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

Abstract

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.A LLORECOGNITION genetic systems, which provide the ability to distinguish self from nonself, play important functional roles in microbial and multicellular organisms. These systems range from restriction endonucleases in bacteria (Meselson and Yuan 1968) to somatic histocompatibility in protocordates (De Tomaso et al. 2005), self-infertility in plants (Nasrallah 2005), and innate immunity in vertebrates (Medzhitov and Janeway 2002) (reviewed by Aanen et al. 2008;Nydam and De Tomaso 2011;Rosengarten and Nicotra 2011). Allorecognition operates widely in filamentous fungi in both the sexual and the vegetative growth phases (Saupe 2000). The role of the mating-type locus in controlling sexual recognition and promoting outbreeding in yeast and filamentous fungi is well understood (reviewed by Coppin et al. 1997). It is also known that somatic or vegetative fusion of fungal cells (termed "anastomosis") occurs at a high frequency within and between individuals promoting network formation (Rayner 1996) and facilitating foraging, the pooling of resources (Rayner 1996), ...

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

“…Independent gene disruption analysis of 12 genes associated with these loci (22, 23) provided formal confirmation that five of the six loci contribute to restriction of virus transmission; an allelic difference at vic4 was shown previously not to restrict virus transmission (24). Moreover, all but one of these mutations resulted in no observable phenotypic change other than increased virus transmission or loss of the incompatibility reaction (22,23). We now report the use of sequential vic gene disruption (23,25) and mating approaches to engineer C. parasitica super hypovirus donor strains.…”

“…Moreover, all but one of these mutations resulted in no observable phenotypic change other than increased virus transmission or loss of the incompatibility reaction (22,23). We now report the use of sequential vic gene disruption (23,25) and mating approaches to engineer C. parasitica super hypovirus donor strains. The results demonstrate the feasibility of global modulation of fungal host allorecognition systems to remove restrictions to mycovirus transmission.…”

“…Subsequent comparative genomic analyses (22, 23) identified multiple polymorphic and idomorphic genes at the different vic loci. For example, the vic3 locus contains two polymorphic genes, vic3a and vic3b, designated vic3a-1 and vic3b-1 for allele 1 and vic3a-2 and vic3b-2 for allele 2, respectively (see Table 1 for current list of vic-associated genes).We previously demonstrated that disruption of individual polymorphic vic genes at the different vic loci resulted in both asymmetric and symmetric effects on virus transmission (22,23). For example, disruption of gene vic6-2 within the vic6 locus resulted in unrestricted virus transmission when paired with a vic6 heteroallelic strain only if the mutant strain was the recipient whereas disruption of the adjacent gene pix6-2 resulted in unrestricted virus transmission only when the mutant strain served as the donor.…”

“…Genetic analyses have defined six diallelic vic genetic loci for C. parasitica (21). These loci and associated genes were recently identified at the molecular level through a comparative genomics approach (22,23) (Table 1). Independent gene disruption analysis of 12 genes associated with these loci (22,23) provided formal confirmation that five of the six loci contribute to restriction of virus transmission; an allelic difference at vic4 was shown previously not to restrict virus transmission (24).…”

“…These loci and associated genes were recently identified at the molecular level through a comparative genomics approach (22,23) (Table 1). Independent gene disruption analysis of 12 genes associated with these loci (22,23) provided formal confirmation that five of the six loci contribute to restriction of virus transmission; an allelic difference at vic4 was shown previously not to restrict virus transmission (24). Moreover, all but one of these mutations resulted in no observable phenotypic change other than increased virus transmission or loss of the incompatibility reaction (22,23).…”