Importance of MAP kinases during protoperithecial morphogenesis in Neurospora crassa

Abstract

In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia.

Figure 1. Protoperithecial morphogenesis of N. crassa wild type.

LTSEM of the main stages of protoperithecial development. (A) Two ascogonial coils differentiated from the vegetative mycelium of a two day-old culture. These two coils have formed on branches (bh) off the main arterial trunk hyphae (th). Some of the surrounding branches have fused with each other, they are therefore considered to be fusion hyphae (fh). Vegetative hyphal fusion is instrumental in the establishment of a fully co-operative interconnected mycelium. Scale bar, 50 µm. (B) Higher magnification of the ascogonial coil boxed in (A). On careful inspection a septum can be seen on the lower part of the coil (aligned with arrowheads). Scale bar, 5 µm. (C) A slightly expanded ascogonial coil again formed on a side branch of a trunk hypha, the coil is being wrapped around by enveloping hyphae. Scale bar, 20 µm. (D) A slightly later stage where enveloping hyphae (arrowheads) originating from the ascogonium have wrapped around the central ascogonial coil (ac). These enveloping hyphae exhibit septation and branching. The ‘parent hypha’ (ph) of the ascogonial coil can be clearly defined, and is separated from the developing fruitbody by a basal septum (bs). (E) The subspherical shape of the protoperithecium becomes evident after additional enveloping hyphae have formed a protective casing around the ascogonium. Trunk hyphae (th), their branches (bh) and fusion hyphae (fh) can be clearly distinguished. Scale bar, 5 µm. (F) Mature protoperithecium, with visible ECM secretion ‘gluing’ enveloping hyphae together, and a trichogyne (arrowhead) emerging from its center. Scale bar, 20 µm. (G) Enlarged view of the boxed area in (D) showing ECM strands between hyphae (arrowhead). Scale bar, 2 µm. (H) Enlarged view of the boxed area in (B) showing ECM strands (arrowhead) between the tightly attaching revolutions of the ascogonial coil. Scale bar, 1 µm. (I) Enlarged view of the boxed area in (F) showing the surface hyphae of the protoperithecium evenly covered in ECM. Scale bar, 5 µm.

Figure 2. Simulation of the transition from two-dimensional hyphal growth into a three-dimensional helical object representing the ascogonial coil.

(A) Mathematically drawn model of an ascogonial coil, helix (cos t, sin t, t) from t = 0 to 6π (3 full circles). The ascogonial mother-cell is shown as a cylinder and the hyphal tip of the coiling branch is represented as a hemisphere. (B) A vertical cross-section through the coil shown in (A) where the distance between the centers of each circular cross-section is equal to the hyphal diameter (2r). (C) Diagram indicating the angle 2.4 rad (∼137.5°). (D) Position of septa from microscopical observations of numerous ascogonial coils in N. crassa. Septa are usually observed around two thirds of a revolution (240°) apart, after the coil-tip has made more than one complete revolution (2π rad or 360°). The angle between projected septa is likely to be optimized around 2.4 rad for maximum structural strength and this is represented here in the cut-away sections (i) of the coil shown in (A). (ii) The positions of the subsequent septa are shown in top view. The angle between ‘septa’ approximates to 2π–2.4 rad (∼222.5°). (E) Diagrammatic representation of an unwrapped-coil (not to scale), showing septation (black vertical lines) and branching of successive enveloping hyphae: (a), (b), and (c) (paler grey) of the coil. Branching is assumed here to occur equidistant between septa, although, in vivo the branching sometimes appears nearer to one septum. A stalk-cell is often observed in vivo. The diagram illustrates this with a basal septum (0) making a ‘stalk-cell’ compartment (s) next to the ascogonial mother-cell (am). (F) Extrapolated representation of a vertical cross-section of a simulated ascogonial coil, which has been wrapped by enveloping hyphae that would have originated from the septated compartments shown in (E). Note that the resulting coiled structure (not to scale) is approaching that of a sphere (represented by the dashed outer-circle). N.B. In vivo, enveloping hyphae tend to be narrower in diameter than the ascogonial mother-cell.

Figure 3. Main stages of protoperithecial development.

