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Mycologia, 95(2), 2003, pp. 251-261.
© 2003 by The Mycological Society of America

Characterization of Bc-hch, the Botrytis cinerea homolog of the Neurospora crassa het-c vegetative incompatibility locus, and its use as a population marker


Elisabeth Fournier 1
Caroline Levis
Dominique Fortini

     PMDV, INRA, Route de Saint-Cyr, F-78026 Versailles cedex, France

Pierre Leroux

     Phytopharmacie et Médiateurs Chimiques, INRA, Route de Saint-Cyr, F-78026 Versailles cedex, France

Tatiana Giraud

     ESE, Bâtiment 362, Université de Paris-Sud, F-91405 Orsay cedex, France

Yves Brygoo

     Phytopharmacie et Médiateurs Chimiques, INRA, Route de Saint-Cyr, F-78026 Versailles cedex, France

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

The Botrytis cinerea homolog (Bc-hch) of Nc-het-c and Pa-hch (vegetative incompatibility loci of Neurospora crassa and Podospora anserina respectively) was cloned and sequenced. The gene structure of Bc-hch is very close to those of Nc-het-c and Pa-hch. A PCR-RFLP approach on a 1171 bp fragment was used to screen polymorphism at this locus among 117 natural isolates of B. cinerea. Restriction patterns by the restriction enzyme HhaI fell into two allelic types. Moreover, haplotypes at the Bc-hch strictly corresponded to the resistance phenotypes to fenhexamid, a novel Botryticide. The use of Bc-hch as a population marker thus reveals a new structuring of B. cinerea natural populations into two groups (I and II). This result was confirmed by genic differentiation tests performed with five other markers on a sample of 132 B. cinerea isolates from the French region of Champagne.

Key words: gray mold, PCR-RFLP, vegetative compatibility groups


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Vegetative compatibility is the ability to form a viable heterokaryon by hyphal fusion. Vegetative compatibility groups have proved to be useful to characterize population diversity in several fungal species (e.g., Katan et al 1991Citation, Elena 1999Citation). This phenomenon, unique to filamentous fungi, is governed by particular genes called het (or vic) genes: Two strains are incompatible when they differ in specificity at one or more het loci. Genetic and molecular characterization of het genes has been carried out in several species (Glass and Kuldau 1992Citation, Leslie 1993Citation, Bégueret et al 1994Citation, Kahman and Bölker 1996Citation, Glass et al 2000Citation). The Neurospora crassa (Nc)-het-c gene, encoding a glycin-rich signal peptide protein (Saupe 2000Citation, Glass et al 2000Citation), presents three allelic forms, the allelic specificity being due to non-silent polymorphism situated in a particular 5' region (Saupe and Glass 1997Citation). Moreover, this polymorphism appears to be transpecific, indicating that the Nc het-c locus might be subject to balancing selection (Wu et al 1998Citation). A homolog of the Nc-het-c gene recently was characterized in Podospora anserina (Saupe et al 2000Citation), but no polymorphism was found at this Pa-hch locus in the 5' specificity region.

The question then arises of the evolutionary age of the polymorphism at this specific locus. We thus investigated the species complex Botrytis cinerea, another ascomycete whose evolutionary branching is anterior to the separation between Podospora and Neurospora. Moreover, the occurrence of vegetative incompatibility in this species and its consequences on population structure and resistance to fungicides is poorly known (Beever and Parkes 1993Citation). We found the homolog of Nc-het-c and Pa-hch in a Botrytis cinerea expressed sequence tags library constructed in 1995 and named it Bc-hch (for Botrytis cinerea het-c homolog). B. cinerea (teleomorph Botryotinia fuckeliana) is a haploid, filamentous, heterothallic ascomycete, responsible for gray mold on a wide range of host plants, including grapevine. The present study aimed to answer two specific questions: Does Bc-hch have the same structure as Nc-het-c and Pa-hch and is this locus polymorphic?

