Thomas J. Leonard

Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus.

Under limiting growth conditions,Aspergillus nidulans produces a carcinogenic secondary metabolite related to aflatoxin and called sterigmatocystin (ST). The genes for ST biosynthesis are co-ordinately regulated and are all found within an approximately 60-kilobase segment of DNA. One of the genes within this region is predicted to encode a CX2CX6CX6CX2CX6CX2 zinc binuclear cluster DNA-binding protein that is related to theAspergillus flavus andAspergillus parasiticus aflatoxin regulatory geneaflR. Deletion of theA. nidulans aflR homolog resulted in an inability to induce expression of genes within the ST gene cluster and a loss of ST production. BecauseA. nidulans aflR mRNA accumulates specifically under conditions that favor ST production we expect that activation of ST biosynthetic genes is determined byA. nidulans aflR. In support of this hypothesis, we demonstrated that induced expression of theA. flavus aflR gene inA. nidulans, under conditions that normally suppress ST gene expression, resulted in activation of genes in the ST biosynthetic pathway. This result demonstrates that AflR function is conserved betweenAspergillus spp. and thataflR expression is sufficient to activate genes in the ST pathway.

Cytology of the life-cycle of Morchella

Various stages of the morel life-cycle are studied cytologically. Photomicrographic evidence demonstrates that the average number of nuclei per cellular compartment in vegetative hyphae of Morchella is 10–15 and that hyphal fusions are quite frequent. The resting structures, the sclerotia, are actually pseudosclerotia which form from the repeated branching and enlargement of terminal hyphae from either primary (homokaryotic) or secondary (heterokaryotic) hyphae. Photomicrographs also depict the development of fruiting body primordia. Photomicrographs of ascus development demonstrate autogamy rather than de novo heterokaryon formation by hyphal fusion in the subhymenial layer of the fruiting body. For the first time a comprehensive life-cycle diagram of the morel is introduced.

Three independent genetic systems that control initiation of a fungal fruiting body

SummaryThe initiation of monokaryotic fruiting in the basidiomycetous fungus Schizophyllum commune has been observed to occur spontaneously, in response to biochemical substances, and following mechanical injury. The responses to these three stimuli are genetically separable and under polygenic control.

Experimental studies on the morel. I: Heterokaryon formation between monoascosporous strains of Morchella

Three lines of evidence are reported for heterokaryon formation in Morchella esculenta and related species. Cultural studies demonstrated a genetic basis for different types of interactions between mycelia from sister and non-sister spores. Cytological studies of the mycelial interaction between non-sister monoascosporous isolates demonstrated nuclear pairing in presumptive heterokaryons. Two different mutations isolated in this study were used to show genetic complementation in these heterokaryons.

Monokaryotic fruiting in Schizophyllum commune: Genetic control of the response to mechanical injury

SummaryMonokaryotic fruiting is used as a tool to study mushroom development and differentiation in Schizophyllum commune. This paper reports data which further elucidate the genetic control of the monokaryotic fruiting response to mechanical injury. Models relating the various genes implicated in monokaryotic fruiting body production are proposed and evaluated on their ability to explain the observed data. A minimum estimate is made of the number of genes involved in the initiation of monokaryotic fruiting in response to mechanical injury.

Monokaryotic Fruiting in Schizophyllum commune: Survey of a Population from Wisconsin 1

The ability to produce monokaryotic fruiting bodies in culture was exam- ined in the progeny from 41 dikaryotic fruiting bodies of Schizop,hyllum commune collected in Wisconsin, All of the dikaryotic fruits produced some progeny which formed monokaryotic fruits spontaneously and/or in response to mechanical injury. All but two of the dikaryotic fruits produced -progeny which fruited in response to biochemical induction. Just as the three fruiting traits could be observed among the progeny from a single fruiting body, so could a monosporous colony exhibit any combination of the three fruiting responses, These three rnonokaryotic fruiting phenotypes are widely distributed in a natural population and not simply the product of selection among laboratory strains.

Nuclear Control of Monokaryotic Fruiting in Schizophyllum Commune

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Monokaryotic Fruiting in Schizophyllum Commune: Phenoloxidases

Phenoloxidases (PO) are enzymes which are known to be correlated with the initiation of reproductive structures in a variety of fungi including Panus tigrinus (Bull. ex Fr.) Sing. (Faro, 1972), Podospora anserina (Ces.) Niessl (Esser, 1968), Neurospora crassa Shear & Dodge (Hirsch, 1954), and Schizophyllum commune Fr. (Leonard, 1971, 1972; Leonard and Phillips, 1973). Phenoloxidases are coppercontaining enzymes and are generally divided into two major groups based upon their substrate specificity: the tyrosinase type and the laccase type. Those in S. commune are of the laccase type (Leonard, 1971). When PO activity was measured in dikaryotic colonies throughout a complete fruiting cycle, enzyme activity was observed to increase until the onset of sporulation and then to decrease to a nearly undetectable level (Leonard and Phillips, 1973). Previous work (Leonard, 1971, 1972) has shown that PO activity is always detectable in mycelia that were forming fruiting bodies; PO activity may or may not be detected in dikaryotic mycelia that were unable to produce fruiting structures. These observations suggested that PO activity was necessary, but not sufficient, for formation of the fruiting structure. Recently, we conducted a series of genetic studies on fruiting in S. commune (Leslie, 1979; Leslie and Leonard, 1979), and during the course of these experiments a large number of monokaryotic progeny were generated. Crosses of fruiter x fruiter, fruiter x nonfruiter, and nonfruiter x nonfruiter were made. All of these crosses produced mono-

Production of Specialty Mushrooms in North America: Shiitake and Morels

There is increasing interest in the American marketplace for mushrooms other than the common white button mushroom. The trend is toward species with more flavor. Among the new mushrooms making common appearances are the oyster mushroom and shiitake, more formally known as Pleurotus spp. and Lentinula (=Lentinus) edodes, respectively. A third type of mushroom, although less common, is the morel, Morchella spp, which is just beginning to be developed commercially. Since morels and shiitake are the more flavorful of the three mushrooms and are more difficult to produce, we focus our discussion on commercial cultivation practices for these two mushrooms and the challenges ahead for making them more readily available.

Somatic recombination of themnd chromosomal region in diploids and dikaryons ofSchizophyllum commune

Abstract Somatic recombination was compared in genetically identical diploid and dikaryotic mycelia heteroallelic for the recessive morphological marker,mnd, and six other genetic markers. Although recombination including the chromosomal segment containingmnd was detected in both types of somatic cells, the underlying process appears to be different. The diploids showed parasexual recombination associated with sectoring, whereas the dikaryon typically showed, with rare exception, a peculiar type of internuclear transfer resulting in a single genetic alteration in themnd chromosomal region. Some of the haploidmnd end products recovered from diploids produced unusual and unstable dikaryotic transformed mound tissue when mated tomnd+ strains. The instability produced normal dikaryotic sectors which proved upon analysis to be revertants to nonmounds. The results suggest that these “transformed” dikaryotic mound cells contained a donormnd nucleus and a recipient nucleus which is somehow heteroallelic [mndmnd+]. Reversion from a mound to a nonmound cell is envisioned as loss of the previously acquiredmnd allele. These results suggest that unstable mounds formed in dikaryotic colonies do not occur by a somatic recombination mechanism involving allelic substitution, as previously hypothesized, but rather by the addition of genetic material to themnd+ nucleus without genetic loss. Whatever the precise mechanism for stable and unstable mound development in dikaryons, the studies detailed within suggest that a diploid nucleus is an unlikely intermediate in the somatic recombination process.