Heterokaryon Incompatibility in Fungi More Than Just Another Way to Die

J. Goner., Vol. 75, Number 3, December 1996, pp. 415-424. $]) Indian Academy of Sciences

Heterokaryon incompatibility in fungi more than just another way to die

JOHN F. LESLIE* and KURT A. ZELLER Department of Plant Pathology, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, Kansas 66506-5502, USA

Abstract. In filamentous fungi heterokaryon (vegetative) compatibility is regulated by a number of different loci. Vegetative incompatibili~:yis most often detected as the inability to form a prototrophic heterokaryon under forcing conditions, or as the formation of a barrage when two incompatible strains interact. Vegetative compatibility has been used as a multilocus phenotype in analysis of fungal populations. In some highly clonal populations the vegetative-compatibility phenotype is correlated with pathogenicity. The molecular basis for vegetative compatibility is not well understood. Four he~ loci have been cloned from Neurospora crassa or Podospora anserina, but no two are alike and it is clear that the her genes themselves do not encode the gone products that are directly responsible for cell death. We suggest that a broader view of vegetative compatibility would include genes that are responsible for profusion, fusion, and posffusion activities. Postfusion activities could include the fungal apoptotie apparatus since microscopic observations of cell death resemble those in higher plants and animals.

Keywords. Apoptosis; fungi;vegetative compatibility.

1. Introduction

The ready formation of a heterokaryon involving hyphal fusion and nuclear mixing is common in fungi and is an integral part of their life cycle. To the inexperienced it may appear that any pair of strains are capable of forming a heterokaryon. Upon closer inspection, however, it quickly becomes obvious that heterokaryon formation is a complex process, and that not all pairs of strains are capable of forming heterokaryons. Heterokaryons may form vegetatively or as a part of the sexual reproduction process. In some species a difference at a single gone, determining mating type, simultaneously precludes formation of a vegetative heterokaryon between two strains and is essential for formation of a sexual heterokaryon between the same two strains. Although the phenomenology of this process is well understood, its molecular basis remains obscure. Linking the molecular basis of compatibility to its corresponding phenomenology should provide new insights into basic cellular mechanisms and identify relationships between compatibility and previously described processes. Neurospora crassa has played a major role in the study of vegetative (heterokaryon) compatibility. A genetic basis for the vegetative-compatibility phenomenon was first described by Beadle and Coonradt (1944), who determined that differences at the mating-type locus were sufficient to prevent formation of a vegetative heterokaryon. Efforts to separate the sexual and the vegetative compatibility functions of the mating-type locus by recombination were unsuccessful (Newmeyer and Taylor 1967;

*For correspondence 415 416 John F. Leslie and Kurt A. Zeller

Newrneyer et aI. 1973), although these functions can be inactivated independently by mutation (Griffiths and Delange 1978; Griffiths 1982). At least one allele at the tot locus can suppress the vegetative-corapatibility interaction at the mating-type locus (New- meyer 1970) even though tol neither reduces sexual fertility nor affects interactions between alleles at any of the other known her loci (Newmeyer 1970; Perkins 1975; Johnson 1979; Leslie and Yamashiro 1997). In N. tetrasperma, tol plays a crucial role in establishment and maintenance of the pseudohomothallic life style (Jacobson 1992). Three loci involved exclusively in vegetative compatibility were identified by Garnjobst (1953, 1955) and Wilson and Garnjobst (1966). One of these loci, her-C, has recently been cloned (Saupe et al. 1996). Mylyk (1975) expanded the number of her loci to ten by identifying regions that contained one, or more, heterozygous het loci through phenotypic differences in partial aneuploids. Genetic evidence consistent with existence of multiple alleles at individual bet loci has been reported (Howlett et al. 1993), but final confirmation of these results will require cloning the putative alleles and testing their fhnctional relatedness. The involvement of loci other than her loci in the vegetative- compatibility phenomenon has been recently described (Arganoza et at. 1994). Studies of vegetative compatibility in filamentous fungi have expanded to numerous other species, including species of Aspergiltus, Colletotrichum, Cryphonectria, Fusarium, Ophiostoma and Podospora. Three primary areas of study are: (i) descriptions of phenomenology, (ii) identification of population and evolutionary implications of heterokaryon formation, and (iii) elucidation of molecular mechanisms. Readers who want greater depth than is presented here should consult recent review articles by B6gueret et al. (1994), Glass and Kuldau (1992), and Leslie (1993, 1996).

