produce few secondary hyphae in comparison with the wild-type strain and, furthermore, the setting up of these structures is delayed 1–2 days, leading mostly to abortive lesion development. Accordingly, the clnr1 mutants are non-pathogenic despite a few occasional lesions occurring on whole plantlets. These results are different from the results of the inactivation of the global nitrogen regulatory genes nut1 and nrf1 from the hemibiotrophic plant fungal pathogen M. grisea and from the biotrophic plant fungal pathogen C. fulvum, which did not result in a significant decrease in the virulence of these two fungi on rice and tomato, their respective hosts (Froeliger and Carpenter, 1996; Pérez-Garcia et al., 2001). The occurrence of additional regulatory gene(s) involved in nitrogen metabolism in the genome of M. grisea has been invoked to account for the pathogenicity of the nut1 mutant. The existence of additional global nitrogen regulatory gene(s) in the genome of M. grisea is strongly suggested by the npr1 and npr2 mutants that were isolated via chlorate resistance (Lau and Hamer, 1996). These two mutants are unable to use a wide array of nitrogen sources and display a significant reduction in pathogenicity, whereas the nut1 mutant is still fully pathogenic. NPR1 and NPR2 may act as AREA/NIT2-like regulators in M. grisea, but the npr1 and npr2 mutants have complex phenotypes, and their genetic defects are not fully characterized.

Distinct but non-exclusive hypotheses can be proposed to explain the non-pathogenic phenotype of the clnr1 mutants on common bean. A first explanation for the difficulties that the clnr1 mutants display in the setting up of secondary hyphae is that they are unable to use the nitrogen sources available in planta at the end of the biotrophic phase. Indeed, C. lindemuthianum probably encounters drastic nitrogen starvation during the period covering the end of the biotrophic phase and the beginning of the necrotrophic phase. This nitrogen starvation probably begins from the very early phases of its infection cycle. However, during the penetration stage and the biotrophic phase, the fungus probably uses readily available nitrogen sources, the utilization of which does not require the role of the clnr1 gene and, therefore, the clnr1 mutants are able to develop functional appressoria, as well as functional infection vesicles and primary hyphae. These sufficient nitrogen sources could originate from (i) the nutrients present in the conidia for the build up of appressoria and/or (ii) glutamine, glutamate, aspartate and asparagine, which are the dominant components in the total free amino acid pool in chlorophyll-containing organs of most leguminous plants (Lam et al., 1996) for the build up of infection vesicles and primary hyphae. Accordingly, in vitro tests showed that clnr1 mutants are still able to use glutamate, aspartate and asparagine, although not up to wild-type levels (Table 2). Within this hypothesis, nitrogen starvation that occurs during the early phases of the infection cycle of C. lindemuthianum is not a signal for the regulation of genes that are required during these phases either in the setting up of the different specialized structures or in their functionality. Then, at the beginning of the necrotrophic phase, C. lindemuthianum may no longer find sufficient readily available nitrogen sources to develop the numerous secondary hyphae that result in the sudden increase in fungal biomass that occurs at this stage. At that crucial point, the fungus probably requires the clnr1 gene function for the induction of several nitrogen catabolic pathways compulsory for the uptake and the utilization of other host-derived nitrogen-containing compounds. Thus, whereas the clnr1 mutants would be able to use the scarce readily available nitrogen compounds (glutamate, aspartate and asparagine) as during the biotrophic phase, these compounds would probably be present in insufficient amounts to produce the fungal biomass that is required to pursue the infection cycle by the differentiation of numerous secondary hyphae. Local heterogeneous availability of higher amounts of readily available nitrogen sources close to primary hyphae at the end of the biotrophic phase and/or local higher basal expression in primary hyphae of specific fungal genes involved in nitrogen metabolism might explain the differentiation of few secondary hyphae by the clnr1 mutants. Subsequently, infrequent amounts of nutrients, probably released by fungal cell wall-degrading enzymes (Centis et al., 1997) and/or proteases from secondary hyphae, would allow the clnr1 mutants to complete the infection cycle progressively until the conidiation stage. In this respect, the time required for C. lindemuthianum to enter the necrotrophic phase might be a crucial point for the distinct phenotype displayed by the clnr1 mutants of C. lindemuthianum compared with other fungal pathogens. Indeed, the biotrophic phase of M. grisea is a fleeting step in comparison with that of C. lindemuthianum (Talbot, 1995). Thus, unlike the clnr1 mutants of C. lindemuthianum, the nut1 mutant of M. grisea might require less readily available nitrogen sources during the biotrophic phase to enter the necrotrophic phase efficiently, which would explain the full pathogenicity of the nut1 mutant of M. grisea.

