Proteomic investigation of interhyphal interactions between strains of Agaricus bisporus
Introduction
Unicellular fungi from the Ascomycota phylum, such as the yeasts Candida albicans and Saccharomyces cerevisiae, form adhesions for cell–cell communication, biofilm formation, pathogenesis, commensalisms and primary phases of saprophytic interactions composed of mannoproteins covalently bound to the cell wall (Lipke, 2018). Adhesion and signalling domains are critical for the innovation from unicellularity to complex multicellularity (CM). Phylogenomic and genomic evidence suggests that CM has independently evolved in five eukaryotic groups including the fungi (Knoll, 2011). Within the fungi it occurs in most major clades and displays at least 8 and perhaps as many as 11 independent origins (Nagy et al., 2018). A variety of complex micropore structures bridge intracellular connections of multicellular ascomycete and basidiomycete hyphae (Markham, 1994). Hyphae are the structural units, segmented by septa (Harris, 2001), of vegetative growth in filamentous fungi. They are tubular in shape and have polarised extensions through apical growth mediated by high pressure and vesical transport of a multitude of important enzymes for cell wall biosynthesis. These enzymes remain inactive in cytoplasmic transit until buried in the transmembrane of apical regions, whereby the synthesis of β-1-3-glucan and chitin can occur and recruitment of cytosolic-derived glycoproteins from the endoplasmic reticulum-to-Golgi pathway for cell wall biogenesis (Riquelme et al., 2016). In fast growing hyphal tips, a cytoskeletal-derived structure known as the Spitzenkörper, both fuses and extends the cell wall of the apex by recruitment of transport vesicles (Bartnicki-Garcia et al., 1989) and is responsible for the directionality of hyphal growth (Reynaga-Pena et al., 1997). Hyphae both extend at their apex and form sub-apical growth of branching hyphae commonly in a bifurcating fashion (Girbardt, 1957, Girbardt, 1969, Löpez-Franco and Bracker, 1996, Riquelme and Bartnicki-Garcia, 2004). Anastomosis (hyphal fusion) of branching and apical hyphae takes place to form the reticulating network architecture known as the mycelium (Fig. 1).
The process of anastomosis allows the mycelium to form large single-unit colonies for purposes of heightening exocytosis coverage for chemotactic activity and hydrolysing proteins allowing for physical expansions in ecosystems. Mating of combinations of different cultures was first evaluated when it was found that mixing cultures of the model fungus Aspergillus nidulans created parasexual recombinants (Pontecorvo et al., 1953). Parasexuality in filamentous fungi allows heterokaryons with different genotypes to undergo anastomosis and form a new hybrid heterokaryotic mycelium with cytoplasmic exchange (plasmogamy) and novel nuclear types, conferring genetic advantages to species, particularly those that may have low rates of meiosis or recombination (Glass and Fleissner, 2006, Pontecorvo, 1956, Swart et al., 2001). To prevent anastomoses of incompatible hyphae or hyphae that may incur deleterious interactions, a vegetative incompatibility complex (vic) and a sexual incompatibility (het) system, mediated by mating loci, are found in filamentous fungi. Incompatible fusions of fungal hyphae can trigger inhibited growth and even programmed cell death (Biella et al., 2002, Garnjobst and Wilson, 1956, Labarere and Bernet, 1977, Sarkar, 2002). These systems can act as protective mechanisms where anastomosis with non-self hyphae could be disadvantageous. An example of such is fusion with a foreign mycelium harbouring infectious intracellular mycoviruses (Chu et al., 2002, Grogan et al., 2004, Kashif et al., 2019, Romaine et al., 1993, van Diepeningen et al., 1998).