2. Phenomenology

The phenotypes of vegetative-incompatibility interactions and the methods used to detect them are critical to the understanding of vegetative compatibility. The most common phenotypes are (i) inability to form a prototrophic heterokaryon under forcing conditions, and (ii) formation of a barrage when two incompatible strains interact. The intensity of the interaction is known to vary depending on the locus, the alleles, and the number of gene differences involved (Mylyk 1975; Anagnostakis and Waggoner 1981). Most studies focus only on allelic interactions, i.e. incompatibility resulting fron~ interactions of different alleles at the same locus. However, nonallelic interactions, in which two alleles at different loci interact to give a vegetative- incompatibilityinteraction, are known in P. anserina (see Glass and Kuldau E1992] and B6gueret et al. [19941 for recent reviews). Existence of nonallelic interactions in other fungi has not been demonstrated. Heterokaryons resulting from protoplast fusion of otherwise vegetatively incompatible strains are often quite different from similar heterokaryons formed following hyphal anastomosis (Adams et al. 1987; Stasz et aI. 1989). The killing reaction seen in heterokaryons formed via hyphal fusion may be lacking in those formed following protoplast fusion (Dales and Croft 1977; Ferenczy et al. 1977; Peberdy and Ferenczy 1985; Moln~tr et al. 1990). These data, when combined with information on some of the cloned her loci, suggest that cell-wall or cell-lnembrane components are responsible for triggering the kitling reaction associated with vegetative incompatibility. Such an explanation could also be used to explain the survival of heterozygous Heterokaryon incompatibility in Jhngi 417 partial diploids in Neurospora that carry both het alleles in the same nucleus (Mylyk 1975). If the vegetative-incompatibility reaction requires an interaction between two different cell walls (or cell membranes), the partial-duplication method of identifying het loci may miss some loci. An additional difficulty in characterizing hot genes is in cloning of the loci. Direct selection for transformants is possible in systems where barrages can be observed if a transformation results in a change fi'om one vegetative-compatibility group to another. Transformants would produce a barrage with neighbouring colonies of a different compatibility group and should be relatively easy to pick out. Cloning het genes in other systems is more difficult since the only obvious phenotype of heterokaryons for alleles at a her locus is cell death. With individual clones it may be possible to transform strains that differ at a single hot locus, e.g. het-x ~ and het-xtL If the transforming DNA carries the het-x" allele, then a hot-x" recipient should grow normally, while a het-x u recipient should have at best the inhibited growth seen in a heterozygous partial duplication of the same composition. Successful cloning at- tempts have usually relied on a chrornosonae-walking technique to identify the initial library clones that are then subcloned to identify the hot allele.

3. Population considerations

Under laboratory conditions, stable heterokaryons form only when the stratus are genotypically identical at all of the her loci. Under field conditions these different genotypes are called vegetative-compatibility groups (VCGs), and strains in one VCG are genetically isolated from strains in other VCGs during asexual reproduction. VCGs serve as a natural means to subdivide populations of fungi that spend a large fraction of their life cycle reproducing asexually (Leslie and Klein 1996). If selection acts to maintain a la'ge number of VCGs within a population, perhaps due to values of individualism (Rayner 1991) or to reduce the spread of infectious agents (Caten 1972; Hartl et at. 1975), then frequency- dependent selection may play an important :role in maintaining the array of VCGs and in keeping a large number of het loci heterozygous within the population. Population studies rely heavily on the ease with which strains can be assigned to a VCG. Barrage reactions are the fastest and easiest to assess since all that is required is to place two wild-type strains adjacent to one another on an appropriate medium and then score their interaction at a later date. Heterokaryon forcing techniques are somewhat more laborious than the simple assessment of barrage interactions. If heterokaryons are to be forced, then the sirnplest possibility is to use a doubly marked system such as that described by Leslie (1993). In this system a laboratory strain carries two mutations and can grow on a defined medium only when it makes a heterokaryon with a wild-type strain, Nitrate non-utilizing (nit) mutants, which can be isolated as spontaneous chlorate-resistant sectors, have also been used as forcing markers in heterokaryons to identify VCGs in nmnerous fi.mgal populations (Correll et aI. 1987; Leslie 1993). VCGs provide a multilocus phenotype that is well suited for measuring genotypic diversity. Compared to some other multilocus techniques, such as DNA fingerprint probes, VCGs require less laboratory sophistication, but may not sample as many loci and the data obtained may be more difficult to interpret. Although VCG techniques provide a powerful tool for determining clonality, they cannot be used to ascertain any 418 John F. Leslie and Kurt A. Zeller relationship other than identity or non-identity. Since VCGs arise primarily through recombination, strains in different VCGs are always different from each other. Strains in the same VCG are usually assumed to be clones, but the more frequent recombination is within a population, the more likely this assumption is wrong. In practice, VCG distribution can be used to estimate the amount of recombination that is occurring, if it is assumed that individual her alleles and VCG phenotypes are selectively neutral (Milgroom 1996). At present it is not possible to determine frequencies of alleles at individual her loci; however, as more her loci are cloned it should be possible to develop specific probes or primers, or both, that can identify alleles and allow analyses of gene frequencies, as well as genotype frequencies, with respect to het loci. Field populations of fungi are usually diverse with respect to VCG, with the exception of some highly clonal pathogen populations (Mylyk 1976; Leslie 1993, 1996). In some cases pathogenic and nonpathogenic strains appear to be closely related, e.g. in Fusarium oxysporum f. sp. cycIaminis (Woudt et aI. 1995), but more often the nonpathogenic strains appear quite diverse and are not particularly closely related either to each other or to the pathogenic strains. If the population is diverse with respect to VCG, then studies that track the release of individual strains are possible and can be especially useful in studies of epidemic development or of persistence and spread of a strain fi'om a defined source, if only limited VCG variability is present, then VCGs may be useful as a diagnostic tool. To da~e, all evidence suggests that vegetative compatibility is merely correlated with but has no direct effect on pathogenicity. Thus any observed correlations should be experimentally verified with a large number of strains fi'om diverse sources before firm pronouncements about the diagnostic utility of a VCG are made.