Overall, a second explanation for the difficulties displayed by the clnr1 mutants in setting up the secondary hyphae could be that, in response to nitrogen starvation, CLNR1 regulates not only genes related to nitrogen nutrition in planta but also developmental genes involved in the differentiation of secondary hyphae. Several genes with no indication of involvement in nitrogen metabolism of plant pathogenic fungi were reported to be expressed in planta during the infection process as well as being induced in vitro during nitrogen starvation. The C. fulvum avr9 gene that is expressed in planta during the infection process is induced in vitro under nitrogen starvation. This regulation is under the control of the global nitrogen regulator NRF1 and is also observed in planta (Pérez-Garcia et al., 2001). Similarly, the induction of the M. grisea mpg1 gene in vitro under nitrogen starvation is under the control of both the suspected additional nitrogen regulators NPR1 and NPR2 (Lau and Hamer, 1996), and there is no indication that either the avr9 gene or the mpg1 gene is directly involved in nitrogen metabolism. Consequently, in the C. lindemuthianum/common bean interaction, we could not exclude the possibility that CLNR1 regulates in planta genes involved in processes other than nitrogen metabolism that are required for the necrotrophic phase. Besides the avr9 gene, it is not infrequent that genes of plant pathogenic fungi that are expressed in planta during the infection process exhibit GATA motifs in their promoter sequences that are the core recognition sequences for binding of all AREA/NIT2-like proteins studied so far. Thus, this may imply that (i) promoter sequences of fungal genes devoted to the development and functionality of specific structures of the infection process have evolved from promoter sequences of genes specifically involved in nitrogen utilization in fungal saprophytic growth and/or (ii) fungal nitrogen regulators have acquired diverse functions other than the regulation of genes involved in nitrogen metabolism, most probably through interaction with other regulators. Interestingly, fungal GATA factors have been reported to be involved in the activation or inactivation of genes in response to signal(s) other than nutritional deficiency (Lowry and Atchley, 2000).

The global nitrogen regulator AREA of the fungal saprophyte A. nidulans can efficiently complement CLNR1 deficiency during the infection process of C. lindemuthianum. Thus, according to our two hypotheses, AREA is able to activate C. lindemuthianum genes that are required for nitrogen nutrition in planta as well as putative genes not related to nitrogen metabolism but involved in the setting up of the necrotrophic phase, whatever the pathways operating upstream and downstream from the level of the global nitrogen regulator.

Finally, the non-pathogenic phenotype of clnr1 mutants resulting from mostly abortive setting up of secondary hyphae is novel for the C. lindemuthianum/common bean interaction. Another previously characterized non-pathogenic mutant of C. lindemuthianum, the H433 mutant, is also able to differentiate wild type-like penetration and biotrophic structures but, unlike the clnr1 mutants, it is completely unable to differentiate secondary hyphae (Dufresne et al., 2000). This mutant is genuinely blocked at the switch between the biotrophic phase and the necrotrophic phase. The clta1 gene, the inactivation of which is responsible for such a phenotype, encodes a putative GAL4-like transcriptional activator that is believed to be a crucial regulator of genes specifically involved in a developmental programme leading to the differentiation of the secondary hyphae and/or the necrotrophic phase. Uncovering the genes that are under the control of each activator and finding among them the particular genes that are responsible for the two distinct mutant phenotypes will be of great interest for understanding the infection process of C. lindemuthianum. The contribution of both CLNR1 and CLTA1 to the control of this sensitive step of the infection cycle of C. lindemuthianum will be revealed by future studies.

