PMID: 10919801 Tandem Repeat of a Transcriptional Enhancer Upstream of the Sterol 14α-Demethylase Gene (CYP51) in Penicillium digitatum

[MPiovesana]

Curated by @MPiovesana.
https://canto.phi-base.org/curs/1e8f09364def576c#

[MPiovesana]

In this papers authors identify a tandem repeat in the promoter region of the CYP51 gene of DMI-resistant strains of Penicillium digitatum. This tandem repeat includes five copies of a 126 bp sequence, which is present as a single copy in the promoter of DMI-sensitive strains, such as PD5. Authors test whether the expression of extra copies of the CYP51prom::CYP51 amplified from both PD5 and a DMI-resistant strain (LC2) is capable of inducing increased resistance to fungicides when introduced in PD5 cells.

[MPiovesana]

Uniprot ID: accession number AB030178 provided by the authors is used to locate the CYP51 from PD5 in Uniprot (as PD5 is the reference and recipient strain). ID Q9P340 is located and selected for curation session.

[MPiovesana]

UPDATED FOLLOWING TRANSFORMANT ALLELE DISCUSSIONS:

Genotypes and annotations:
Genotypes were created as described:
CYP51prom::CYP51+ [Ectopic]: expression of CYP51prom::CYP51 from PD5 in PD5 cells; allele type other was selected as transformant is not suitable here (origin of the sequence same as recipient strain);

CYP51prom::CYP51 transformant (LC2-CYP51prom(5x126bp tandem repeat)::CYP51) [Ectopic]: expression of CYP51prom::CYP51 from LC2 in PD5 cells; 126 bp tandem repeats in promoter included in allele description.

I considered choosing Ectopic as expression level to describe the expression of the extra copies of the gene; however, authors quantify expression levels of CYP51 and observe a slight increase for CYP51prom::CYP51+ and a more significant increase for lines expression CYP51prom::CYP51 transformant, so I believe overexpression is appropriate here. Endogenous CYP51 present was added to background.

[MPiovesana]

Genotypes were annotated with terms:
resistance to fenarimol
resistance to triflumizole (suggested)
resistance to bitertanol (suggested)
resistance to pyrifenox (suggested)

[MPiovesana]

Curation completed pending review.

[CuzickA]

Genotypes

Changed to

[CuzickA]

AE alteration_in _archetype

Questions for Nichola

I'm not sure about adding the AE alteration in archetype here, need to follow up with Nichola.

I could use
Non-coding region; Cyp51A; ASPEFU
or
Non-coding region; Cyp51B; SEPTTR

It is unclear as Penicillium digitatum gene is 'Cyp51'.
The alteration in the promoter can be captured using 'Non-coding region'

[CuzickA]

New terms added, session approved. leaving ticket open as need to follow up with Nichola about adding the AE alteration in archetype here (see comment above).

[CuzickA]

Use FRAST to determine if Cyp51A or B

[CuzickA]

From the publication
'Nucleotide sequence accession numbers. The sequences of PdCYP51 from
PD5 and LC2 are available under GenBank accession numbers AB030178 and
AB030179, respectively.'

Look up AB030178 in Genbank gives https://www.ncbi.nlm.nih.gov/nuccore/AB030178

Created AA FASTA file in notepad.

Tried to use FRAST option for 'automatic' input to see whether PdCYP51 closer to Cyp51 A or B. However, FRAST didn't work. No results were shown. Double checked method using info from #51 and this example did work.

We still don't know whether to use Cyp51A or Cyp51B.

[CuzickA]

After discussion with Nichola

1. Paper https://academic.oup.com/g3journal/article/12/11/jkac249/6709346 states that Pd has Cyp51A and Cyp51B.
2. Find genbank ids for Pd Cyp51A and Cyp51B in this paper and obtain AA sequences
3. For each, do Blast2seq to compare against Cyp51 in curated publication. As same species, expect either Cyp51A or B to have 100% match to curated publication Cyp51.

[CuzickA]

Supplementary Table 2 from https://academic.oup.com/g3journal/article/12/11/jkac249/6709346 PMID: 36130263

Pd Cyp51A XP_014532172.1 https://www.ncbi.nlm.nih.gov/protein/XP_014532172.1
Pd Cyp51B XP_014538931.1 https://www.ncbi.nlm.nih.gov/protein/XP_014538931.1

[CuzickA]

Blast2seq
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq

Compare

1. 'PdCyp51 PMID 10919801' with 'PdCyp51A PMID 36130263'
2. 'PdCyp51 PMID 10919801' with 'PdCyp51B PMID 36130263'

Enter
'PdCyp51 PMID 10919801' AA FASTA file (copy and paste) in 'Enter query sequence' and 'PdCyp51A PMID 36130263' AA FASTA file (copy and paste) in 'Enter Subject Sequence'. No change to any settings, hit 'BLAST'

Enter
'PdCyp51 PMID 10919801' AA FASTA file (copy and paste) in 'Enter query sequence' and 'PdCyp51B PMID 36130263' AA FASTA file (copy and paste) in 'Enter Subject Sequence'. No change to any settings, hit 'BLAST'

Conclusion

PdCyp51 from PMID 10919801 is PdCyp51A

[CuzickA]

No need for FRAST as genotype has an alteration in the promoter which can be captured using 'Non-coding region'

Two genotypes
CYP51+[Overexpression]
CYP51 transformant(LC2-CYP51promoter(5x126bp tandem repeat)::CYP51)[Overexpression]

AE added to both
'Non-coding region; Cyp51 (Cyp51A); ASPEFU'

Next steps

1. Check AE with Nichola
2. Then session ready for approval

[CuzickA]

AE checked by Nichola - looks fine.

Approving session and closing ticket.