The Aspergillus nidulans rcoA Gene Is Required for veA-Dependent Sexual Development

Abstract

The Aspergillus nidulans rcoAΔ mutant exhibits growth and developmental defects. We show that the rcoAΔ mutant lacks cleistothecia and is self-sterile. In crosses with wild-type strains, rcoAΔ nuclei do not contribute to the cleistothecial walls. Furthermore, sexual development resulting from veA overexpression is rcoA dependent, indicating that rcoA lies downstream of veA in the sexual development pathway.

ASPERGILLUS (Emericella) nidulans undergoes asexual development to produce spores (conidia). As growth proceeds, sexual development occurs, producing presumed nurse cells called Hülle cells (Eidam 1883; Ellis et al. 1973) and closed fruiting bodies (cleistothecia) containing unordered red ascospores in asci (Eidam 1883; Benjamin 1955; Zonneveld 1988). Each cleistothecium contains up to 10,000 ascospores that are the meiotic progeny of a single dikaryotic ascogenous hypha (Pontecorvo et al. 1953). The balance between vegetative growth, asexual development, and sexual development is tightly controlled (Tsitsigiannis et al. 2004, 2005). The rcoA gene is required for normal vegetative growth and asexual development (Hicks et al. 2001). We show here that rcoA is a pivotal sexual development regulator and lies downstream of veA in the sexual development pathway.

rcoA is required for sexual development and nuclear contribution to the cleistothecium wall:

A. nidulans is homothallic and therefore does not require a partner for production of selfed cleistothecia. We observed that the rcoAΔ mutation affects sexual development as rcoAΔ mutant homokaryons do not form cleistothecia and are therefore self-sterile. Heterokaryons formed between two individuals produce selfed cleistothecia, where both nuclei forming the zygote are from the same parent, and hybrid cleistothecia, which arise from a zygote derived from cross-karyogamy (fusion of one nucleus from each parent) (Pontecorvo et al. 1953). We studied the behavior of rcoAΔ nuclei during sexual development in [rcoAΔ + rcoA⁺] heterokaryons. We used, as a marker for nuclear contribution to the cleistothecium wall, the blue ascus (blA1) mutation (Apirion 1963), which acts cell autonomously to confer blue cleistothecial walls and in a cell-nonautonomous fashion to cause blue ascospores irrespective of ascospore genotype. As expected, in wild-type × blA1 crosses selfed and hybrid cleistothecia containing either blue or red ascospores were observed (Table 1). Cleistothecia with blue ascospores arise from two nuclei from the blA1 parent contributing to the cleistothecial wall, whereas cleistothecia with red ascospores occur when the cleistothecial wall is generated from a dikaryon in which one or, more commonly, both nuclei arise from the wild-type parent (Zonneveld 1988; Bruggeman et al. 2003). We reasoned that in crosses between the blA1 mutant and a mutant unable to contribute nuclei to the cleistothecial wall, all of the cleistothecia (either selfed or hybrid) would contain blue ascospores because the blA1 strain is the only parent able to contribute to the wall. In rcoAΔ × blA1 crosses blue selfed and hybrid cleistothecia but not red cleistothecia were observed (Table 1). Thus rcoAΔ nuclei participated in zygote formation but were not observed to contribute to the cleistothecium, suggesting that rcoA is required for nuclear contribution to the cleistothecial walls.

TABLE 1.

The rcoAΔ mutant does not contribute nuclei to the cleistothecium wall

	Ascospore colorb	No. of cleistothecia
Crossa			Selfedc	Crossed	Total
Wild type × blA1	Red	12	4	16
	Blue	2	1	3
rcoAΔ × blA1	Red	0	0	0
	Blue	18	2	20

^aCrosses were carried out using the heterokaryon technique (Pontecorvo et al. 1953). Crosses using the blA1 marker for contribution to the cleistothecial wall were based on the method of Apirion (1963). Briefly, cleistothecia were cleaned and physically ruptured in 100 μl of sterile water in individual wells of microtiter trays and ascospore and/or cleistothecial wall color was scored using a dissecting microscope. The genotypes of the strains used are: wild type (MH1: biA1; veA1), blA1 (A268: yA2; wA3 thiA4 cnxE16 adD3 blA1; veA1), and rcoAΔ (MH9748: biA1; rcoAΔ; veA1). MH9748 was derived from an outcross of AnrcoA (biA1; rcoAΔ argB2; methG1) (Hicks et al. 2001). Crosses used segregating conidial color markers to distinguish hybrid and selfed cleistothecia by the appearance of color recombinants among the progeny. Three microliters of each ascospore suspension was spread on Aspergillus complete media (Cove 1966), incubated for 2 days, and scored by visual inspection for conidial color.

^bAscospore color phenotype in the cleistothecium.