Experimental procedures

Fungal strains and culture conditions

Colletotrichum lindemuthianum strain UPS9 (Fabre et al., 1995) is used as the reference strain. All C. lindemuthianum strains, wild type and transformants, were grown at 22°C in Petri dishes on solid malt extract agar medium (15 g l−1) (3N+) (Duchefa). For nitrogen growth property assays, C. lindemuthianum strains were grown at 22°C in Petri dishes on solid minimal medium containing 20 g l−1d-glucose, 1 g l−1 KH2PO4, 0.5 g l−1 MgSO4, 0.5 g l−1 KCl, 0.15 g l−1 CaCl2(2H2O), 3 mg l−1 FeSO4(7H2O), 3 mg l−1 ZnSO4(7H2O), 1.25 mg l−1 CuSO4(5H2O), 350 µg l−1 MnSO4(H2O), 250 µg l−1 Na2MoO4(2H2O), 6.25 × 10−6µg l−1 biotin and 1.25 mg l−1 thiamine, supplemented with each of the various nitrogen-containing compounds as the sole nitrogen source at a 5 mM final concentration. Ten-day-old 3N+ plate cultures were used to prepare conidia suspensions to inoculate liquid culture for mycelia growth. Mycelia were grown in Roux's flasks (Fabre et al., 1995) containing 50 ml of complete medium: 20 g l−1d-glucose (Labosi), 5 g l−1 mycopeptone (Oxoid), 1 g l−1 yeast extract (Difco), 1 g−1 of casamino acids (Difco) or 50 ml of minimal medium. For both genomic DNA and total RNA extractions, the mycelium was collected after incubation of liquid cultures for 48 h at 22°C. Mycelium was frozen in liquid nitrogen and conserved at −80°C until nucleic acid extraction.

The A. nidulans strain CS 1028 pabaA1 (C. Scazzocchio's laboratory, Université de Paris-Sud, France) is the wild-type strain, and the A. nidulans strain CS 2529 areAr170 areA1601 biA1 pantoB100 (C. Scazzocchio's laboratory) is the areA loss-of-function mutant strain. A. nidulans strains are grown at 37°C on solid complete medium (Pontecorvo, 1953).

Aspergillus nidulans areA loss-of-function mutant

areA 1601 mutant was grown on solid complete medium supplemented with ammonia at a 20 mM final concentration. For mycelial growth, 50 ml of liquid minimal medium containing 10 g l−1d-glucose (Labosi), 0.5 g l−1 KCl, 0.5 g l−1 MgSO4(7H2O) and 1.5 g l−1 KH2PO4, supplemented with trace elements [2 mg l−1 Na2B4O7(10H2O), 20 mg l−1 CuSO4, 36 mg l−1 FePO4, 36 mg l−1 MnSO4(H2O), 40 mg l−1 Na2MoO4(2H2O) and 400 µg l−1 ZnSO4(7H2O)] were inoculated with conidia of A. nidulans strains at a final concentration of 106 conidia ml−1 and incubated for 15 h at 23°C in cultures on an orbital shaker at 200 r.p.m. For nitrogen growth property assays, A. nidulans strains were grown at 37°C in Petri dishes on solid minimal medium supplemented with the various nitrogen-containing compounds as the sole nitrogen source at a 5 mM final concentration.

Isolation of the clnr1 gene, the areA/nit-2 orthologous gene of C. lindemuthianum

The two degenerate oligonucleotide primers areA1 (5′-TGTACNAAYTGYTTYACNCA-3′) and areA2 (5′-TTCTTRAT NACRTCNGTYTT-3′), designated on the conserved region coding for the zinc finger domain of the areA and nit-2 nitrogen regulatory genes of A. nidulans and N. crassa, respectively (Haas et al., 1995), were used in a PCR experiment on 50 ng of genomic DNA from C. lindemuthianum UPS9 strain. DNA amplification was performed according to the manufacturer's instructions in 50 µl mixtures using 3 units of Taq DNA polymerase (GoldStar Red DNA polymerase, Eurogentec). Amplification conditions were as follows: 35 cycles of 15 s denaturation at 94°C, 30 s annealing at 50°C and 30 s polymerization at 72°C. The PCR resulted in a 141 bp fragment, which was cloned into the pGEM-T vector (Promega) and sequenced. The 141 bp PCR product was used as a homologous probe for the screening of the C. lindemuthianum UPS9 genomic library (Dufresne et al., 1998). About 5 × 106 recombinant phages of the C. lindemuthianum UPS9 genomic library were plated on Escherichia coli strain Q358 according to standard procedures (Sambrook et al., 1989). Plaques were blotted on Nytran membrane filters (Schleicher & Schuell) according to the manufacturer's instructions and screened by plaque hybridization. Seven hybridization plaques identified as positive clones were selected, plated and screened in two other rounds of hybridization for purification. All clones, analysed by restriction analysis, exhibited similar restriction patterns as well as similar hybridization patterns when the 141 bp PCR fragment was used as a probe. A 7.1 kb XhoI–XhoI genomic fragment encompassing the entire areA/nit-2 orthologue of C. lindemuthianum was subcloned in the pBlueScript-II KS+ vector (Stratagene), resulting in the plasmid pclnr1, and was subsequently sequenced.

