PMID:27558019 Development of a novel strategy for fungal transformation based on a mutant locus conferring carboxin-resistance in Magnaporthe oryzae

[MPiovesana]

Curated by @MPiovesana https://canto.phi-base.org/curs/e19bd7df7425b419

[MPiovesana]

In this paper, authors develop a transformation strategy for Magnaporthe oryzae using the carboxin resistance conferred by a mutant allele of Mosdi1 gene (succinate dehydrogenase) as a selection marker. To test whether a point mutation previously described in the literature in other fungal species could confer carboxin resistance in M. oryzae, authors replace the endogenous wild type copy of Mosdi1 from wild type strain Guy11 with a mutant allele harbouring the H245L mutation. The resulting mutant is resistant to carboxin. Authors then proceed to develop a transformation strategy where gene replacement of Mosdi1 and introduction of a transgene adjacent to the Mosdi1 locus take place simultaneously, which is proven to work. After careful reading through the whole paper, I believe the only genotype and phenotype of interest for curation in PHI-Canto is that of the Guy11-derived gene replacement mutant showing resistance to carboxin.

[MPiovesana]

Uniprot ID: using the accession number provided by the authors (XP_003718958), Uniprot ID G4NE45 was retrieved.

Strain: wild type strain Guy11 was selected.

[MPiovesana]

Genotype creation: an amino acid substitution allele type was created to record the H245L mutation in Mosdi1 which confers resistance to carboxin. Mosdi1Δ added to background to reflect gene replacement.

[MPiovesana]

Genotype annotation: genotype was annotated with PHIPO term resistance to carboxin.

[MPiovesana]

Curation completed pending review.

[CuzickA]

Annotation looks fine.

Next Can I add Sdh archetype info for this annotation?

[CuzickA]

AE alteration in archetype

Magnaporthe oryzae Mosdi1(aaH245L)[Not assayed]

Could not find an entry in Nichola S file or Mair et al.

Try FRAST

[CuzickA]

https://www.ncbi.nlm.nih.gov/protein/XP_003718958

[CuzickA]

In FRAST not sure whether to align to SdhB , C or D.

The automatic search for Blast N is not providing any information.

Need to follow up with Nichola.

[CuzickA]

Nichola also could not get the automatic search for Blast N in FRAST to provide any information.

Nichola did a protein-protein blast in genbank gives 100% match of sdi1 = sdhB

To do 1) repeat the blast search and document 2) FRAST align sdi1 with SdhB archetype

[CuzickA]

[CuzickA]

Next FRAST align sdi1 with SdhB archetype

Magnaporthe oryzae Mosdi1(aaH245L)[Not assayed]

[CuzickA]

Magnaporthe oryzae Mosdi1(aaH245L)[Not assayed]

Confirm above sequence snip for Mosdi1 contains 'H' at position 245.

Input sequence 245 aligns with archetype sequence at position 277

Therefore, Magnaporthe oryzae Mosdi1(aaH245L)[Not assayed] would be 'H277L; Sdi1 (SdhB); PYRNTE'

[CuzickA]

Next step

1) Check AE with Nichola

Then session will be ready for approval.

[CuzickA]

Checked AE with Nichola. Looks fine.

Approving session and closing ticket.