We therefore cloned and sequenced the entire Bc-hch gene, and we screened for polymorphism within a 1171 bp region by PCR-RFLP among 117 field isolates.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
B. cinerea strains – Lesions caused by B. cinerea were collected in the field during Summer and early Autumn. Colonies were grown on PDA medium. After monospore purification, isolates were preserved in 80% glycerol at -80 C. The monospore isolates were cultured on plates containing 20 mL of NY medium (20 g/liter malt, 20 g/liter agar, 2 g/liter yeast extract) from an inoculum of 1 cM2 at 25 C.

Giraud et al (1997)Citation suggested that B. cinerea populations were structured as two genetically isolated subgroups characterized by the presence or absence of the transposable elements Boty and Flipper. B. cinerea var transposa strains possess both Boty and Flipper, whereas B. cinerea var vacuma strains lack both. For the cloning of Bc-hch, we used the T4 transposa reference strain isolated from tomato and the K1 vacuma reference strain isolated from kiwi fruit. A total of 117 isolates from different host plants and different regions were used to screen Bc-hch for polymorphism (Table I).


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  		TABLE I. Geographic origin, Bc-hch haplotypes, responses to fenhexamid (HydS for sensitive and HydRI for resistant) and vacuma/transposa types (? = not determined) of 117 B. cinerea field isolates

 

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  		TABLE I. Continued

 

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  		TABLE I. Continued

 
DNA extraction – The mycelium was cultured as described by Diolez et al (1994)Citation. Genomic DNA was isolated by the method of Möller et al (1992)Citation.

Cloning of Bc-hch – An EST library was built in 1995 from strain T4, as well as two BAC libraries from strains T4 and K1 (Lévis and Brygoo, unpubl data). The B. cinerea EST clone containing the Nc-het-c homolog was picked out by sequence confrontation with international databases using a BLAST search (Genbank accession number of this Bc clone: AL 113429). After purification, the corresponding plasmid was 32P-labelled and hybridised on high-density filters, onto which BAC library of strains T4 and K1 had been spotted. BACs carrying the Nc-het-c homolog were purified. The entire Bc-hch gene then was cloned by chromosomic walk (Universal Genome Walker Kit, Clontech) in strain T4. The genomic DNA of interest was first digested with several restriction enzymes, then linkers provided in the kit were ligated to the fragments' extremities. Two nested PCR assays then were performed. The first PCR was done with one primer in the linker and one primer designed in a known sequence of the gene to be cloned; the second PCR was realized with two more internal primers, also designed in the linker and in the known sequence, to amplify preferentially the fragment of interest. This fragment then was sequenced with an ABI Prism 7700 (Applied Biosystems).

PCR amplifications and restrictions – From the Bc-hch sequence of strain T4, two primers were designed bracketing the region of potential allelic specificity as defined in N. crassa (Saupe and Glass 1997Citation): primer 262: 5'-AAGCCCTTCGATGTCTTGGA-3'; primer 520L: 5'-ACGGATTCCGAACTAAGTAA-3'. These primers amplified a 1171 bp fragment between positions 701 and 1871 of the Bc-hch gene. The PCR assay was done in a total volume of 50 µL containing 5 µL DNA, 5 mL reaction buffer, 4 µL MgCl2, 2.5 µL of each primer and 0.2 µL of EUROGENTEC Taq polymerase. The PCR assay used 30 cycles of 30 s at 94 C, 1 min 30 s at 55 C and 1 min at 72 C. Restriction digests were done for 1.5 h at 37 C, in a total volume of 20 µL containing 5 µL PCR product, 2 µL reaction buffer, and 1 U restriction enzyme HhaI (GIBCO).

Antifungal assays – The effect of fenhexamid toward the mycelial growth of B. cinerea isolates was assessed as described in Leroux et al (1999)Citation.

