Combination of driselase and lysing enzyme in one molar potassium chloride is effective for the production of protoplasts from germinated conidia of Fusarium verticillioides

Ramamoorthy, V.; Govindaraj, Lavanya; Dhanasekaran, Madhumitha; Vetrivel, Sharmilee; Kumar, K. K.; Ebenezar, E. G.

doi:10.1016/j.mimet.2015.02.010

Cited by 13 publications

(7 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(de Bekker et al, 2009) and Fusarium spp. (Ramamoorthy et al, 2015). Because cell wall composition varies strongly across fungal species and culture conditions (Reilly and Doering, 2010;Li et al, 2017), cell wall lysis protocols need to be carefully optimized for each condition and fungal strain.…”

Section: Introductionmentioning

confidence: 99%

Expansion Microscopy for Cell Biology Analysis in Fungi

Götz¹,

Panzer²,

Trinks³

et al. 2020

Front. Microbiol.

View full text Add to dashboard Cite

Super-resolution microscopy has evolved as a powerful method for subdiffractionresolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

show abstract

Section: Introductionmentioning

confidence: 99%

Expansion Microscopy for Cell Biology Analysis in Fungi

Götz¹,

Panzer²,

Trinks³

et al. 2020

Front. Microbiol.

View full text Add to dashboard Cite

show abstract

“…The maximum release of protoplasts was obtained from a combination of 0.5% Novozyme 234 and 5% Glucanex (Cheng and Bélanger, 2000). Ramamoorthy et al (2015) evaluated various cell wall degrading enzymes for the production of protoplasts in Fusarium verticillioides and concluded that a combination of lysing enzyme and driselase was effective for protoplast production. In the present study, we determined that the combination of 2% lysing enzyme and 0.8% driselase in 1 M NH 4 Cl with 3 h incubation was optimal for protoplast release in C .…”

Section: Discussionmentioning

confidence: 99%

Optimization of Polyethylene Glycol-Mediated Transformation of the Pepper Anthracnose Pathogen Colletotrichum scovillei to Develop an Applied Genomics Approach

et al. 2019

View full text Add to dashboard Cite

“…It is worth noting that 3–5 h were required to gain sufficient protoplasts in F. virguliforme and F. brasiliense compared to the 2 h reported for F. graminearum [29]. A detailed comparison between protoplasting buffers and enzyme compositions also concluded that potassium chloride was optimal for protoplast generation in the fungal pathogen F. verticillioides , and the addition of chitinase helps generate protoplasts in other Fusarium species [34].…”

Section: Discussionmentioning

confidence: 99%

A protoplast generation and transformation method for soybean sudden death syndrome causal agents Fusarium virguliforme and F. brasiliense

Roth

Chilvers

2019

Fungal Biol Biotechnol

View full text Add to dashboard Cite

Background Soybean production around the globe faces significant annual yield losses due to pests and diseases. One of the most significant causes of soybean yield loss annually in the U.S. is sudden death syndrome (SDS), caused by soil-borne fungi in the Fusarium solani species complex. Two of these species, F. virguliforme and F. brasiliense , have been discovered in the U.S. The genetic mechanisms that these pathogens employ to induce root rot and SDS are largely unknown. Previous methods describing F. virguliforme protoplast generation and transformation have been used to study gene function, but these methods lack important details and controls. In addition, no reports of protoplast generation and genetic transformation have been made for F. brasiliense . Results We developed a new protocol for developing fungal protoplasts in these Fusarium species and test the protoplasts for the ability to take up foreign DNA. We show that wild-type strains of F. virguliforme and F. brasiliense are sensitive to the antibiotics hygromycin and nourseothricin, but strains transformed with resistance genes displayed resistance to these antibiotics. In addition, integration of fluorescent protein reporter genes demonstrates that the foreign DNA is expressed and results in a functional protein, providing fluorescence to both pathogens. Conclusions This protocol provides significant details for reproducibly producing protoplasts and transforming F. virguliforme and F. brasiliense . The protocol can be used to develop high quality protoplasts for further investigations into genetic mechanisms of growth and pathogenicity of F. virguliforme and F. brasiliense . Fluorescent strains developed in this study can be used to investigate temporal colonization and potential host preferences of these species. Electronic supplementary material The online version of this article (10.1186/s40694-019-0070-0) contains supplementary material, which is available to authorized users.

show abstract

Combination of driselase and lysing enzyme in one molar potassium chloride is effective for the production of protoplasts from germinated conidia of Fusarium verticillioides

Cited by 13 publications

References 24 publications

Expansion Microscopy for Cell Biology Analysis in Fungi

Expansion Microscopy for Cell Biology Analysis in Fungi

Optimization of Polyethylene Glycol-Mediated Transformation of the Pepper Anthracnose Pathogen Colletotrichum scovillei to Develop an Applied Genomics Approach

A protoplast generation and transformation method for soybean sudden death syndrome causal agents Fusarium virguliforme and F. brasiliense

Contact Info

Product

Resources

About