^cThe genotype of selfed cleistothecia corresponded in all cases to the genotype of the parent with the same color ascospores.

Introduction of additional copies of rcoA promotes sexual development by suppression of the veA1 phenotype:

Multicopy rcoA strains were generated by introduction of the rcoA gene into MH10447 (biA1 pyrG89 veA1). Transformants containing additional copies of rcoA formed normal asexual and sexual developmental structures but produced more cleistothecia than control transformants (Table 2). Most laboratory A. nidulans strains, including the recipient, carry the veA1 mutation, which confers reduced sexual development and increased asexual development (Käfer 1965). The multicopy rcoA transformants resembled veA⁺ colonies, with sexual development favored over asexual development, suggesting that increased expression of rcoA partially suppresses the veA1 phenotype (Table 2). veA⁺ strains show increased conidiation in the presence of a light source compared with dark-grown cultures (Mooney and Yager 1990). Some, but not all, multicopy rcoA transformants showed significant light-dependent conidiation. Therefore additional copies of rcoA suppress the veA1 phenotype and drive sexual development. Similarly, A. nidulans transformants carrying more than two copies of the Penicillium marneffei rcoA homolog tupA, which complements the growth and developmental phenotypes of the A. nidulans rcoAΔ mutant (Todd et al. 2003), show increased sexual development and reduced asexual development (data not shown).

TABLE 2.

Multiple copies of rcoA promote sexual development

		Conidia (×106)c		Cleistotheciad
Straina	Copies of rcoAb	Lighte	Darkf	Light	Dark
MH1: veA1	1	36.5 (2.7)	21.8 (1.1)	71.8 (4.1)	81.7 (3.3)
WIM126: veA+	1	29.0 (0.9)	0.5 (0.2)	164.8 (4.2)	119.3 (3.8)
EV1: veA1	1	41.5 (2.6)	9.9 (1.1)	100.8 (5.1)	87.3 (3.7)
EV2: veA1	1	52.3 (4.4)	14.8 (0.9)	91.5 (6.6)	87.3 (3.2)
RMC1: veA1	2	40.2 (0.6)	8.6 (0.8)	104.7 (3.6)	106.7 (4.8)
RMC2: veA1	2	41.5 (5.2)	8.8 (0.7)	130.2 (3.9)	160.3 (7.4)
RMC3: veA1	2	8.6 (1.2)	0.3 (0.0)	149.2 (3.9)	172.5 (5.6)
RMC13: veA1	3	21.7 (1.1)	0.6 (0.2)	185.8 (6.5)	184.8 (6.4)
RMC5: veA1	10–15	44.7 (1.9)	18.7 (2.6)	166.7 (9.6)	177.5 (5.9)
RMC6: veA1	10–15	27.5 (1.6)	15.2 (0.7)	175.5 (2.4)	147.8 (4.0)

^aThe rcoA gene (−1142 to +2299; GenBank accession no. AF197225) was subcloned as a 3.4-kb PstI fragment from pW03D10 (Hicks et al. 2001) into pALX223, which contains the A. nidulans pyrG selectable marker in the backbone of pBluescriptSK−. The resulting plasmid, pRT5253, was transformed, using the protocol of Andrianopoulos and Hynes (1988), into MH10447 (biA1 pyrG89 veA1), and PyrG prototrophs were selected. The transformants RMC1, RMC2, RMC3, RMC13, RMC5, and RMC6 were chosen for analysis. MH1 (biA1), WIM126 (yA2 pabaA1; veA⁺), and two strains (EV1 and EV2), generated by transformation of MH10447 with pALX223, were used as controls.

^brcoA copy number was estimated using a 2.6-kbp SalI fragment of the rcoA plasmid as a probe in Southern hybridization (Sambrook et al. 1989) of genomic DNA isolated according to the method of Lee and Taylor (1990).

^cFor each strain, ∼10⁶ spores were spread on Aspergillus nitrogen-free minimal medium (ANM) (Cove 1966) plus 10 mm nitrate plus supplement plates and grown at 37° in the presence or absence of a light source for 48 hr. Conidia were harvested from two circular regions (20 mm diameter), and counted using a hemocytometer. The average number of conidia/10 mm² calculated from two experiments is shown with the standard error in parentheses.

^dStrains were grown on ANM plus 10 mm nitrate plus supplement plates at 37° in the presence or absence of a light source for 2 days, sealed, and then incubated for a further 7 days. Photographs were taken from three regions of growth, each representing an area of 6.75 mm², and the number of cleistothecia were counted. The average number of cleistothecia/6.75 mm² with standard error in parentheses for two independent experiments is shown.

^eCultures were incubated in the presence of a light source.

