stress responses. Strain E, which was also not as susceptible to MVX, also mounted an entirely different genetic response to that of Strain C. Thus A. bisporus strains in breeding program- mes could be targeted and mined for their anti-viral response genes (this work will be published shortly).
FISH-ing for viruses
Fluorescence In-Situ Hybridisation (FISH) is a method that allows you to locate and visualise elements inside living cells. It is a difficult tech- nique and has only once been used with fungal viruses with limited success. Most virus detec- tion methods are destructive, using a crushed up sample of mushroom or mycelium, which is then tested for the presence or absence of specific viruses. The FISH method is different in that it uses a living culture. A fluorescent tag is added to a probe for a specific virus. This solution is applied to the mycelium, where the probes enter the mycelium, find the viruses and bind to them in situ. The mycelium can then be examined with a microscope and the viruses can be visuali- sed due to the fluorescence. We developed this technique for the AbV16 and AbV6 viruses within infected A. bisporus mycelium with excellent results (O’Connor et al, 2020b). Fluorescent- ly-labelled AbV16 showed high intensity and distribution within the network of hyphae deep within the culture in a highly compartmentalised manner (Figure 4). The AbV6 virus on the other hand was located more at the periphery of the culture and could also be detected at very low levels at the hyphal tips (Figure 5). This differen- tial localisation of the two viruses adds to the complexity of the virus–host interaction.
Wild A. bisporus genome The whole genome of the wild A. bisporus cul- ture - ARP23 - was assembled and the final genome was found to be 33.49 Mb in length and had significant levels of similarity to other sequenced A. bisporus strains. Overall, 13,030 genes were annotated. Relative to other A. bis- porus genome sequences, A. bisporus ARP23 is the largest in terms of gene number and genome size and thus represents an important resource for future genetic studies and breeding programmes (O’Connor et al, 2019).
The MVX complex has confounded growers and researchers over two decades. Developing an understanding of the complexity and interaction of these viruses with their mushroom host is critical to creating effective control strategies. Combining classical techniques including vege- tative incompatibility with technologies such as fluorescent “tagging” of the virus particles has provided new insight into the distribution and
transmission of the viruses. Ultimately we hope this work will help guide efforts to develop cul- tures that are either tolerant or resistant to infection by this virus complex.
References and acknowledgements: 1. O’Connor et al. 2019. Whole genome sequence of the com- mercially relevant mushroom strain Agaricus bisporus var. bisporus ARP23. G3: Genes, Genomics, Genetics https://doi. org/10.1534/g3.119.400563
2. O’Connor et al. 2020a. Proteomic investigation of inter- hyphal interactions between strains of Agaricus bisporus. Fungal Biology. In Press.
https://doi.org/10.1016/j.fun- bio.2020.02.011
3. O’Connor et al. 2020b. FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridization. Journal of Microbiologi- cal Methods. In Press.
https://doi.org/10.1016/j. mimet.2020.105913
Mr. Eoin O Connor was funded by a Teagasc Walsh Scholar- ship. We acknowledge the DJEI/DES/SFI/HEA Irish Centre for High-End Computing (ICHEC) for the provision of computati- onal facilities and support. Mass spectrometry facilities were funded by Science Foundation Ireland (SFI 12/RI/ 2346(3)). This was the work of Eoin O Connor, a PhD student at Maynooth University, Ireland. Eoin recently defended his PhD thesis and will be conferred with his doctorate later in the year. We thank Sylvan for providing a number of different A. bisporus strains for us to work on in these studies.
Different strains respond
differently to infection with
MVX at a genetic and protein level
Figure 4. AbV16 virus (green fluorescence) detected in certain hyphae within the mycelial network of an A. bisporus culture using the FISH method.
Figure 5. AbV6 virus (green fluorescence) detected at the margins of an A. bisporus culture using the FISH method.
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