Isolation of Fungal Homokaryotic Lines from Heterokaryotic Transformants by Sonic Disruption of Mycelia

Abstract

Fungal hyphae--and in some cases, spores--are multi-nucleate. During genetic transformation of these spores or mycelia, only one nucleus generally receives the transferred T-DNA generating heterokaryotic colonies. Characterization of genetic changes, such as the effects of gene disruption in the transformants, requires purified homokaryotic lines. Hyphal tip transfer has conventionally been used to isolate homokaryons. We developed an alternative method for purification of fungal homokaryons from transformed heterokaryotic lines of Sclerotinia sclerotiorum. Ultrasound pulses were used to generate bi-septate, unicellular hyphal fragments that regenerate under selection to produce homokaryotic lines that can be easily identified using colony PCR. This technique facilitates the purification of transformed lines, which allows for routine genome manipulation, and should be adaptable for other filamentous fungi.

The Sclerotinia sclerotiorum Slt2 mitogen-activated protein kinase ortholog, SMK3, is required for infection initiation but not lesion expansion

“…Sclerotinia sclerotiorum strain 1980 (ATCC18683) was used throughout this study. The generation and selection of homokaryotic Smk3υ disruption mutant lines derived from 1980 has been described previously (Bashi et al 2010b) and were cultured under the same conditions.…”

“…The transition between these phases is determined by numerous signals, including contact with hard surfaces (Bashi et al 2012), glucose levels (Vautard-Mey et al 1999), cAMP levels (Rollins and Dickman 1998;Girard et al 2004;Jurick and Rollins 2007), ambient pH (Rollins and Dickman 2001;Cotton et al 2003;Rollins 2003;Favoron et al 2004), and oxidative stress (Kim et al 2011;Papapostolou et al 2014). Furthermore, coordination of these signaling pathways may involve various protein kinases (Vautard- Mey and Fevre 2000;Vacher et al 2003;Chen et al 2004;Jurick et al 2004;Harel et al 2005;Erental et al 2008;Bashi et al 2010b;Duan et al 2013) and phosphatases (Harel et al 2006;Erental et al 2007). An increasing number of extra-cellular effectors have also been shown to affect host-pathogen interactions and disease progression (Liang et al 2013;Zhu et al 2013;Guyon et al 2014;Xiao et al 2014;Zhang et al 2014;Lyu et al 2015Lyu et al , 2016Pan et al 2015).…”

The eukaryotic protein kinase superfamily of the necrotrophic fungal plant pathogen, Sclerotinia sclerotiorum

“…This was attributed to alterations in mycelial apical dominance, leading to the inability to aggregate and form infection cushions. The mutation also caused a loss of the ability to produce sclerotia, increased aerial mycelia and altered mycelial hydrophobicity (Bashi et al ., , unpublished results). The corresponding protein in Candida albicans , Mkc1p, is involved in the regulation of surface contact‐dependent processes leading to filamentous invasive growth (Kumamoto, ).…”

Biomass measurement by flow cytometry during solid‐state fermentation of basidiomycetes

“…Wet disruption processes do not require sample drying and thus save time during pretreatment. Ultrasonic disruption and disruption using microwaves were regarded as promising wet disruption methods due to literature reports concerning the use of ultrasound to extract bacterial and fungal DNA (53,54) for PCR and the use of microwaves to extract fungal DNA (49,55). Both methods are very fast-for example, the suggested protocol for microwave disruption involves treatment times of 15 s to 1 min (56).…”

“…In addition, the reproducibility of the results obtained using these methods may be limited by inhomogeneities in their energy input and the formation of hotspots within the sample despite same geometry of the set-up and same material of the sample container. The microwave treatment was performed due to the rapidity and successful application according to the literature (47,53,54). However, this method includes many sources of error resulting in inhomogeneities of energy and temperature distribution and inconsistent and weak nuclei signals.…”