^fCultures were incubated in complete darkness.

rcoA is required for veA-dependent sexual development:

We constructed an rcoAΔ veA⁺ strain by meiotic crossing (Figure 1). The rcoAΔ veA⁺ strain showed poorer growth on complete medium than the rcoAΔ veA1 strain (Figure 1A) and the difference in growth was enhanced in the presence of 1.0 m sorbitol (Figure 1B). Therefore, veA1 partially suppresses the growth phenotype of the rcoAΔ mutant. This suggests a role for VeA in regulating growth in addition to its role in development. Neither the rcoAΔ veA⁺ strain nor the rcoAΔ veA1 strain formed cleistothecia. As the veA⁺ allele promotes sexual development in an rcoA⁺ background (Kim et al. 2002), this suggests that the rcoAΔ mutation confers self-sterility rather than reduced fertility.

Figure 1.—

The growth phenotype of the rcoAΔ is partially suppressed by the veA1 allele. A. nidulans rcoA⁺ veA1 (MH1: biA1; veA1), rcoA⁺ veA⁺ (WIM126: yA2 pabaA1; veA⁺), rcoAΔ veA1 (MH9748: biA1; rcoAΔ; veA1), and rcoAΔ veA⁺ (MH10291: rcoAΔ; veA⁺) strains were grown at 37° for 3 days on (A) complete medium or (B) complete medium plus 1.0 m sorbitol.

To distinguish whether veA and rcoA act in separate pathways or whether veA acts upstream of rcoA to regulate sexual development, we overexpressed veA under the control of the nitrate-inducible niiA promoter in the rcoAΔ mutant. The niiA(p)-veA plasmid, pVEL-nA (Kim et al. 2002), was introduced into a control strain (MH10447: pyrG89 veA1) by cotransformation with the pyrG selectable marker plasmid pAB4626 (Oakley et al. 1987; Borneman et al. 2002). Pyrimidine prototrophic transformants were inspected for veA-dependent sexual development using the method of Kim et al. (2002), following 7 days growth at 37° under conditions inducing (0.3% sodium nitrate) or not inducing (0.2% ammonium tartrate) veA expression. The niiA(p)-veA fusion gene conferred veA-dependent sexual development under inducing (0.3% sodium nitrate) conditions in 30% (3/10) of the transformants, consistent with previous findings that forced expression of veA drives sexual development (Kim et al. 2002). Cotransformation of the niiA(p)-veA plasmid and the pyrG selectable marker plasmid into the rcoAΔ mutant (MH10198: pyrG89 rcoAΔ veA1) produced transformants, 71% (61/86) of which showed reduced growth on complete medium (containing 10 mm ammonium tartrate) in the presence of 1.0 m sorbitol compared with a rcoAΔ veA1 strain. Five transformants, randomly chosen from the class showing reduced growth analyzed by Southern hybridization, contained intact copies of niiA(p)-veA (data not shown). None of three randomly chosen transformants with growth identical to the rcoAΔ veA1 strain contained copies of niiA(p)-veA (data not shown). Therefore the niiA(p)-veA fusion complemented the vegetative growth phenotype of the veA1 mutation. Surprisingly, this occurred in the presence of ammonium, suggesting that only very low levels of niiA(p)-veA expression are required. Transformants containing the niiA(p)-veA fusion gene were assessed for sexual development under conditions inducing (0.3% sodium nitrate) veA expression. No cleistothecia were observed after 7 days of growth at 37°, indicating that the niiA(p)-veA fusion gene did not drive sexual development in the rcoAΔ mutant. These data are consistent with a model in which veA acts upstream of rcoA in the regulation of sexual development. Overall, the self-sterility of the rcoAΔ mutant in veA⁺, veA1, and veA1 niiA(p)-veA backgrounds and the suppression of the sexual development phenotype of the veA1 mutation by additional copies of rcoA indicate that rcoA is required for sexual development.

We have shown that the rcoA deletion mutant is unable to self and in heterozygous crosses rcoAΔ nuclei do not contribute to the female cleistothecium wall tissue but do participate in zygote formation. Our crosses are not informative with respect to whether rcoAΔ nuclei can be contributed to the zygote from the female and male parent. The rcoA gene encodes a WD40 repeat protein with sequence similarity to the pleiotropic transcriptional repressors RCO1 from Neurospora crassa and TUP1 from Saccharomyces cerevisiae (Hicks et al. 2001). RCO1 is required for female sexual development (Yamashiro et al. 1996) and TUP1 is required for MATα sexual function (Mukai et al. 1991). Cryptococcus neoformans TUP1 also functions in mating (Lee et al. 2005). Therefore rcoA homologs act as key regulators of sexual development in fungi. Our data place rcoA in the veA sexual development pathway. veA homologs exist in the genomes of a number of fungi, including N. crassa and C. neoformans. However, the relationship of these veA homologs with their cognate rcoA homologs remains to be determined.