Obtention and analysis of C. lindemuthianum and A. nidulans transformants

Preparation and transformation of C. lindemuthianum protoplasts were carried out as described by Rodriguez and Yoder (1987) with slight modifications as described by Dufresne et al. (1998). The fungal transformation vector pCB1003 containing the hygromycin resistance hph gene (hph cassette) under the control of A. nidulans promoter trpC sequence (Carroll et al., 1994) was used to construct the gene replacement vector to replace the clnr1 gene. In order to obtain a clnr1 replacement vector, the 4.3 kb EcoRI–EcoRI fragment of the plasmid pclnr1 was removed and replaced by the 1.4 kb EcoRI–EcoRI fragment containing the hph cassette originating from the plasmid pCB1003, leading to the vector pR-clnr1 (Fig. 2A). In the replacement vector pR-clnr1, the hph cassette is flanked on the left and right sides by 1.3 kb and 1.5 kb, respectively, of homologous sequences to the clnr1 locus. The pR-clnr1 vector was digested with XhoI, and the resulting 4.2 kb XhoI–XhoI fragment was used for the transformation of the C. lindemuthianum wild-type protoplasts. In order to screen for the replacement of the clnr1 gene, the genomic DNA of 52 independent C. lindemuthianum transformants was digested with XhoI and analysed through Southern blot hybridization using the 1.5 kb EcoRI–XhoI fragment indicated as a probe in Fig. 2A. For clnr1-replaced strains, the switch of the 7.1 kb hybridizing fragment specific to the wild-type locus to a 4.2 kb hybridizing fragment is observed (Fig. 2B). Subsequently, the clnr1-replaced strains were tested for their growth properties on minimal medium supplemented with various nitrogen sources.

To confirm that the phenotype displayed by the clnr1-replaced strains on the various nitrogen sources results from the inactivation of the clnr1 gene, complementation of the clnr1-replaced strain R8 was done with the 4.5 kb XhoI–BglII fragment of the 7.1 kb genomic insert (from 360 bp upstream of the initiation codon to 570 bp downstream of the stop codon). The genomic DNA of 19 C. lindemuthianum transformants was digested with EcoRI and analysed through Southern blot hybridization using the 1.7 kb EcoRI–EcoRI fragment (from 890 bp downstream of the initiation codon to 653 bp upstream of the stop codon of the clnr1 gene) and screened for the presence of the 1.7 kb EcoRI–EcoRI fragment. Four resulting complemented strains (2.13, 2.17, 2.118 and 2.119) were tested for their growth properties on minimal medium supplemented with various nitrogen sources.

For complementation of C. lindemuthianum with the A. nidulans areA gene, the clnr1-replaced strain R8 was transformed with the pKFA plasmid containing a 4.6 kb KpnI fragment corresponding to the wild-type A. nidulans areA gene (Langdon et al., 1995). The genomic DNA of 19 C. lindemuthianum transformants was digested with EcoRI, which does not cut in the wild-type areA gene, and analysed through Southern blot hybridization using the 4.6 kb areA-containing KpnI fragment. Three resulting complemented strains (4.2, 4.16, 4.19) carrying a single copy of the areA gene were tested for their growth properties on minimal medium supplemented with various nitrogen sources.

Transformation of A. nidulans was performed as described previously by Tilburn et al. (1983). For complementation of A. nidulans with the clnr1 gene, the A. nidulans areA loss-of-function mutant areA 1601 was transformed with the plasmid pclnr1. The genomic DNA of 10 A. nidulans transformants was digested with PstI, which does not cut in the clnr1 gene, and analysed through Southern blot hybridization using the 1.7 kb EcoRI–EcoRI fragment (from 890 bp downstream of the initiation codon to 653 bp upstream of the stop codon of the clnr1 gene). Four A. nidulans transformants (1.1, 2.1, 3.2, 5.1) containing a single copy of the clnr1 gene and one A. nidulans transformant (4.1) containing two copies of the clnr1 gene were analysed for their growth properties on minimal medium supplemented with various nitrogen sources.