Genic differentiation tests – To confirm the genetic structuring inferred from the Bc-hch locus, 132 isolates (66 isolates sensitive to fenhexamid, 66 isolates resistant to fenhexamid) from a single region (Champagne, France) were chosen from the sample scored by Giraud et al (1997)Citation. Five markers were retained for this analysis: 3 PCR-RFLP markers (ATP/ADP) translocase, IGS, Nitrate reductase; contrarily to Giraud et al (1997)Citation, each locus was considered as a single genetic marker, even if several restriction enzymes were used), and 2 markers of sensitivity to fungicides (vinchlozolin and antimicrotubules; Leroux 1985Citation). Genetic differentiation between the two groups of strains was calculated using the GENEPOP program (Raymond 1995Citation; the null hypothesis to test for genetic differentiation was H0: "the allelic distribution is independent across populations").


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Cloning of Bc-hch – The sequence of one EST clone coming from the reference strain T4 was found to match the downstream half of the Nc-het-c gene. Hybridization of this clone as a probe on BAC libraries of strains T4 and K1 revealed strong hybridization signals to 9 spots for strain K1 and only 1 low intensity signal for strain T4. We therefore used the K1 genomic DNA for the cloning of the entire gene. For this we used the GenomeWalker protocol of chromosomic walk. Following this protocol, a 900 bp fragment was amplified, matching the 5' part of the Nc-het-c gene up to Position 241. The corresponding fragment of the gene was cloned and sequenced in strain T4. The cloning of the entire gene then was achieved by a further chromosomic walk step on genomic DNA of strain T4, allowing the amplification of a 2 kb fragment corresponding to the 5' region and flanking region of the gene.

The sequencing of these fragments showed that the Bc-hch gene (accession number AY032846) encodes a 795 amino-acid putative protein (Bc-HCH, Fig. 1). It contains a putative 51 bp intron between amino acids 102 and 103, and a 48 bp intron between amino acids 525 and 526. We compared the Bc-HCH putative amino-acid sequence with Pa-HCH, the putative homologous protein in P. anserina (accession number: AF169793) and with the three allelic forms of HET-C, the putative homologous protein in N. crassa (Nc-HETCOR, accession number: AF206700; Nc-HETCPA, accession number: AF195874; Nc-HETCGR, accession number: AF196305) (Fig. 1). Percent identity between the five putative proteins is summarized in Table II; the best match is found between Bc-HCH and Pa-HCH. In Nc-HET-C, the region between residues 247 and 284 has been described as the specificity region (Saupe and Glass 1997Citation). This region is highly divergent between Bc-HCH, Pa-HCH and the three allelic classes Nc-HET-COR, PA and GR, whereas the conservation is very strong upstream and downstream this specificity region.



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  		 FIG. 1. Alignment of the gene products of the OR, PA and GR het-c alleles of Neurospora crassa, the Pa-hch of Podospora anserina, and the Bc-hch of Botrytis cinerea. The alignment was performed with ClustalX (Thompson et al 1997Citation). Primers 262 and 520L are underlined with horizontal arrows; putative specificity is framed in gray; black triangles indicate the positions of introns; the position of the HhaI polymorphic restriction site in Bc-hch is indicated by a black ellipse

 

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  		TABLE II. Identities among the putative proteins Bc-HCH of B. cinerea, Pa-HCH of P. anserina, and Nc-HETCOR, Nc-HETCGR and Nc-HETCPA of N. crassa. Alignments were performed with Multalin software (Corpet, 1988)