A. bisporus is the most extensively cultivated mushroom in the world and is grown commercially on a pasteurised compost substrate most commonly composed of wheat straw, horse and/or poultry manure and gypsum (Van Griensven, 1987, Vedder, 1978). In commercial practice, two main phases precede the formation of mushrooms; the spawn run phase and casing phase. Focusing on the former, the success of the spawn run phase is dependent on the compost substrate being heavily colonised by A. bisporus hyphae (Kabel et al., 2017), a process that involves mass breakdown of aromatic lignins, cellulose, hemicellulose and nitrogen sources (including bacterivorous nutrient acquisition (Fermor et al., 1991)). To begin this process, pasteurised compost is ‘seeded’ with A. bisporus-coated spawn grains that instigates the process of compost colonisation. These isolated colonies must undergo self-recognition and anastomose with other colonies. While studies have focused on molecular mechanisms governing how A. bisporus breaks down commercial compost (Pontes et al., 2018, Wood and Thurston, 1991, Yague et al., 1997), little attention has been paid to the impact of colony recognition/anastomosis in this process. To address this, we have analysed the proteomic response of three different strains of A. bisporus in-vitro to build an understanding of the molecular mechanisms governing inter-hyphal interactions. Particular focus is paid to the globally cultivated present day white-hybrid mushroom strains, as they have been almost exclusively used in commercial mushroom industries for nearly three decades due to their commercial appeal. There is very little genetic variation with these white-hybrid strains (Sonnenberg et al., 2017), so they are all susceptible to the same diseases worldwide, including disease-causing mycoviruses, the transmission of which is governed by anastomosis of infected mycelium with healthy mycelium (Grogan et al., 2003). Breeding research that focuses attention on novel hybrids that have vegetative incompatibility with present day white-hybrids, and which do not readily form hyphal fusions, would pave the way for ‘virus-resistant’ varieties. This is exemplified by a control method once used against mushroom virus disease, which required growing a “virus-breaker” strain such as A. bitorquis, instead of A. bisporus, as they do not readily anastomose, thereby preventing transmission of mycoviruses to the new crop (Van Zaayen, 1978, Fletcher and Gaze, 2007), although hyphal anastomoses between them may still occur. Three A. bisporus are included in this study which include a present day white hybrid Sylvan A15, a novel experimental fourth generation hybrid strain, referred herein as CWH, which has shown heightened resistance to mushroom virus X (data unpublished) and ARP23, a wild isolate of the ARP collection (Callac et al., 1996). Wild strains of A. bisporus have also shown promise in terms of disease resistance (Glass et al., 2000, Glass and Kuldau, 1992, Leslie, 1993) due to their lack of vegetative compatibility (in heterokaryotic terms) with commercial white strains.
Inter-hyphal fusion in the commercial button mushroom fungus A. bisporus is a key area of interest due to its importance in successful compost substrate colonisation and the roles it plays in the spread of deleterious mycoviral diseases. In this study, phenotypic evidence shows A15 anastomoses more readily with itself than with CWH or ARP23 strains. By using a combination of three distinct strains of A. bisporus, an untargeted approach was taken to elucidate the proteomic response of anastomosis.
Section snippets
Strains and culture conditions
Three strains of A. bisporus were used in this study: (1) commercial strain A15, (2) a novel experimental fourth generation hybrid strain (CWH), and (3) a wild strain ARP23 from the Agaricus Resource Program (ARP) collection (Callac et al., 1996), all obtained from Sylvan Inc., France. All strains were grown on complete yeast media (CYM) containing 2 g proteose peptone, 2 g yeast extract, 20 g glucose, 0.5 g MgSO4, 0.46 g KH2PO4, 1 g K2HPO4, 10 g agar in 500 ml dH20. Once molten media had
Interactions of three strains of A. bisporus
Observations were made between anastomoses of A15 with A15 (A15-A15), A15 with CWH (A15-CWH) and A15 with ARP23 (A15-ARP23). A clear distinction was evident between the interactions of A15-A15 (self) and the other combinations. When A15 was paired with itself, a plethora of hypha–hypha fusion ensued upon interaction of the two colonies growing in the same trajectory (positive tropism) (Fig. 2A). However, a characteristic demarcation zone of interaction was still evident pertaining to a level of
Discussion
Many studies into the outcomes of fungal interactions and anastomosis have been carried out in the ascomycetes and basidiomycetes, ranging from biocontrol of economically-damaging plant pathogens to understanding wood decomposition fungal community structures (Ainsworth and Rayner, 1986, Boddy, 2000, Van Bael et al., 2009, Schöneberg et al., 2015, El Ariebi et al., 2016, Hiscox et al., 2017). Many studies have also been conducted into the hyphal fusion process fungi undertake to form mycelium
Conclusion
This is the first study to characterise the proteomic response of three interacting A. bisporus strains ranging from full vegetative compatibility to incompatibility. New insights into pathways and candidate proteins vital to anastomosis have been discussed. Our analyses shows that vegetative compatible interactions are represented by high levels of carbohydrate metabolism in the form of cell wall biogenesis, modification, and expansion. With respect to co-cultures, A15-CWH represents less of
Acknowledgments
EOC is funded by a Teagasc Walsh Fellowship Scheme (grant reference number 10564231). We acknowledge the DJEI/DES/SFI/HEA Irish Centre for High-End Computing (ICHEC) for the provision of computational facilities and support. Mass spectrometry facilities were funded by Science Foundation Ireland (SFI 12/RI/2346(3)).
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