The ascogonium forms as a specialized, coiled, hyphal branch from a ‘parent hypha’ of the vegetative mycelium. The coil expands, adheres to itself, septates and branches. It sends out more branches, which envelop it. Additional, enveloping hyphae from neighboring areas of the vegetative mycelium, aggregate, reinforce and expand the protective casing around the ascogonium. Secretion of ECM is a precursor to hyphal adhesion during this process, which potentially also involves hyphal fusion. Continued fruitbody expansion, cellular differentiation through septation, branching and cell conglutination (conglutinate cells are those that have adhered to each other), melanization and emergence of the trichogyne mark the final stages of protoperithecium maturation. Mating-cell fusion leading to fertilization and dikaryon formation mark the transition into perithecial development. Autonomous developmental stages of the protoperithecium are highlighted with grey shading. Table 3 summarizes the range of sizes observed for these main developmental stages observed during protoperithecium morphogenesis.

Figure 4. Colony morphology of MAP kinase mutants.

All MAP kinase mutants showed macroscopic colony phenotypes clearly distinct from the wild type and between the three MAP kinase pathways, but highly conserved within each cascade. (A) CWI-MAP kinase mutants (Δmik-1, Δmek-1 and Δmak-1) typically showed increased autolysis resulting in rosette-like colony growth, and slow colony extension even on nutrient rich media. (B) MAP kinase mutants of the PR pathway (Δnrc-1, Δmek-2 and Δmak-2) were characterized by short aerial hyphae and conidiation starting from the colony center. (C) Colony phenotypes of OS-MAP kinase mutants (Δos-4, Δos-5 and Δos-2) comprised reduced aerial hyphae in the colony center, elevated carotenoid biosynthesis and intense production of ‘sticky’ aerial hyphae and macroconidiophores were foremost at the plate edge. (D) Wild type controls, and the ‘old’ Δos-2 strain FGSC11436, which displayed a colony phenotype different to that of the genuine os mutants (see Figure S4 for a more detailed genotypic and phenotypic comparison between the two Δos-2 mutants FGSC11436 and FGSC17933).

Figure 5. Protoperithecial development in MAP kinase gene-deletion mutants.

In comparison to the wild type, which formed regular, subspherical protoperithecia 40–80 µm in diameter, only mutants of the PR- and CWI-MAP kinase cascades formed protoperithecial-like structures of similar appearance. These however, did vary in size, shape and degree of pigmentation and were not clearly discernable as protoperithecia even to an experienced microscopist using the stereomicroscopy technique shown here. It was these observations that warranted investigations using more powerful microscopic techniques, as used for Figures 1 and 6–8. Protoperithecial-like structures could not be observed in any of the newly generated OS-MAP kinase mutants. In contrast to the other os mutants, Δos-2 FGSC11436 showed disorganized mycelial architecture, typical of hyphal fusion defects. The Δnrc-1 strains generated from FGSC18162 by vegetative homokaryon selection (HS) showed no phenotypic differences compared to Δnrc-1 FGSC11466. In order to calibrate the results, all strains were inoculated onto cellophane over LSA medium (and SCM for comparison), and incubated for 5–7 days at 25°C dependent on the rate of developmental of the mutant strain. By cutting out cellophane squares carrying mycelium the same samples as shown here were subsequently prepared for LTSEM. Finally, these female cultures were fertilized with opposite mating type conidia of the wild type to confirm female sterility. All scale bars, 50 µm.

Figure 6. ECM and hyphal adhesion seem essential for the organized assembly of enveloping hyphae into protoperithecia.

(OS) Despite several attempts, ascogonial coils, let alone protoperithecial-like structures, could not be identified in mycelia of the three OS-MAP kinase mutants. Large areas of the mycelium were collapsed, indicating extensive lysis of vegetative hyphae. Hyphal loops (a.k.a. hyphal coils or lassoes), as shown here in Δos-2 (arrowhead in E) were occasionally observed in all three mutants. These structures are frequently found in the wild type, and although their function is unknown, a connection to sexual development seems unlikely (see discussion). Scale bars: (A) 100 µm; (B, C, E) 50 µm; (D, F) 25 µm. (CWI) Δmik-1, Δmek-1 and Δmak-1 strains initiated ascogonial coils and differentiated enveloping hyphae. The assembled multicellular structures, however, remained loose hyphal aggregations and ECM was absent, suggesting that hyphal adhesion was not sufficient to form subspherical protoperithecia. Scale bars: (G) 10 µm; (H, I, K, L) 25 µm; (J) 50 µm. (PR) Δnrc-1, Δmek-2 and Δmak-2 strains produced ECM, and hyphal aggregations resembled better-organized and more spherical ‘early-stage’ protoperithecia. Nevertheless, trichogynes have not been observed in these strains, and sexual development did not progress beyond this stage. Scale bars: (M–R) 25 µm.

