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.     

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.     

Programmed cell death correlates with virus transmission in a filamentous fungus

Programmed cell death (PCD) is an essential part of the defence response in plants and animals against pathogens. Here, we report that PCD is also involved in defence against pathogens of fungi. Vegetative incompatibility is a self/non-self recognition system in fungi that results in PCD when cells of incompatible strains fuse. We quantified the frequency of cell death associated with six vegetative incompatibility (vic) genes in the filamentous ascomycete fungus Cryphonectria parasitica. Cell death frequencies were compared with the effects of vic genes on transmission of viruses between the same strains. We found a significant negative correlation between cell death and virus transmission. We also show that asymmetry in cell death correlates with asymmetry in virus transmission; greater transmission occurs into vic genotypes that exhibit delayed or infrequent PCD after fusion with an incompatible strain. Furthermore, we found that virus infection can have a significant, strain-specific, positive or negative effect on PCD. Specific interactions between vic gene function and viruses, along with correlations between cell death and transmission, strongly implicate PCD as a host-mediated pathogen defence strategy in fungi.     

Vegetative Incompatibility Loci with Dedicated Roles in Allorecognition Restrict Mycovirus Transmission in Chestnut Blight Fungus

Vegetative incompatibility (vic), a form of nonself allorecognition, operates widely in filamentous fungi and restricts transmission of virulence-attenuating hypoviruses in the chestnut blight fungus Cryphonectria parasitica. We report here the use of a polymorphism-based comparative genomics approach to complete the molecular identification of the genetically defined C. parasitica vic loci with the identification of vic1 and vic3. The vic1 locus in the C. parasitica reference strain EP155 consists of a polymorphic HET-domain-containing 771-aa ORF designated vic1a-2, which shares 91% identity with the corresponding vic1a-1 allele, and a small (172 aa) idiomorphic DUF1909-domain-containing ORF designated vic1b-2 that is absent at the vic1-1 locus. Gene disruption of either vic1a-2 or vic1b-2 in strain EP155 eliminated restrictions on virus transmission when paired with a vic1 heteroallelic strain; however, only disruption of vic1a-2 abolished the incompatible programmed cell death (PCD) reaction. The vic3 locus of strain EP155 contains two polymorphic ORFs of 599 aa (vic3a-1) and 102 aa (vic3b-1) that shared 46 and 85% aa identity with the corresponding vic3a-2 and vic3b-2 alleles, respectively. Disruption of either vic3a-1 or vic3b-1 resulted in increased virus transmission. However, elimination of PCD required disruption of both vic3a and vic3b. Additional allelic heterogeneity included a sequence inversion and a 8.5-kb insertion containing a LTR retrotransposon sequence and an adjacent HET-domain gene at the vic1 locus and a 7.7-kb sequence deletion associated with a nonfunctional, pseudo vic locus. Combined gene disruption studies formally confirmed restriction of mycovirus transmission by five C. parasitica vic loci and suggested dedicated roles in allorecognition. The relevance of these results to the acquisition and maintenance of vic genes and the potential for manipulation of vic alleles for enhanced mycovirus transmission are discussed.     

Genetics of self/nonself recognition in Serpula lacrymans

This study provides an analysis of the vegetative incompatibility system in Serpula lacrymans (Basidiomycota), a genetic system used to recognize nonself in fungi. Seventy-five worldwide isolates could be grouped into eight vegetative compatibility (VC) types, some of them distributed on different continents. Mating studies combined with vegetative incompatibility analyses revealed that the vegetative incompatibility response between isolates mainly could be explained by two biallelic vegetative incompatibility (vic) loci. The frequency distributions of the interpreted vic alleles do not seem to support the idea of frequency-dependent or balancing selection acting on the vic loci. We find little genetic variation at the vic loci and in one of the loci there was a significant heterozyote deficiency among strains in the overall material. The results may be explained by a recent worldwide dispersal of a few S. lacrymans isolates and, correspondingly, only a few vic alleles are being maintained in these populations.     

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.     

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.     

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.     