 
Bc-hch PCR-RFLP haplotypes and resistance phenotypes to fenhexamid – We designed the primers 262 and 520L to amplify a 1171 pb fragment corresponding to the region of the Bc-hch gene that is homologous to the specificity region in N. crassa het-c. We used these primers for screening this region for PCR-RFLP polymorphism. Four restriction enzymes (EcoRI, HaeIII, HhaI, RsaI) were tried on four isolates, but only HhaI revealed some polymorphism. Two restriction patterns were detected (Fig. 2), differing by one restriction site. We sequenced both alleles (Fig. 3) and verified that the Bc-hch2 1171-bp fragment contains 5 HhaI restriction sites (positions 119, 274, 283, 367, 884), whereas the Bc-hch1 allele contains only 4, the site in Position 367 being mutated. The two restriction profiles thus differ in the size of the upper band (Bc-hch2: 6 bands of size 517, 287, 155, 119, 84 and 9 bp; Bc-hch1: 5 bands of size 601, 287, 155, 119, 84 and 9 bp; see Fig. 2).



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  		 FIG. 2. PCR products from genomic DNA of 6 B. cinerea isolates, obtained with primers 262–520L (left), and (right) restriction profiles after digestion of these products with the enzyme HhaI. "2": allele Bc-hch2; "1": allele Bc-hch1

 


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  		 FIG. 3. Alignment of the nucleotidic sequence of Isolate T4 (carrying allele Bc-hch2), with the sequence of the PCR product obtained with primers 262/520L from Isolate 945 carrying allele Bc-hch1. Primers 262 and 520L are underlined with horizontal arrows. The putative specificity region is framed. The black ellipse indicates the position of the polymorphic HhaI restriction site. The putative amino-acid product is indicated below

 
The 1171-bp fragment therefore systematically was amplified and digested with HhaI for 117 natural isolates (Table I). All these isolates also were scored for their resistance to fenhexamid. This novel Botryticide inhibits the 3-keto reductase involved in the sterol C-4 demethylation (Debieu et al 2001Citation, Leroux et al 2000Citation). The sample contains 43 vacuma isolates resistant to fenhexamid (36.5%), 17 vacuma isolates sensitive to fenhexamid (14.5%), 49 transposa isolates sensitive to fenhexamid (42%), and 8 (sensitive or resistant) isolates whose vacuma/transposa types could not be determined (7%). These 117 isolates came from different host plants: grape (91), strawberry (13), tomato (4) and pea (2). In all strains, fenhexamid resistance was associated strickly to allele Bc-hch1, whereas all strains carrying allele Bc-hch2 were sensitive to this fungicide. Moreover, we observed that all strains resistant to fenhexamid and carrying allele Bc-hch1 were of type vacuma, whereas sensitive strains carrying allele Bc-hch2 were either vacuma or transposa. As observed previously, the fenhexamid resistant strains (HydR1, Table I) exhibited an increased sensitivity to fenpropimorphe (Leroux et al 2000Citation).

B. cinerea strains thus were divided in two groups: Group I containing isolates carrying allele Bc-hch1, resistant to fenhexamid and all of vacuma type, versus Group II containing isolates carrying allele Bc-hch2, sensitive to fenhexamid and vacuma or transposa types.

Genic differentiation test within the Champagne sample – To test for the strength of this genetic structuring, we re-analysed part of the data on isolates collected and scored by Giraud et al (1997)Citation. We chose 132 isolates from the French region of Champagne (66 sensitive and 66 resistant to fenhexamid), also including vacuma and transposa types; 56 isolates came from grape, 3 from Rubus sp., 3 from Geranium sp., 2 from Brassica sp., 1 from Rosa sp. and 1 from Trifolium sp. Bc-hch alleles were not scored with the PCR-RFLP method for these isolates but were inferred from their resistance to fenhexamid, which was known from earlier studies. In total, the sample contains 66 vacuma resistant (50%), 14 vacuma sensitive (10.6%) and 52 tranposa sensitive (39.4%) isolates. Genetic differentiation tests performed for the 5 selected markers were highly significant (Table III), showing that the allelic frequencies were significantly different between Group I and Group II, and therefore that these groups are genetically isolated. Giraud et al (1997)Citation showed that the same markers also differentiated significantly between vacuma and transposa types in this Champagne sample. However, since Group I contains only vacuma types, and Group II contains vacuma and transposa types, the present test reveals that the genetic structuring might be more complicated than previously thought.


