Genetic control of horizontal virus transmission in the chestnut blight fungus, Cryphonectria parasitica

Vegetative incompatibility in fungi has long been known to reduce the transmission of viruses between individuals, but the barrier to transmission is incomplete. In replicated laboratory assays, we showed conclusively that the transmission of viruses between individuals of the chestnut blight fungus Cryphonectria parasitica is controlled primarily by vegetative incompatibility (vic) genes. By replicating vic genotypes in independent fungal isolates, we quantified the effect of heteroallelism at each of six vic loci on virus transmission. Transmission occurs with 100% frequency when donor and recipient isolates have the same vic genotypes, but heteroallelism at one or more vic loci generally reduces virus transmission. Transmission was variable among single heteroallelic loci. At the extremes, heteroallelism at vic4 had no effect on virus transmission, but transmission occurred in only 21% of pairings that were heteroallelic at vic2. Intermediate frequencies of transmission were observed when vic3 and vic6 were heteroallelic (76 and 32%, respectively). When vic1, vic2, and vic7 were heteroallelic, the frequency of transmission depended on which alleles were present in the donor and the recipient. The effect of heteroallelism at two vic loci was mostly additive, although small but statistically significant interactions (epistasis) were observed in four pairs of vic loci. A logistic regression model was developed to predict the probability of virus transmission between vic genotypes. Heteroallelism at vic loci, asymmetry, and epistasis were the dominant factors controlling transmission, but host genetic background also was statistically significant, indicating that vic genes alone cannot explain all the variation in virus transmission. Predictions from the logistic regression model were highly correlated to independent transmission tests with field isolates. Our model can be used to estimate horizontal transmission rates as a function of host genetics in natural populations of C. parasitica.     

Selective acquisition of novel mating type and vegetative incompatibility genes via interspecies gene transfer in the globally invading eukaryote Ophiostoma novo-ulmi

The Dutch elm disease fungus Ophiostoma novo-ulmi, which has destroyed billions of elm trees worldwide, originally invaded Europe as a series of clonal populations with a single mating type (MAT-2) and a single vegetative incompatibility (vic) type. The populations then rapidly became diverse with the appearance of the MAT-1 type and many vegetative incompatibility types. Here, we have investigated the mechanism using isolates from sites in Portugal at which the rapid evolution of O. novo-ulmi populations from clonality to heterogeneity was well established. We show by genetic mapping of vic and MAT loci with AFLP markers and by sequence analysis of MAT loci that this diversification was due to selective acquisition by O. novo-ulmi of the MAT-1 and vic loci from another species, Ophiostoma ulmi. A global survey showed that interspecies transfer of the MAT-1 locus occurred on many occasions as O. novo-ulmi spread across the world. We discuss the possibility that fixation of the MAT-1 and vic loci occurred in response to spread of deleterious viruses in the originally clonal populations. The process demonstrates the potential of interspecies gene transfer for facilitating rapid adaptation of invasive organisms to a new environment.     

Heterokaryon incompatibility function of barrage-associated vegetative incompatibility genes (vic) in Cryphonectria parasitica

Six vegetative incompatibility (vic) loci have been identified in Cryphonectria parasitica based on barrage formation during mycelial interactions. We used hygromycin B- and benomyl-resistance as forcing markers in C. parasitica strains to test whether heteroallelism at each vic locus prevents heterokaryon formation following mycelial interactions. Paired strains that had allelic differences at any of vic1, 2, 3, 6 or 7 but not vic4 displayed heterokaryon incompatibility function, as recognized by slow growth or aberrant morphology. While clearly forming barrages in mycelial interactions, paired strains with different alleles at vic4 formed stable heterokaryons. With examples from other fungi, this inconsistency at vic4 suggests that barrage formation and heterokaryon incompatibility are not different manifestations of the same process. Rather, the evidence indicates that heterokaryon incompatibility represents a component of a vegetative incompatibility system that may also use cell-surface or extracellular factors to trigger programmed cell death to modulate nonself recognition in fungi.     

Molecular characterization of vegetative incompatibility genes that restrict hypovirus transmission in the chestnut blight fungus Cryphonectria parasitica

Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.     