Inheritance of Restriction Fragment Length Polymorphisms in Agaricus Brunnescens

The cultivated mushroom, Agaricus brunnescens, is secondarily homothallic; most basidia produce only two basidiospores, each of which receives two of the four post meiotic nuclei. The segregation of restriction fragment length polymorphisms (RFLPs) detected by four plasmid probes carrying single-copy nuclear DNA of Agaricus was followed in seven parental strains including commercial, wild-collected, and artificially synthesized heterokaryons. Of a total of 367 single-spore progeny examined, 351 (95.6%) were heteroallelic at all RFLP loci heteroallelic in the respective parents. Of the 16 segregant isolates, ten (2.7% of the total) were homoallelic at all segregating loci assayed, suggesting that these isolates were most probably derived from rare spores that had received only a single postmeiotic nucleus. Some of these ten isolates had recombinant genotypes. Only five isolates (1.4% of the total) showed homoallelism at one of the loci heteroallelic in the parent, while remaining heteroallelic at other loci. These five genotypes suggest that a crossover had occurred between a marker locus and its respective centromere. Taken together, the results suggest that meiosis in A. brunnescens is accompanied by low levels of recombination and that nonsister nuclei are preferentially incorporated into basidiospores after meiosis II.     

Evidence for interspecies transmission of viruses in natural populations of filamentous fungi in the genus Cryphonectria

Interspecies transmission is a significant evolutionary event that has allowed a variety of pathogens to invade new host species. We investigated interspecies transmission of viruses between the chestnut blight fungus, Cryphonectria parasitica, and a sympatric unidentified Cryphonectria species in Japan. Two isolates of Cryphonectria sp. were found to contain Cryphonectria hypovirus 1 (CHV-1), which has been typically found in C. parasitica. Three lines of evidence support the hypothesis of interspecies transmission of CHV-1. First, host species occur sympatrically and therefore have the opportunity to come into physical contact. Second, we transmitted CHV-1 between species experimentally in the laboratory. Third, phylogenetic analysis of 476 bp of the ORF B region of CHV-1 showed that sequences from Cryphonectria sp. were more closely related to those from C. parasitica than to each other. Local geographical subdivision of virus sequences from both host species argues against the alternative hypothesis of independent evolution of CHV-1 since speciation of their hosts. Based on these findings, we rule out the hypotheses that CHV-1 diverged from viruses in a common ancestor of the hosts, or that ancestral polymorphisms in CHV-1 persisted in the two host taxa. Estimating the direction and frequency of interspecies transmission in nature will require more extensive samples of CHV-1 from both host species.     

Transmission of Fusarium boothii mycovirus via protoplast fusion causes hypovirulence in other phytopathogenic fungi

There is increasing concern regarding the use of fungicides to control plant diseases, whereby interest has increased in the biological control of phytopathogenic fungi by the application of hypovirulent mycoviruses as a possible alternative to fungicides. Transmission of hypovirulence-associated double-stranded RNA (dsRNA) viruses between mycelia, however, is prevented by the vegetative incompatibility barrier that often exists between different species or strains of filamentous fungi. We determined whether protoplast fusion could be used to transmit FgV1-DK21 virus, which is associated with hypovirulence on F. boothii (formerly F. graminearum strain DK21), to F. graminearum, F. asiaticum, F. oxysporum f. sp. lycopersici, and Cryphonectria parasitica. Relative to virus-free strains, the FgV1-DK21 recipient strains had reduced growth rates, altered pigmentation, and reduced virulence. These results indicate that protoplast fusion can be used to introduce FgV1-DK21 dsRNA into other Fusarium species and into C. parasitica and that FgV1-DK21 can be used as a hypovirulence factor and thus as a biological control agent.     

Virulence not only costs but also benefits the transmission of a fungal virus

Current theory suggests that cost-benefit relationships govern the evolution of parasite virulence. The cost of virulence is expected to be high for fungal viruses, which are obligate parasites and completely dependent on their hosts. The majority of fungal viruses infect their hosts without any apparent symptoms. Cryphonectria hypovirus 1 (CHV-1), in contrast, is virulent and debilitates its host, Cryphonectria parasitica. However, the virulence of CHV-1 is associated with high costs for virus transmission, such as an attenuated fungal growth and reduced production of the fungal spores spreading the virus. In this study, we tested the hypothesis that virulence may not only have costs but also benefits for transmitting CHV-1 across vegetative incompatibility barriers between fungi. We investigated viruses with low, medium, and high virulence, and determined their transmission rate per host-to-host contact (transmissibility). The average transmission rate across all combinations tested was 53% for the most virulent virus, 37% for the virus with intermediate virulence, and 20% for the virus with lowest virulence. These results showed that increased virulence was strongly correlated with increased transmissibility, potentially counterbalancing virulence costs. This association of virulence and transmissibility may explain why CHV-1 spread widely and evolved higher virulence than most other fungal viruses.     