Isolation and analysis of nucleic acids

Plasmid DNA, propagated in E. coli strain DH5α, was isolated using the QIAprep Spin miniprep kit (Qiagen). C. lindemuthianum genomic DNA extractions were performed according to the non-grinding method for DNA isolation (Ross, 1995). DNA restriction, agarose gel fractionation and transfer to nylon membranes (Hybond N; (Amersham Life Science) were performed according to manufacturer's instructions. Total RNAs from fungal mycelia were extracted using the Extract-All reagent according to the manufacturer's instructions (Eurobio). Southern hybridization was done overnight at 65°C in 5× SSC (3 M NaCl and 0.3 M sodium citrate), 0.5% SDS and 5× Denhardt's (0.1% Ficoll, 0.1% PVP, 0.1% BSA). Two washes of 30 min were performed at 65°C in 2× SSC, 0.1% SDS and in 0.1× SSC, 0.1% SDS, respectively, before autoradiography exposure.

RT-PCR experiments

In order to demonstrate the genuine existence of the two putative introns revealed by consensus sequence comparisons between the clnr1 gene and the five areA/nit-2 orthologous genes, RT-PCR experiments were performed using the Access RT-PCR kit according to the manufacturer's instructions (Promega). Primers clnr i1g (5′-CTTGGCCACCCAG GTGTGGA-3′), clnr i1d (5′-ATGAAGTCGTCAAGGTTCAT-3′) for intron 1 and clnr RT1 (5′-CCGTAAAACGAGTATTGACGA-3′) and clnr i2d (5′-CGCATCCAGGTCATTAGTTGT-3′) for intron 2 were designed in the clnr1 coding sequence for differential size amplification of the clnr1 transcript. RT-PCR experiments were done on 1 µg of total RNA extracted from UPS9 mycelium grown in liquid minimal medium supplemented with 10 mM sodium nitrate.

DNA sequencing

DNA sequencing of the clnr1 gene was performed using oligonucleotides deduced from the DNA sequence and using the Big Dye dideoxy chain terminator cycle sequencing kit on a 373A DNA sequencer (Perkin-Elmer, Applied Biosystems).

Infection assays

The common bean (Phaseolus vulgaris) cv. La Victoire (Tezier) was used as the susceptible cultivar in all pathogenicity assays. Plantlets of common bean cv. La Victoire were grown as described previously (Dufresne et al., 1998). For pathogenicity assays on detached leaves, cotyledonary leaves were detached, placed in Petri dishes on water-moistened filter paper, and the lower surface of the leaf was inoculated by spray inoculation. Ten-day-old 3N+ plate cultures of C. lindemuthianum were used to prepare conidia suspensions at a concentration of 106 conidia ml−1 for spray inoculation for pathogenicity assays on both detached leaves and whole plantlets. Detached leaves and whole plantlets were incubated in the same conditions as described previously by Dufresne et al. (1998). Eight leaves and 10 plantlets were used per C. lindemuthianum strain, and pathogenicity assays were repeated three times.

Microscopy

Cytological observations were done on strips of stem epidermal tissue from whole plantlets inoculated as described in the infection assays. Preparation of strips of epidermal tissue was performed as follows: (i) strips were treated twice for 10 min at 70°C with 1 M KOH; (ii) cleared samples were stained with methyl blue (0.01% methyl blue in 0.067 M K2HPO4). Specimens were observed with an Axioscope microscope (Zeiss) equipped with epifluorescence (filter set BP365/12, FT395 and LP397).

Acknowledgements

We thank L. Lechat for excellent technical assistance, C. Scazzocchio (Université Paris-Sud XI, France) for providing the A. nidulans wild-type strain and the A. nidulans areA loss-of-function mutant, M. X. Caddick (University of Liverpool, UK) for providing the pKFA plasmid containing the wild-type A. nidulans areA gene, M.-H. Lebrun for providing the pCB1003 plasmid (CNRS/Aventis CropScience, France), A. Apostolaki (Université Paris-Sud XI, France) for technical support in obtention and analysis of A. nidulans transformants, O. Roche (Université Paris-Sud XI, France) for technical support in microscopy, and R. Boyer (Université Paris-Sud XI, France) for photographs and help in figure layout. This research was supported by the Ministère de la Recherche et de l’Education Nationale.

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