4. Molecular mechanisms

The molecular mechanism(s) underlying the vegetative-incompatibility reaction has been of interest since the phenomenon was first described over 40 years ago. Garnjobst and Wilson (1956) described the microscopic symptoms of cell death due to vegetative incompatibility, which resemble those now attributable to apoptotic cell death. Wilson etaI. (1961) demonstrated that cytosolic factors, probably proteins, could trigger cell death. These cytosolic factors retained activity after DNAase or RNAase treatments, but were inactivated by heating, denaturing conditions, or treatment with proteases. Williams and Wilson (1966) demonstrated that the active component(s) may be associated with RNA molecules. They also determined that either the reactive incompatibility factor, or its temporary binding site, was closely associated with the cell membrane, and that initiation of cell death required saturation of a receptor molecule. Microscopic observations of incompatibility-reaction phenotypes in N. crassa are similar to the response observed in Podospora (see Garnjobst and Wilson 1956). In P. anserina vegetative incompatibility initiates synthesis of proteases and a phenoloxidase (B6gueret 1972; B~gueret and Bernet 1973) from existing mRNAs; these enzymes have been implicated as the effectors of cell death. Competitive protease inhibitors attenuate the effects of these proteases, and inhibit the killing reaction in Podospora (Delettre et cd. 1978), confirming that these cellulytic enzymes are directly involved in the death process. Similar mechanisms are likely to occur in N. crassa. Heterokaryon incompatibility in J~,n,gi 419

In N. crassa partial-aneuploid strains heterozygous for individual her loci are characterized by slow initial growth with little to no aerial hyphae, and by the production of a characteristic pigment (dark agar) on phenylalanine/tyrosine media. Growth is more inhibited in some partially heterozygous combinations than in others (Mylyk 1975), and both growth and pigment production may be ahnost completely inhibited. Despite the presence of incompatible het loci in the same cytoplasm, however, partial heterozygotes do not self-lyse (Perkins 1975). The phenotype of incompatible N. crassa het-I allele interactions is different from those described for other her interactions (Pittenger and Brawner 1961). Instead of a cell-death reaction, heterokaryons that contain incompatible het-I alleles gradually lose one of the two nuclear types. No simnilar genes have been identified in filamentous ascomycetes, and the mechanisms involved are unknown at present.

4.1 Cloned genes from N. crassa

Alleles at two loci that affect vegetative compatibility have been cloned fl'om N. crassa. The first locus cloned was mating type (Glass et al. 1990; Staben and Yanofsky 1990), at which there are two functional alleles (idiomorphs). Sexual and vegetative compatibility are known to be encoded by different portions of the same protein (Philley and Staben 1994), but little else is known about their vegetative-compatibility function. The second locus that has been cloned is het-C. Incompatible interactions at this locus are characterized by a slow-growing, curly, aconidial phenotype that is less severely debilitated than the incompatibility phenotypes attributed to differences at other het loci (for illustrations see Mylyk 1975). het-C encodes a protein of 966 amino acids (Saupe et al. 1996). The amino-terminal segment contains a region with high sequence similarity to functional signal peptides, indicating that this protein could enter a secretory pathway; however, the protein contains no strongly hydrophobic regions to suggest a transmem- brane structure. Other regions within the het-C polypeptide are similar to sequences commonly involved in protein-protein interactions, and the carboxy-terminal region is similar to sequences coml-nonly associated with cell-envelope proteins. Null rnutations at het-C have no apparent phenotypic effect, other than allowing vegetative compatibility with both het-C and het,-c alleles (Saupe et al. 1996). These data suggest that het-C has a nonessential function associated with the cell wall, and that it is involved in a protein- protein interaction. How the different alleles recognize each other and how this recognition leads to cell death remains unknown.