Widespread vegetative compatibility groups in the dry-rot fungus Serpula lacrymans

Serpula lacrymans is the most notorious decayer of wooden buildings in temperate regions. The occurrence of geographically widespread vegetative compatibility groups (VCG) in S. lacrymans in Europe is demonstrated in this study. Among 22 heterokaryotic isolates of S. lacrymans, five VCG were found. The most widespread VCG included isolates from Belgium, south and central Norway, separated by more than 1500 km. No other genetic variation, measured as DNA sequence variation or ISSR polymorphisms, was detected between the investigated S. lacrymans isolates, whereas a considerable level of genetic variation was found among five European isolates of the sister taxon, S. himantioides. It is hypothesised that isolates of S. lacrymans have lost their ability to recognize self from nonself due to sharing of similar VC alleles, caused by a recent genetic bottleneck during the establishment in northern Europe. Isolates re-isolated from overlapping mycelial zones between different compatible isolates had significantly slower growth than that of the original isolates and the different isolates within a VCG had different growth morphology, indicating that isolates within a single VCG may belong to different genets.     

Genetics of Vegetative Incompatibility in Cryphonectria parasitica

Vegetative incompatibility in the chestnut blight fungus, Cryphonectria parasitica, in Europe is controlled by six unlinked vic loci, each with two alleles. Four previously identified vic loci (vic1, vic2, vic3, and vic4) were polymorphic in European vegetative compatibility (vc) types. Two new loci, vic6 and vic7, also were identified among European vc types. In one cross, vic genes segregated independently at five loci, and 194 progeny were assigned to 32 vc types; none of these loci were linked. A total of 64 vc types were identified from all crosses. All 64 genotypes possible from six vic loci, each with two alleles (2⁶ = 64), were identified and assigned to vc types. Based on our model, vc types v-c 5 and v-c 10, which had been used in previous genetic studies, differ by only five vic genes. Future studies of vc types in C. parasitica can use knowledge of vic genotypes for analysis of population genetic structure based on vic allele frequencies and to determine the effect of each vic gene on virus transmission between vc types.     

Ultraviolet Light-Induced Heterokaryon Formation and Parasexuality in Cryphonectria parasitica

Rizwana, R., and Powell, W. A. 1995. Ultraviolet light-induced heterokaryon formation and parasexuality in Cryphonectria parasitica. Experimental Mycology 19, 48-60. The effect of ultraviolet-light on heterokaryon formation, vegetative compatibility, and parasexuality in Cryphonectria parasitica was examined. Heterokaryons of complementary auxotrophic strains could not be made by hyphal anastomosis if the strains belonged to different vegetative compatibility groups. Protoplast fusions overcame incompatibility of strains differing in the alleles of a single but not multiple vegetative incompatibility loci. Fusion of protoplasts from ultraviolet light-treated complementary auxotrophs increased heterokaryon formation by 10⁴ to 10⁵ using the strains differing in alleles of a single vegetative incompatibility gene but had no detectable effect on strains differing in multiple vegetative incompatibility genes. Vegetative compatibility tests of single conidial isolates resolved from these heterokaryons suggest that diploids had formed followed by the loss of one of the VIC alleles. Presence of both auxotrophic markers in some of these single conidial isolates confirms the occurrence of a parasexual cycle. These experiments demonstrate that ultraviolet-light can enhance heterokaryon formation and parasexuality in C. parasitica .     

Multilocus self-recognition systems in fungi as a cause of trans-species polymorphism

Trans-species polymorphism, meaning the presence of alleles in different species that are more similar to each other than they are to alleles in the same species, has been found at loci associated with vegetative incompatibility in filamentous fungi. If individuals differ at one or more of these loci (termed het for heterokaryon), they cannot form stable heterokaryons after vegetative fusion. At the het-c locus in Neurospora crassa and related species there is clear evidence of trans-species polymorphism: three alleles have persisted for approximately 30 million years. We analyze a population genetic model of multilocus vegetative incompatibility and find the conditions under which trans-species polymorphism will occur. In the model, several unlinked loci determine the vegetative compatibility group (VCG) of an individual. Individuals of different VCGs fail to form productive heterokaryons, while those of the same VCG form viable heterokaryons. However, viable heterokaryon formation between individuals of the same VCG results in a loss in fitness, presumably via transfer of infectious agents by hyphal fusion or exploitation by aggressive genotypes. The result is a form of balancing selection on all loci affecting an individual's VCG. We analyze this model by making use of a Markov chain/strong selection, weak mutation (SSWM) approximation. We find that trans-species polymorphism of the type that has been found at the het-c locus is expected to occur only when the appearance of new incompatibility alleles is strongly constrained, because the rate of mutation to such alleles is very low, because the number of possible incompatibility alleles at each locus is restricted, or because the number of incompatibility loci is limited.     

