Hello everyone,
A non-model filamentous fungi (Rhizoctonia solani) which I studied is hard to transformed via protoplast, electroporation, and agrobacterium tumefaciens method.
Now I have obtained some genes maybe associated with sclerotia formation by RNA-seq procedure.
Can I study these genes in a model fungi like Botrytis cinerea?
Rhizoctonia solani and Botrytis cinerea both produce sclerotia, although they are different.
Can you kindly give me some suggestions?
Thank you.
Regards,
Canwei
The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, a CRISPR-Cas9 based system was developed & adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, a script has developed that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered.
You can try PEG-4000 mediated transformation of your fungus.. As this method is very simple. if you have obtained protoplast in enough quantity and they can be regenerated then there is a possibility that PEG-4000 can be successful in your case. If it's still not working then you should check for your genes homologous in other fungus you want to check in.. Wish you good luck.. If you need protocol for PEG-4000 mediated protoplast transformation, I can provide you.
Dear Canwei,
I would like to know which plasmid you are using to transform Rhizoctonia solani and how are you linearizing it prior to transformation into the protoplasts.
Dear Srijita,
Thank you for your suggestion. I think we should start to sut up CRISPR.
At present, we used a traditional method only.
Hi Latif,
Yes, we have tried PEG mediated method for more than one year.
The munber of protoplast we used per transformation is more than 1,000,000.
Because we failed to obtain positive transformants by targeting a gene, we try to transform a raw plasmid (pSilent1).
Unfortunately, some transformants which grown up on the regeneration medium stop grow on the seleted medium (amend with ) hygromycin.
Is that right the plasmid has not been transformed?
Regards,
Canwei
Hello, Shada,
The plasmid which we used is pSilent 1.
To linearize this plasmid, we cut the plasmid at a single enzyme digestion site and check it has been linearized.
At the same time, we also use a un-linearized plasmid in the experiment.
But, some transformants which grown up on the regeneration medium stop grow on the seleted medium (amend with ) hygromycin.
Can you give more suggestion?
Regards,
Canwei
Hello Canwei,
Thanks for letting me know your plasmid. pSilent 1 is a plasmid which is having promoters from the ascomycetes fungus, Aspergillus nidulans. Since Ascomycetes and Basideomycetes are two different phylum in the kingdom fungi, therefore, it is very likely that the ascomycete promoter is not going to work in a basideomycetes. This is the main reason why its not working in your case. I am working on the basideomycetes fungus Piriformospora indica and I earlier tried to transform it using the same promoters that you have used. It never worked. But when I switched to P. indica's own promoters it worked very well.
I hope my answer will be of some help to you.
All the best.
Shadab.
Dear Shada,
Thank you for your valuable suggestions.
If so, can I replace the promoter region of pSilent 1 using a Rhizoctonia solani promoter, like GPD promoter?
Thanks again,
Cheers,
Canwei
Dear Canwei,
For sure you can replace the Promoter or pSilent1 with that of R. solani. GPD is good choice as we are also using that. In addition, I will suggest you to use TEF promoter of R. solani as well. Better to check both TEF and GPD promoter and check how they are working for you.
http://www.sciencedirect.com/science/article/pii/S1087184509000759
This is the paper that was published using GPD and TEF promoter for P. indica.
All the best.
Shadab.
You can also try to transform the protoplast with a plasmid expressing GFP/RFP. As it's very easy to observe whether the transformation is successful or not? in case of V. dahliae, we observe the GFP fluorescence after 16-18 hours of transformation by culturing in Regeneration broth..
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