FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridisation
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.
Section snippets
Strains and culture conditions
MVX-infected A. bisporus cultures MVX-1283, MVX-2735, and MVX-1153 were derived from mushroom samples that were taken from symptomatic crops in the UK and Ireland between 2000- and 2016 (Table 1). Presence of AbV16 RNA 1 was confirmed by RT-PCR according to (Fleming-Archibald et al., 2015). Presence of AbV6 RNA 2 was confirmed using the same methodology, and PCR primers designed specifically for AbV6 RNA 2 (F: GGCAGGAGCAGATGAACATT R: ACCTGGAACAGCAGCAAAAC; product size 305 bp; Fleming-Archibald,
Permeabilisation of A. bisporus hyphae and FISH controls
Permeabilisation was necessary to facilitate FISH probe penetration of fungal cell walls. Porousness of cells was achieved using the nuclear stain DAPI (Fig. 2). DAPI was added to every culture preparation as quality control to reduce the likelihood of false negatives i.e. where no fluorescent signal is a result of inefficient cell permeabilisation and not the absence of the target virus. Additionally, RNase-cocktails were used to introduce negative controls. Specificity of probes to bind to
Discussion
The method for FISH was adapted from previous studies involving this technique on filamentous fungi (Teertstra et al., 2004; Villa et al., 2009), and was used in a novel application to target two disease phenotype-associated viruses of MVX in A. bisporus mycelium. This approach facilitated in situ cultivation, permeabilisation, hybridisation of FISH probes and visualisation by fluorescence light microscopy of mycelium on a singular platform, without the need for cutting, embedding, or any form
Conclusion
In this work, FISH was successfully adapted to target two viruses within the mycelium of A. bisporus. The full FISH workflow was achieved on singular microscope slides, with a reproducible, cost-effective and non-destructive method. The detection, localisation and patterns of distribution of two different mycoviruses, AbV6 and AbV16, at both high and low levels of presence within the host mycelium, were characterised. The localisation patterns observed may suggest movement of viruses through
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Acknowledgments
EOC is funded by a Teagasc Walsh Scholarship Scheme (grant reference number 10564231). This work was also funded by the Teagasc Overseas Travel Award (2017).
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