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), ...

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.M ycovirus infections have been reported to reduce virulence (hypovirulence) for a wide range of plant pathogenic fungi, providing potential for biological disease control (1-6). For hypovirulence to be effective, the virulence-attenuating viruses must be efficiently transmitted from infected hypovirulent strains to uninfected virulent strains (5, 7). Mycoviruses generally have evolved exclusive intracellular lifestyles (8). With very few exceptions (9), mycovirus infections cannot be initiated by exposure of uninfected hyphae to cell extracts or secretions from infected fungal isolates. Transmission is limited to intracellular mechanisms, vertical transmission through asexual spores, and horizontal transmission through anastomosis (fusion of hyphae).Horizontal mycovirus transmission to uninfected isolates of the same fungal species is complicated by nonself allorecognition genetic systems, termed "heterokaryon" or "vegetative incompatibility" (vic), which operate widely in filamentous fungi (10). Interactions between genetically distinct individuals of the same species result in an incompatible reaction that triggers localized programmed cell death (PCD), forming a line of demarcation, or barrage, along the zone of contact (10-12) and restricting cytoplasmic transmission of viruses and other cytoplasmic elements (1,(13)(14)(15).A negative correlation between vic diversity and virus transmission has been reported for several fungal hosts (1, 16), but has most extensively been demonstrated for the chestnut blight fungus Cryphonectria parasitica infected with virulence-attenuating hypoviruses (7,(17)(18)(19)(20). 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) ...

Neotyphodium uncinatum and Neotyphodium siegelii are fungal symbionts (endophytes) of meadow fescue (MF; Lolium pratense), which they protect from insects by producing loline alkaloids. High levels of lolines are produced following insect damage or mock herbivory (clipping). Although loline alkaloid levels were greatly elevated in regrowth after clipping, loline-alkaloid biosynthesis (LOL) gene expression in regrowth and basal tissues was similar to unclipped controls. The dramatic increase of lolines in regrowth reflected the much higher concentrations in young (center) versus older (outer) leaf blades, so LOL gene expression was compared in these tissues. In MF-N. siegelii, LOL gene expression was similar in younger and older leaf blades, whereas expression of N. uncinatum LOL genes and some associated biosynthesis genes was higher in younger than older leaf blades. Because lolines are derived from amino acids that are mobilized to new growth, we tested the amino acid levels in center and outer leaf blades. Younger leaf blades of aposymbiotic plants (no endophyte present) had significantly higher levels of asparagine and sometimes glutamine compared to older leaf blades. The amino acid levels were much lower in MF-N. siegelii and MF-N. uncinatum compared to aposymbiotic plants and MF with Epichloë festucae (a closely related symbiont), which lacked lolines. We conclude that loline alkaloid production in young tissue depleted these amino acid pools and was apparently regulated by availability of the amino acid substrates. As a result, lolines maximally protect young host tissues in a fashion similar to endogenous plant metabolites that conform to optimal defense theory.