Clonal population structure of the chestnut blight fungus in expanding ranges in southeastern Europe

Expanding populations are often less genetically diverse at their margins than at the centre of a species' range. Established, older populations of the chestnut blight fungus, Cryphonectria parasitica, are more variable for vegetative compatibility (vc) types than in expanding populations in southeastern Europe where C. parasitica has colonized relatively recently. To test whether vc types represent clones, we genotyped 373 isolates of C. parasitica from southern Italy, Romania, Bulgaria, Macedonia, Greece and Turkey using 11 sequence-characterized amplified region (SCAR) markers. Ten SCAR loci and six vegetative incompatibility (vic) loci were polymorphic in these samples. These populations are clonal by all criteria tested: (i) among 373 isolates, we found only eight multilocus haplotypes, and the same haplotypes were found in multiple countries, sometimes separated in time by as much as 12 years; (ii) the number of haplotypes observed was significantly less than expected under random mating; (iii) populations are in linkage disequilibrium; (iv) the two sets of independent markers, SCARs and vc types, are highly correlated; and (v) sexual structures of C. parasitica were found only in Bulgaria and Romania. One mating type (MAT-1) was found in 98% of the isolates sampled. In contrast, a population in northern Italy, in the central part of the range in Europe, had 12 multilocus haplotypes among 19 isolates. The spread of a few clones could be the result either of founder effect and restricted migration, or these clones have greater fitness than others and spread because they are better adapted to conditions in southeastern Europe.     

A Single Amino Acid Difference Is Sufficient to Elicit Vegetative Incompatibility in the Fungus Podospora Anserina

Vegetative incompatibility is known to limit heterokaryosis in filamentous fungi. It results from genetic differences between incompatible strains at specific loci. The proteins encoded by the two incompatible alleles het-s and het-S of the fungus Podospora anserina differ from each other by 14 amino acids. Two approaches have been used to identify how many and which of these differences are necessary to elicit incompatibility. Twelve alleles of the het-s locus of wild-type isolates of P. anserina and of the related species Podospora comata have been sequenced to determine the extent of the variability of genes controlling s and S specificities. Expression of hybrid het-s/het-S genes and site-specific mutagenesis revealed that the specificities of het-s and het-S are under the control of a limited number of amino acid differences. The results show that vegetative incompatibility between s and S strains can be attributed to a single amino acid difference in the proteins encoded by the het-s locus.     

The homologue of het-c of Neurospora crassa lacks vegetative compatibility function in Fusarium proliferatum

For two fungal strains to be vegetatively compatible and capable of forming a stable vegetative heterokaryon they must carry matching alleles at a series of loci variously termed het or vic genes. Cloned het/vic genes from Neurospora crassa and Podospora anserina have no obvious functional similarity and have various cellular functions. Our objective was to identify the homologue of the Neurospora het-c gene in Fusarium proliferatum and to determine if this gene has a vegetative compatibility function in this economically important and widely dispersed fungal pathogen. In F. proliferatum and five other closely related Fusarium species we found a few differences in the DNA sequence, but the changes were silent and did not alter the amino acid sequence of the resulting protein. Deleting the gene altered sexual fertility as the female parent, but it did not alter male fertility or existing vegetative compatibility interactions. Replacement of the allele-specific portion of the coding sequence with the sequence of an alternate allele in N. crassa did not result in a vegetative incompatibility response in transformed strains of F. proliferatum. Thus, the fphch gene in Fusarium appears unlikely to have the vegetative compatibility function associated with its homologue in N. crassa. These results suggest that the vegetative compatibility phenotype may result from convergent evolution. Thus, the genes involved in this process may need to be identified at the species level or at the level of a group of species and could prove to be attractive targets for the development of antifungal agents.     

The Use of Duplication-Generating Rearrangements for Studying Heterokaryon Incompatibility Genes in Neurospora

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N. crassa are difficult because in commonly used stocks differences are present at several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.—Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had previously been established by conventional heterokaryon tests—het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplication-generating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.—The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.—The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests.     