Recombination and migration of Cryphonectria hypovirus 1 as inferred from gene genealogies and the coalescent

Genealogy-based methods were used to estimate migration of the fungal virus Cryphonectria hypovirus 1 between vegetative compatibility types of the host fungus, Cryphonectria parasitica, as a means of estimating horizontal transmission within two host populations. Vegetative incompatibility is a self/non-self recognition system that inhibits virus transmission under laboratory conditions but its effect on transmission in nature has not been clearly demonstrated. Recombination within and among different loci in the virus genome restricted the genealogical analyses to haplotypes with common mutation and recombinational histories. The existence of recombination necessitated that we also use genealogical approaches that can take advantage of both the mutation and recombinational histories of the sample. Virus migration between populations was significantly restricted. In contrast, estimates of migration between vegetative compatibility types were relatively high within populations despite previous evidence that transmission in the laboratory was restricted. The discordance between laboratory estimates and migration estimates from natural populations highlights the challenges in estimating pathogen transmission rates. Genealogical analyses inferred migration patterns throughout the entire coalescent history of one viral region in natural populations and not just recent patterns of migration or laboratory transmission. This application of genealogical analyses provides markedly stronger inferences on overall transmission rates than laboratory estimates do.     

Mixed mating in natural populations of the chestnut blight fungus, Cryphonectria parasitica

As in plants, fungi exhibit wide variation in reproductive strategies and mating systems. Although most sexually reproducing fungi are either predominantly outcrossing or predominantly selfing, there are some notable exceptions. The haploid, ascomycete chestnut blight pathogen, Cryphonectria parasitica, has previously been shown to have a mixed mating system in one population in USA. In this report, we show that both selfing and outcrossing occur in 10 additional populations of C. parasitica sampled from Japan, Italy, Switzerland and USA. Progeny arrays from each population were assayed for segregation at vegetative incompatibility (vic) and DNA fingerprinting loci. Outcrossing rates (t(m)) were estimated as the proportion of progeny arrays showing segregation at one or more loci, corrected by the probability of nondetection of outcrossing (alpha). Estimates of t(m) varied from 0.74 to 0.97, with the lowest rates consistently detected in USA populations (0.74-0.78). Five populations (four in USA and one in Italy) had t(m) significantly less than 1, supporting the conclusion that these populations exhibit mixed mating. The underlying causes of variation in outcrossing rates among populations of C. parasitica are not known, but we speculate that--as in plants--outcrossing is a function of ecological, demographic and genetic factors.     

Variation in tolerance and virulence in the chestnut blight fungus-hypovirus interaction

Chestnut blight, caused by the fungus Cryphonectria parasitica, has been effectively controlled with double-stranded RNA hypoviruses in Europe for over 40 years. The marked reduction in the virulence of C. parasitica by hypoviruses is a phenomenon known as hypovirulence. This virus-fungus pathosystem has become a model system for the study of biological control of fungi with viruses. We studied variation in tolerance to hypoviruses in fungal hosts and variation in virulence among virus isolates from a local population in Italy. Tolerance is defined as the relative fitness of a fungal individual when infected with hypoviruses (compared to being uninfected); virulence is defined for each hypovirus as the reduction in fitness of fungal hosts relative to virus-free hosts. Six hypovirus-infected isolates of C. parasitica were sampled from the population, and each hypovirus was transferred into six hypovirus-free recipient isolates. The resulting 36 hypovirus-fungus combinations were used to estimate genetic variation in tolerance to hypoviruses, in hypovirus virulence, and in virus-fungus interactions. Four phenotypes were evaluated for each virus-fungus combination to estimate relative fitness: (i) sporulation, i.e., the number of asexual spores (conidia) produced; (ii) canker area on field-inoculated chestnut trees, (iii) vertical transmission of hypoviruses into conidia, and (iv) conidial germination. Two-way analysis of variance (ANOVA) revealed significant interactions (P < 0.001) between viruses and fungal isolates for sporulation and canker area but not for conidial germination or transmission. One-way ANOVA among hypoviruses (within each fungal isolate) and among fungal isolates (within each hypovirus) revealed significant genetic variation (P < 0.01) in hypovirus virulence and fungal tolerance within several fungal isolates, and hypoviruses, respectively. These interactions and the significant genetic variation in several fitness characters indicate the potential for future evolution of these characters. However, biological control is unlikely to break down due to evolution of tolerance to hypoviruses in the fungus because the magnitudes of tolerance and interactions were relatively small.     