4.2 Cloned genes from P. anserina

Three loci that affect vegetative compatibility have been cloned fi'om P. anserina. These loci all appear to be different from each other and from the het-C locus of N. crassa, het-s was the first heterokaryon-incompatibility locus to be cloned; both het-S and het-s alleles have been cloned (Turcq et at. 1990). Each allele encodes a polypeptide of 289 amino acids, and the two alleles differ at 14 amino acid resi- dues; however, a single arnino-acid substitution is sufficient to elicit a vegetative- incompatibility response (Deleu et al. 1993). Loss of function at het-S does not affect viability or sexual fertility (Turcq et al. 1990), and the protein currently has no known function in the vegetative-incompatibility interaction process. 420 John F. Leslie and Kurt A. Zeller

Nonallelic vegetative-incompatibility interactions are also known in P. anserina. In these interactions particular alleles at two different loci are involved. Alleles at two loci involved in such a nonallelic interaction have been cloned and characterized in P. anserina. Alleles at the het-e locus encode a polypeptide of 1356 amino acids (Saupe et al. 1995). The carboxy-terminal region is composed of sets of 42-amino-acid repeats similar to those found in fl-subunits of trimeric G proteins, which also have been implicated in a variety of protein-protein interactions. The amino-terminal region aIso is similar to consensus GTP-binding regions of GTPases such as the G proteins (Saupe et aI. 1995). G proteins play an important role in signal transduction pathways, e.g. pheromone responses in S. cerevisiae, and as an information relay step to MAPK (mitogen-activated protein kinase) signal transduction pathways that in turn regulate cell differentiation (Herskowitz 1995). While the similarity between het-e and G proteins suggests a possible signal transduction role for the het-e gene product, it has not yet been determined whether the het-e gene participates in such a signalling interaction. Alleles at the hel-c locus also have been cloned, sequenced, and characterized (Saupe et aI. 1994). het-c encodes a polypeptide of 208 amino acids with homology to proteins involved in glycolipid exchange between cell membranes (Saupe et at. 1994). Unlike the other characterized vegetative-incompatibility genes, inactivation of het-c has pleiotropic effects on nuclear distribution and ascospore size during postmeiotic divisions (Saupe et aI. 1994), suggesting that this gene product is required for proper completion of the sexual cycle in addition to playing a role in vegetative compatibility.

5. An expanded view of vegetative compatibility

Vegetative compatibility has traditionally been viewed from the perspective of the her loci, and questions about vegetative compatibility are often fi'amed in terms of how her alleles could interact directly to produce a killing reaction. With the cloning of several her genes from Neurospora and Podospora it is clear that het genes do not encode the proteins that are directly involved in cell death. Instead het loci are part of a larger group of genes responsible for the establishment and maintenance of a stable heterokaryon. One view of this process is outlined in figure 1. Mechanical and genetic processes leading to her interactions begin before hyphal fusion, are continued through one or more signalling networks, and culminate either in the formation of a stable heterokaryon or in a cell-death process (apoptosis).

Self I Non-Self Maintenance / Initiation Recognition Signaling Rejection (Pre-Fusion~--~ ( Fusion

hsi Genes her Genes su~Genes Apoptotic* Genes

9Proximity Pheromones 9Structural Genes 9Downstream bet Receptors . Proteolytic Enzymes 9Pheromone Receptors 9Self/Non-Self 9Protein Kinases ' DNAses 9 2 ~Branching Deficiencies Recognition Genes 9MAPK-like Signal Cascades 9RNAses 9Fimbriae Deficiencies 9Upstream Effeetors of Apoptosis

Figure 1, A simplified model identifying steps in the vegetative-incompatibility interaction process and some of the genes that might be associated with these steps. Heterokaryon incompatibility in )hncdi 421