Asian origin and rapid global spread of the destructive dry rot fungus Serpula lacrymans

The dry rot fungus Serpula lacrymans (Basidiomycota) is the most damaging destroyer of wood construction materials in temperate regions. While being a widespread aggressive indoor biodeterioration agent, it is only found in a few natural environments. The geographical source of spread and colonization by this fungus in human environments is thus somewhat of an enigma. Employing genetic markers (amplified fragment length polymorphisms, DNA sequences and microsatellites) on a worldwide sample of specimens, we show that the dry rot fungus is divided into two main lineages; one nonaggressive residing naturally in North America and Asia (var. shastensis), and another aggressive lineage including specimens from all continents, both from natural environments and buildings (var. lacrymans). Our genetic analyses indicate that the two lineages represent well-differentiated cryptic species. Genetic analyses pinpoint mainland Asia as the origin of the aggressive form var. lacrymans. A few aggressive genotypes have migrated worldwide from Asia to Europe, North and South America and Oceania followed by local population expansions. The very low genetic variation in the founder populations indicate that they have established through recent founder events, for example by infected wood materials transported over land or sea. A separate colonization has happened from mainland Asia to Japan. Our data also indicate that independent immigration events have happened to Oceania from different continents followed by admixture.     

Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States

Chestnut blight is a devastating disease of Castanea spp. Mycoviruses that reduce virulence (hypovirulence) of the causative agent, Cryphonectria parasitica, can be used to manage chestnut blight. However, vegetative incompatibility (vic) barriers that restrict anastomosis-mediated virus transmission hamper hypovirulence efficacy. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, we have designed PCR primer sets that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. However, incongruences were observed for isolates from multiple locations and suggested that the coculturing assay can overestimate diversity at the six known vic loci. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies.     

Comparative analysis of Pleurotus ostreatus natural isolates

A comparative analysis is performed of the polymorphism of the Pleurotus ostreatus (Fr.) Kumm naturally occurring strains isolated from the natural substrates found in two geographically remote Russian natural preserves, the Central Arboreal Biosphere Tver State Preserve (CABTSP) and the Moscow State University Zvenigorod Biological Station (ZBS, Moscow oblast), and within the city of Moscow. The results of the frequency analysis for the isozyme loci alleles and for the sexual and vegetative incompatibility groups are presented; the genetic structure and the interpopulation relations among 58 P. ostreatus dikaryotic strains are estimated. The natural samples from the Moscow and Tver oblasts are shown to have a high degree of polymorphism with a genetic differentiation of 0.743; in spite of their territorial remoteness, they are, however, actively exchanging genetic material. The natural fungal isolates form two reproductively isolated groups.     

Identification of specificity determinants and generation of alleles with novel specificity at the het-c heterokaryon incompatibility locus of Neurospora crassa

The capacity for nonself recognition is a ubiquitous and essential aspect of biology. In filamentous fungi, nonself recognition during vegetative growth is believed to be mediated by genetic differences at heterokaryon incompatibility (het) loci. Filamentous fungi are capable of undergoing hyphal fusion to form mycelial networks and with other individuals to form vegetative heterokaryons, in which genetically distinct nuclei occupy a common cytoplasm. In Neurospora crassa, 11 het loci have been identified that affect the viability of such vegetative heterokaryons. The het-c locus has at least three mutually incompatible alleles, termed het-c(OR), het-c(PA), and het-c(GR). Hyphal fusion between strains that are of alternative het-c specificity results in vegetative heterokaryons that are aconidial and which show growth inhibition and hyphal compartmentation and death. A 34- to 48-amino-acid variable domain, which is dissimilar in HET-C(OR), HET-C(PA), and HET-C(GR), confers allelic specificity. To assess requirements for allelic specificity, we constructed chimeras between the het-c variable domain from 24 different isolates that displayed amino acid and insertion or deletion variations and determined their het-c specificity by introduction into N. crassa. We also constructed a number of artificial alleles that contained novel het-c specificity domains. By this method, we identified four additional and novel het-c specificities. Our results indicate that amino acid and length variations within the insertion or deletion motif are the primary determinants for conferring het-c allelic specificity. These results provide a molecular model for nonself recognition in multicellular eucaryotes.     