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  		TABLE III. Allelic frequencies of five markers for a sample of 132 isolates of Botrytis cinerea from Champagne (France), previously genotyped by Giraud et al (1997), and genic differentiation calculated for each marker between isolates from groups I and II. The P value is the probability associated with the exact Fischer test for the null hypothesis H0: "the allelic distribution is independent across Group I and Group II"

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
This study of the Bc-hch locus, a homolog of the het-c vegetative incompatibility loci of N. crassa and P. anserina, shows that the gene structure is highly conserved among these three species, with the best match found between Bc-HCH and Pa-HCH. As in N. crassa and P. anserina, the B. cinerea C-terminal region of Bc-hch presents glycin-rich repeats. In the N-terminal part, conservation with Nc-HET-C and Pa-HCH was strong upstream and downstream of the putative specificity region. This region (residues 247 to 284 in Bc-HCH) strongly diverged from the homologous region in Pa-HCH and still more in the three allelic forms of Nc-HET-C (Fig. 1 and Table II). As it is the case for Pa-HCH, the putative specificity region in Bc-HCH lacks a 5–6 amino-acid segment (positions 253 to 258 in Fig. 1) found in all Nc-HET-C alleles analyzed so far (Saupe and Glass 1997Citation, Wu et al 1998Citation, Saupe et al 2000Citation).

The 117 isolates analyzed by PCR-RFLP fell into two groups corresponding to two allelic types at the Bc-hch locus (Table I): Group I for strains carrying allele Bc-hch1; Group II for strains carrying allele Bc-hch2. The 1171 bp fragment was sequenced in a B. cinerea isolate carrying allele Bc-hch2, and compared with the sequence of Isolate T4, carrying allele Bc-hch2 (Fig. 3). All substitutions were silent, except one situation in Position 1090 of the 1171 bp fragment. The putative specificity region is strictly identical in the two alleles. Group I and Group II also differed in their response to fenhexamid: Isolates carrying Bc-hch1 (Group I) were resistant to this fungicide; the others were sensitive. The first hypothesis to explain the strict correspondence between Bc-hch haplotype and fenhexamid resistance phenotype, is that the Bc-hch gene might be the fungicide target. However, it has been shown that resistance to fenhexamid results from its detoxification, probably through a cytochrome P450 monoxygenase (Leroux et al 2000Citation, Suty et al 1999Citation), and this protein cannot be the putative product of Bc-hch. The second hypothesis is that Bc-hch and the fungicide target are tightly linked. However, even in this case the strict concordance between haplotypes at both locus would be highly improbable, because of the high recombination rate in B. cinerea (Giraud et al 1997Citation), except if there has been a recent selective sweep or hitch-hiking process, which is improbable because the fungicide has been slightly and only recently used in the field. Hence, the most parsimonious hypothesis to explain the strict concordance is that groups I and II are sufficiently genetically isolated to have fixed several private alleles, including Bc-hch alleles and alleles related to the resistance phenotype. This hypothesis is strongly confirmed by the genetic differentiation between resistant and sensitive isolates from the same population of Champagne (Table III). Moreover, Leroux (pers comm) reported the abortion of all sexual crosses assays between Group I and Group II isolates, indicating that groups I and II cannot interbreed.

These results indicate that the PCR-RFLP polymorphism observed at the Bc-hch locus rather corresponds to the fixation of two private alleles in two reproductively isolated populations: groups I and II, with no polymorphism observed within each group at this locus. The status of true species for these groups has to be confirmed by multiple gene genealogies. Nevertheless, this indicates that the Bc-hch locus does not fit the balancing selection model proposed by Wu et al (1998)Citation, as well as the P. anserina Pa-hch locus studied by Saupe et al (2000)Citation. These last authors also suggest that in P. anserina, the hch locus does not play any role in vegetative incompatibility. In our case, several confrontation assays were performed in Petri dishes (Fournier, unpubl data): On the one hand, "barrages" were observed systematically between Group I and Group II isolates; on the other hand, barrages also were observed among Group II members, showing that this group contains several VCGs, although it is monomorphic at the Bc-hch locus. This might indicate that Bc-hch does not function as a het gene in B. cinerea; however, other disruption/complementation experiments should be conducted to address this question.