Figure 7. Excessive ECM deposition in OS-MAP kinase mutants and aborted fruitbody development in CWI-MAP kinase mutants.

(A) All hyphal surfaces of Δos-2 (FGSC17933) were covered with punctate clusters of ECM depositions. Scale bar, 10 µm. Magnified view in inset; scale bar, 5 µm. (B) Macroconidiophores of Δos-2 were also heavily covered in ECM material. Scale bars, 20 µm. (C) Granular ECM depositions were not present on the surfaces of matured, detached macroconidia. Scale bar, 2 µm. (D) Higher magnification of the clustered ECM depositions on a mature hyphal surface of Δos-2. Scale bar, 2 µm. (E) Smooth surface of a mature hypha of the wild type control. Scale bar, 2 µm. (F) ECM-covered macroconidiophore of Δos-5. Scale bar, 20 µm. (G) Wild type macroconidiophore. Scale bar, 20 µm. (H) CWI-MAP kinase mutant strains displayed early-onset initiation of fruitbody development at the colony periphery. Inset shows a magnified view of the protoperithecial-like ‘hyphal knot’ formed only about 400 µm behind the leading colony edge of Δmik-1. Scale bar, 100 µm; in inset 10 µm. (I) Immature multicellular structures in the sub-periphery of Δmak-1 colonies aborted, then autolyzed and were subsequently reabsorbed into the mycelium, resulting in little evidence of any recognizable protoperithecial-like structures. Scale bar, 50 µm.

Figure 8. Genetic complementation rescued protoperithecial development in all three MAP kinase mutants.

(A) Young protoperithecium of a rescued Δos-2 transformant (NCAL020) enwrapped by enveloping hyphae. Granulated ECM depositions as seen on Δos-2 hyphae (Figure 7A) could no longer be observed in the rescued Δos-2 transformants, which showed smooth hyphal surfaces evenly covered in ECM (compare to wild type in Figure 7E). Scale bar, 20 µm; in inset 5 µm. (B) Mature protoperithecium of the rescued Δos-2 transformant. Scale bar, 20 µm. (C) VHF (arrowheads) undergone in the rescued Δmak-1 transformant (NCAL010). Scale bar, 20 µm. The inset shows an ascogonial coil of this strain from which a straight hypha emerges which resembles a trichogyne initial (arrowhead). Scale bar, 5 µm. (D) Mature protoperithecium of the rescued Δmak-1 transformant. Scale bar, 20 µm. (E) CAT-mediated cell fusion (arrowheads) in a rescued Δmak-2 transformant (NCAL043). Scale bar, 20 µm. (F) Mature protoperithecium of a rescued Δmak-2 transformant. Scale bar, 20 µm.

Figure 9. Optical sectioning of developing protoperithecia.

Montages of selected optical sections through developing protoperithecia of the rescued Δmak-2 strain expressing MAK-2-GFP (NCAL043). (A) The small dimensions of a late stage ascogonial coil are fully accessible to optical sectioning when labelled with CFW and MAK-2-GFP. Scale bar, 5 µm. (B) With increasing size, CFW dye is unable to penetrate the interior of the developing fruitbody, and consequently cannot be used to optically section the interior of the ascogonium. Fluorescently labelled MAK-2, however, allows visualization of the whole protoperithecium. Scale bar, 10 µm. (C) Optical sectioning of a mature protoperithecium reveals the complex and tightly wound hyphal network comprising this structure. Scale bar, 20 µm. (D) Middle section of a protoperithecium expressing MAK-2-GFP. The corresponding surface plot shows that fluorescence intensity peaks in the central core region, suggesting that MAK-2-GFP accumulates in the ascogonial coil tissue. (E) MAK-1-GFP fluorescence in the rescued Δmak-1 strain (NCAL010) also peaked in the central ascogonium region, however, was not as pronounced as in the case of MAK-2. Scale bar, 10 µm. Movies S5, S6, S7, and S8 show full z-stacks of optically sectioned protoperithecia.