Evolutionary origin,worldwide dispersal,and population genetics of the dry rot fungus Serpula lacrymans

Recent research on the evolution, phylogeography and population genetics of the dry rot fungus Serpula lacrymans is reviewed. The fungus causes severe damage to construction wood in temperate regions worldwide. Multi-locus genetic analyses have shown that S. lacrymans includes two cryptic species currently referred to as var. shastensis and var. lacrymans. Both lineages occur naturally in high altitude regions, but var. lacrymans has also spread from its natural range in Asia, and established itself in indoor environments in temperate regions worldwide. Japan was apparently colonized independently from Europe, North and South America by var. lacrymans. The population found in Australia and New Zealand seems to represent a mixture of the Japanese and the Euro-American lineages. Little genetic variation exists in the founder populations of var. lacrymans worldwide. Due to the introduction of a limited number of vic (vegetative incompatibility) alleles into the founder populations, genetically different individuals often cannot recognize self from non-self through the vegetative incompatibility response. Moreover, only a low number of MAT (mating) alleles seem to be present in Europe. Genetic analyses of the European and Japanese indoor populations have shown that S. lacrymans mainly spreads sexually via basidiospores. Surprisingly, an excess of heterozygotes has been observed in the founder populations. This could be due to heterozygote advantage caused by a limited number of MAT alleles. Recent analyses of the S. lacrymans genome provide new opportunities for further research on the dry rot fungus.     

Linkage of soybean cyst nematode alleles for incompatibility with soybean

Selection and inbreeding of soybean cyst nematodes increased populations' ability to produce cysts on some soybean lines with concurrent decreases in numbers of cysts on other soybean lines: evidence that some alleles for incompatibility were either linked or at the same loci. Some responses could be explained only by linkage of nematode genes for avirulence. Linkage of nematode alleles for incompatibility could be involved when selection increased numbers of cysts on several lines even though the usual interpretation has been that the lines had some of the same genes for resistance. Most of the lines used in this study may have fewer alleles for incompatibility than most "resistant" lines. Use of these lines with fewer genes for resistance should help in the identification of individual alleles for incompatibility necessary for resolving the allelism and/or linkage of these nematode genes.     

HET-E and HET-D belong to a new subfamily of WD40 proteins involved in vegetative incompatibility specificity in the fungus Podospora anserina

Vegetative incompatibility, which is very common in filamentous fungi, prevents a viable heterokaryotic cell from being formed by the fusion of filaments from two different wild-type strains. Such incompatibility is always the consequence of at least one genetic difference in specific genes (het genes). In Podospora anserina, alleles of the het-e and het-d loci control heterokaryon viability through genetic interactions with alleles of the unlinked het-c locus. The het-d2(Y) gene was isolated and shown to have strong similarity with the previously described het-e1(A) gene. Like the HET-E protein, the HET-D putative protein displayed a GTP-binding domain and seemed to require a minimal number of 11 WD40 repeats to be active in incompatibility. Apart from incompatibility specificity, no other function could be identified by disrupting the het-d gene. Sequence comparison of different het-e alleles suggested that het-e specificity is determined by the sequence of the WD40 repeat domain. In particular, the amino acids present on the upper face of the predicted beta-propeller structure defined by this domain may confer the incompatible interaction specificity.     

Vegetative Incompatibility Among Monoconidial Isolates of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis>

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases of cereal crops, such as leaf-spot disease, common root rot, and black point of grain. Because of its great morphological, physiological, and genetic variability, this fungus is difficult to control. The aim of this investigation was to study the variability of isolates of B. sorokiniana by means of vegetative incompatibility. Thirty-five isolates of B. sorokiniana from different geographical regions in Brazil and other countries were used. The vegetative incompatibility between the isolates and the influences of different culture media on these reactions were evaluated. The total protein profile of the isolates was analyzed when the isolates were cultured separately, and in cultures of compatibility and incompatibility reactions. Eighteen of 31 confrontations showed vegetative incompatibility. The results obtained with different culture media for the vegetative compatibility/incompatibility genotypes suggested that the type of substratum influences these reactions. No differences in protein profiles among the isolates were observed. This result suggests that there is no induction of expression of different proteins in vegetative incompatibility reactions.