Saupe (2000)Citation proposed that the non-polymorphic Podospora Pa-hch gene might represent an ancestral allele and suggested that the emergence of het-c incompatibility might be due to the emergence of polymorphism at this locus, before the divergence of Neurospora and Sordaria but after the divergence between Neurospora and Podospora. The present results do not ensure that Bc-hch does not act as a het gene. If this fact was confirmed, then the observed proximity between Bc-HCH and Pa-HCH would agree with Saupe's hypothesis, and would suggest that the het-c allele carried by B. cinerea also might be the ancestral one, because the divergence between Podospora and Botrytis is earlier than the divergence between Neurospora and Podospora.

The Bc-hch locus revealed a new structuring among B. cinerea populations. This structuring does not correspond to the one previously described by Giraud et al (1997)Citation: Group I isolates are all of vacuma type, whereas Group II isolates are either vacuma or transposa. The question then is to know whether the genetic barrier observed by Giraud et al was an artifact, due to the presence of approximately one-third Group I isolates in their sample, or whether vacuma and transposa types still correspond to genetically differentiated subpopulations restricted to Group II. More polymorphic markers are required, such as microsatellite loci, to further study this question. Further phylogenetic analyses, such as multiple gene genealogies, also must be conducted to determine the strength of genetic differentiation between Group I and Group II subpopulations and to determine their true phylogenetic status.


   ACKNOWLEDGMENTS
 
We thank J. Shykoff and J.M. Cornuet for useful comments on previous versions of the manuscript. This work was supported partially by a grant from the Fondation Singer-Polignac to E.F.


   FOOTNOTES
 
1 Corresponding author. Elisabeth.Fournier{at}versailles.inra.fr Back

Accepted for publication August 31, 2002.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Beever R, Parkes SL., 1993 Mating behavior and genetics of fungicide resistance of Botrytis cinerea in New Zealand. New Zealand Journal of Crop and Horticultural Science 21:303-310

Bégueret J, Turcq B, Clavé C., 1994 Vegetative incompatibility in filamentous fungi: het genes begin to talk. Trends in Genetics 10:441-446[Medline]

Corpet F., 1998 Multiple sequence alignment with hierarchical clustering. Nucleic Acids Research 16:10881-10890

Debieu D, Bach J, Hugon M, Malosse C, Leroux P., 2001 The hydroxyanilide fenhexamid, a new sterol biosynthesis inhibitor fungicide efficient against the plant pathogenic fungus Botryotinia fuckeliana (Bortytis cinerea). Pest Management Science 57:1060-1067[Medline]

Diolez A, Marches F, Fortini D, Brygoo Y., 1994 Boty, a long-terminal repeat retroelement in the phytopathogenic fungus Botrytis cinerea. Applied and Environmental Microbiology 61:103-108

Elena K., 1999 Genetic relationships among Verticillum dahliae isolates from cotton in Greece, based on vegetative compatibility. European Journal of Plant Pathology 105:609-616

Giraud T, Fortini D, Lévis C, Leroux P, Brygoo Y., 1997 RFLP Markers show genetic recombination in Botryotinia fuckeliana (Botrytis cinerea) and transposable elements reveal two sympatric species. Molecular Biology and Evolution 14:1177-1185[Abstract]

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Leroux P, Clerjeau M., 1985 Resistance of Botrytis cinerea and Plasmopora viticola in French vineyards. Crop Protection 4:137-160

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