Markers linked to vegetative incompatibility (vic) genes and a region of high heterogeneity and reduced recombination near the mating type locus (MAT) in Cryphonectria parasitica

To find markers linked to vegetative incompatibility (vic) genes in the chestnut blight fungus, Cryphonectria parasitica, we constructed a preliminary linkage map. In general, this map is characterized by low levels of polymorphism, as evident from the more than 24 linkage groups observed, compared to seven expected from electrophoretic karyotyping. Nonetheless, we found markers closely linked to two vic genes (vic1 and vic2) making them candidates for positional cloning. Two markers were found to be linked to vic2: one cosegregated with vic2, i.e., it is 0.0 cM from vic2, the other was at a distance of 4.5 cM; a single marker was found 4.0 cM from vic1. The closest markers linked to three other vic genes (vic4, vic6, and vic7) were >15 cM away; additional markers are needed before efficient positional cloning of these three vic genes can be realized. In contrast to the low levels of polymorphism observed across most of the C. parasitica genome, the linkage group containing the MAT locus appears to harbor an extremely high level of RAPD heterogeneity and reduced recombination. Markers within this highly heterogeneous region are in linkage disequilibrium in some natural populations; however, recombination is clearly evident between this region and the MAT locus.     

The impact of parasitism on resource allocation in a fungal host: the case of Cryphonectria parasitica and its mycovirus,Cryphonectria Hypovirus 1

Parasites are known to profoundly affect resource allocation in their host. In order to investigate the effects of Cryphonectria Hypovirus 1 (CHV1) on the life‐history traits of its fungal host Cryphonectria parasitica, an infection matrix was completed with the cross‐infection of six fungal isolates by six different viruses. Mycelial growth, asexual sporulation, and spore size were measured in the 36 combinations, for which horizontal and vertical transmission of the viruses was also assessed. As expected by life‐history theory, a significant negative correlation was found between host somatic growth and asexual reproduction in virus‐free isolates. Interestingly this trade‐off was found to be positive in infected isolates, illustrating the profound changes in host resource allocation induced by CHV1 infection. A significant and positive relationship was also found in infected isolates between vertical transmission and somatic growth. This last relationship suggests that in this system, high levels of virulence could be detrimental to the vertical transmission of the parasite. Those results underscore the interest of studying host–parasite interaction within the life‐history theory framework, which might permit a more accurate understanding of the nature of the modifications triggered by parasite infection on host biology.     

High diversity of vegetative compatibility types in Cryphonectria parasitica in Japan and China

We found high diversity of vegetative compatibility (vc) types in native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China; almost every isolate was in a unique vc type. In Japan we found 71 vc types in a sample of 79 isolates pooled from six populations. Within two populations in China, all isolates (n=28 and 11) had unique vc types; we found 15 vc types among 25 isolates in a third Chinese population where multiple isolates were collected from some trees. None of the isolates from China and only three isolates in the 71 vc types from Japan were compatible with any of 64 vc type testers from Europe, which have known vegetative incompatibility genotypes. To our knowledge this is the first report of vc type diversity for C. parasitica in Japan or of any comparisons of vc types between Asia and Europe. The most significant result of this survey is the identification fungal isolates for expanding knowledge of the genetics of vegetative incompatibility.     

Persistence of simian immunodeficiency virus Mne variants upon transmission.

In macaques infected with a clone of simian immunodeficiency virus (SIV) Mne, viral variants consistently evolve multiple new potential glycosylation sites in the first variable region (V1) prior to the development of AIDS. In the present study, we asked whether viruses with these glycosylation sites persist when they are transmitted to a naive macaque. Variants that evolved after transmission to a recipient macaque were compared with virus that evolved in the donor, which had been infected by cloned SIV Mne. Upon transmission, the specific serine/threonine-rich motifs potentially encoding novel O-linked glycosylation site(s) in V1 were conserved in virus isolated from lymph node, spleen, and liver tissue from the recipient. There was some accumulation of changes in V3 of envelope in virus from the recipient, whereas changes in this region were not observed in virus from the donor macaque. Some variants detected in the tissue of the recipient at necropsy were most closely related to viruses present in the donor inoculum even though these particular variants were not detected early after infection in the recipient's peripheral blood mononuclear cells. Overall, virus with the predominant V1 sequences associated with progression to disease are transmitted to and persist in the recipient animal.