FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridisation

Highlights

• The technique FISH was used to target viruses within the mycelium of Agaricus bisporus.

• Hyphae was cultured, permeabilised and hybridised with FISH probes on a single platform for fluorescence microscopy.

• Two distinct viruses from the MVX complex were localised within different areas of the mycelium of A. bisporus strains.

• Low levels of virus were detected within non-MVX strain hyphae and high levels were observed in MVX strain hyphae.

Abstract

Agaricus bisporus is a commercial mushroom crop susceptible to a disease caused by a complex of viruses known collectively as mushroom virus X (MVX). Symptoms of MVX include bare patches and mushroom cap discolouration (browning) in the fruiting bodies, phenotypes associated with the viruses AbV6 and AbV16, respectively. Limited understanding exists of the localisation and mobilisation of these viruses within the mycelium of A. bisporus. To this end, a non-destructive fluorescence in situ hybridisation (FISH) method was developed for in situ targeting of AbV6 and AbV16 in A. bisporus mycelium. An MVX strain associated with the bare patch disease phenotype revealed predominantly high signal towards the growing edges of cultures when probed for AbV6, with a ‘halo-effect’ of high signal intensity around putative vacuoles. An MVX strain associated with the browning disease phenotype showed high signal intensities within reticulating networks of hyphae in a highly compartmentalised manner when probed for AbV16. Localisation of the two viruses in MVX-infected cultures appears independent, as both viruses were found in completely discrete areas of the mycelium in differential patterns. FISH detected low level presence of the two viruses, AbV6 and AbV16 in a number of cultures which had tested negative for the viruses by RT-PCR. This suggests that FISH may be more sensitive at detecting viruses at low levels than molecular methods. This study demonstrates that FISH is a powerful tool in the field of mycovirology.

Introduction

Fluorescence in situ hybridisation (FISH) is a method for the hybridisation of targeted nucleic acid sequences using bespoke fluorophore-conjugated nucleic acid probes. A technique developed in the 1980's, the initial intricacies of the technique led to its use being considered challenging and difficult. Innovations in subsequent years have resulted in FISH becoming a more accessible method, with a wide breadth of applications (Huber et al., 2018), with cytogenics arguably reaping the greatest degree in advances (Jiang, 2019).

FISH is a powerful tool in microbiology. For example, it has been used in the detection of community structures in biofilms (Almeida et al., 2011; Aoi, 2002), aquatic microbiome sampling (Dawson et al., 2012; Kurisu et al., 2015; Medlin and Orozco, 2017), cultural heritage conservation (La Cono and Urzi, 2003; Urzi and De Leo, 2001), in clinical samples of blood sera (Da Silva et al., 2015) and many other uses. FISH has been used for the localisation of bacteria, fungi and viruses within respective hosts (reviewed in (Kliot and Ghanim, 2016)). The first use of FISH targeting fungi was in the yeast-like fungus Aureobasidium pullulans (Li et al., 1997). The greatest hurdle in applications of FISH in localisation studies is commonly the issue of probe penetration (Kliot and Ghanim, 2016). This is particularly relevant to fungi, as the fungal cell wall is a complex structure of hydrophobic scaffolds of α-1,3-glucan and chitin encased in layers of convoluted linkages of β-glucans, glycoproteins and α-1,3-glucan (Kang et al., 2018). The lack of or complete absence of permeability of the fungal cell wall, can act as a barrier to probe penetration (Brul et al., 1997; Teertstra et al., 2004). Due to the hydrophobicity and varying degrees of negative charge observed in the fungal cell walls (Free, 2013), non-charged semi-synthetic hybridisation probe alternates may be used, known as peptide nucleic acid (PNA) probes (Nielsen and Egholm, 1999). PNA probes have seen many uses of FISH in fungi (Da Silva et al., 2015; Ferreira et al., 2017; Nakada et al., 2013; Reller et al., 2007; Teertstra et al., 2004). Although, PNA probes are expensive and their use can be highly cost prohibitive. The process or permeabilising the cells of fixed fungal mycelium, without destructive proteases and chitinases, is an effective way of allowing access of DNA probes to their targets and minimizing the dependency on use of PNA technology (Villa et al., 2009).

To date, mycovirology has yet to fully harness the application of FISH. A single study probed a DNA mycovirus in the mycelium of Sclerotinia sclerotiorum, but this was achieved using methods incorporating lytic enzymes (which were avoided in this study) and the resolution of nucleus fluorescence and probed virus fluorescence was limited (Yu et al., 2013).

In this study, the technique of FISH was applied for the investigation of two viruses in the mushroom virus X (MVX) complex. The application of FISH was used to assess whether both viruses could be probed for their detection and for the understanding of their spatial distributions within the mycelium of A. bisporus. The targets were AbV6 and AbV16, which are multipartite and bipartite viruses, respectively (Deakin et al., 2017). As such, the probes designed specifically targeted AbV16 RNA 1(^MVX 1.8 kbp) and AbV6 RNA 2 (^MVX 3.6 kbp) (Grogan et al., 2003). AbV16 RNA 1 is causal for the brown disease phenotype, and present particularly in MVX-infected crops in Ireland (Eastwood et al., 2015; Fleming-Archibald et al., 2015; Grogan et al., 2003). AbV6 was associated with the bare patch disease phenotype, prevalent in diseased crops in the UK in some of the earliest reports of MVX (Grogan et al., 2003) although a definitive correlation with symptoms has not been established. It has been detected in high abundance in AbV16 infected crops with no bare patch symptoms (unpublished data). Low levels were also reported in a ‘non-diseased commercial culture’ (Deakin et al., 2017). The work reported here involves an adapted method of FISH, whereby A. bisporus cultures (with and without MVX viruses) are grown, permeabilised, hybridised and visualised in situ, so as to negate any disturbance to the mycelium. This technique uses non-destructive permeabilization methods (Villa et al., 2009) which allow the use of DNA probes, making the method cost-effective and reproducible. This is the first robust application of FISH on mycoviruses within the mycelium of a fungal host.