Prefusion genes (figure 1), typified by hsi (heterokaryon self-incompatible) genes, are known from several fungal species (Hyakumachi and Ui 1987; Correll et al 1989). One of the best characterized hsi interactions implicates a reduction of interactive fimbriae as a reason for lack of fusion between otherwise genetically cornpatible individuals (Correll et al. 1989). However, genes involved in cell wall synthesis, or hormone production or response could also result in similar phenotypes. These sorts of genes could function in proximity recognition, and in response mechanisms necessary to allow contact and hyphal fusion to proceed. Fusion genes (figure 1) are genes that are expressed in the fused cell. Their protein products may be found in the cell wall, the cell membrane, or in the space separating the two. They may mediate self/nonself recognition and cell fusion through allelic or nonallelic gene interactions and involve (protein?) components fi'om both participating cells. A recognition of 'self' would result in no adverse changes in the cell's metabolism and no new signals would be sent to the signal pathways. In a case of 'nonself' recognition at least two types of interactions are possible. First, a nonself interaction may produce a positive (evolutionarily intended or otherwise coopted) signal that feeds directly into a postfusion signal pathway. Alternatively, a 'nonself' recognition could disrupt the equilibrium in a critical regulatory or maintenance process. This disruption would then indirectly activate the apoptotic response. The data on the her genes characterized to date are insufficient to discriminate between these two hypotheses. It is likely that several mechanisms mediate the self/nonself recognition process since there are numerous her loci, and the alleles at one her locus generally do not interact with those at other her loci. Postfusion genes (figure 1) are much less understood. Conceptually, it is easiest to envisage these genes as those that relay the signal from the her gene interaction to the cell-death apparatus. Evidence for the existence of these genes is now becoming available. In N. crassa, Arganoza et al. (1994) identified a number of mutations that can override heteroallelic incompatibility interactions at one or more her loci. The nature and mode of action of these mutations are presently unknown; however, their existence suggests that individual her gene interactions are interpreted by a downstream network of regulatory genes. The observation that unlinked single-gene mutations can affect incompatibility responses at more than one her locus is consistent with the hypothesis that these signalling genes may accept stimuli from multiple inputs. Similar genes, the rood genes, can alter vegetative compatibility in P. anserina. A double mutant, modA modB, is female sterile and inhibits the nonallelic incompatibility between het-d and het-e (Boucherie and Bernet 1974). These double mutants cannot initiate perithecial or aerial organ formation, processes that are generally thought to be dependent on specific proteases and a phenoloxidase, which are not produced by these double mutants. Another mutation (modD) affects both allelic (Bernet 1992) and nonallelic vegetative-incompatibility interactions (Labar~re and Bernet 1979a; Dur- tens and Bernet 1982). modD has been implicated in initiation of differentiation and exit from stationary phase through the action of its proteolytic activities (Labar~re and Bernet 1979b); however, the relationship of this effcct to vegetative-incompatibility responses is not yet understood. Recently, other loci in Podospora (Bourges et al. 1996), Neurospora (Jacobson et al. 1995) and Fusarium (Kuhn et at. 1996; Zeller and Leslie 1996) that can mitigate the vegetative-incompatibility interaction also have been described. In these cases mutations at the modifying loci all result in broader vegetative compatibility. All of these loci 422 John F. Leslie and Kurt A. Zeller could belong to the postfusion class of genes and could play a role in relaying signals from the initial her interactions to the cell-death apparatus. That many of these mutants do not affect all of the hot gone interactions in the organism suggests that the number of loci in this class is large and that their relationships are complex. The final set of genes are those involved in the apoptotic cell death process (figure 1). Apoptosis is a gene-directed, natural death process used by eukaryotic cells to respond to external and internal stimuli (Williams and Smith 1993), and is composed of several well-defined stages (Vaux and Strasser 1996). Following an initial biotic or abiotic stimulus, a signal is transmitted to the apoptosis apparatus. Proteases and cellulytic enzymes and their positive and negative regulators are activated, leading to cell death in a characteristic manner that is conserved across plant and animal kingdoms (Wang etal. 1996). Many of these characters are similar to the microscopic observations of death resulting from incompatible hyphal fusions in N. crassa (Garnjobst and Wilson 1956; Wilson etal. 1961; Williams and Wilson 1966). There remain many questions to be answered regarding the initiation and maintenance of heterokaryons, and the genetic interactions that are involved. How are self/nonself recognition signals transmitted to downstream response components? How complex and interrelated are response pathways for individual hot genes? Do they share components? Is there a hierarchy of responses? Are differences at some loci more destabilizing than differences at other loci? Through an expanded view of vegetative compatibility it should be possible to integrate it more tightly with our knowledge of fungal physiology and biochemistry and to increase our understanding of the biology of these beguilingly complex microorganisms.

Acknowledgements

Work in J.F.L.'s laboratory is supported by USDA, INTSORMIL, and the Kansas Agricultural Experiment Station. Manuscript no. 97-354-J fi'om the Kansas Agricul- tural Experiment Station, Manhattan.

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