PMID:27242773 C239S Mutation in the β-Tubulin of Phytophthora sojae Confers Resistance to Zoxamide

[MPiovesana]

Curated by @MPiovesana https://canto.phi-base.org/curs/cbee79c9ba230adf

[MPiovesana]

Spontaneous zoxamide-resistant mutants of Phytophthora sojae were isolated in this study from parental strain PsJMS2. Mutants were phenotypically characterised and found to carry a point mutation in the β-Tubulin gene, leading to amino acid substitution C239S. The mutation was back-transformed into wild type strain P6497 and transformants exhibited zoxamide-resistant, as expected. The genotype of the mutant and transformant were curated in this session; this is to allow the creation and comparison of the metagenotype composed of the mutant pathogen genotype compared to the WT PsJMS2 control.

[MPiovesana]

Uniprot ID: after identifying the reference proteome for P. sojae on Uniprot, a gene name search retrieved ID G4ZQS2 for the β-Tubulin gene.

Strains: strains PsJMS2 and P6497 were added to curation session.

[MPiovesana]

Genotype creation: The genotype of the spontaneous mutants derived from strain PsJMS2 was recorded as below: βTub(aaC239S)[Not assayed] = to indicate the aa substitution in the βTub gene.

The genotype of the transformants obtained from strain P6497 was recorded as below: βTub transformant(RZ11-1-βTub(C239S))[Ectopic] = to indicate the introduction of the βTub gene from mutant strain RZ11-1, which harbours the C239S mutation. Expression was selected as ectopic. Endogenous βTub present was added to background.

[MPiovesana]

Genotype annotations: The PsJMS2 genotype was annotated with the following terms: resistance to zoxamide (suggested) normal number of asexual spores normal asexual spore germination frequency normal growth on chlorothalonil (suggested) normal growth on azoxystrobin (suggested) normal growth on cymoxanil (suggested) normal growth on metalaxyl (suggested) normal growth on flumorph (suggested)

[MPiovesana]

Metagenotype creation: a metagenotype was created including the mutant allele in the PsJMS2 background and G. max (cv Williams). As the three isolated mutants with the same genotype showed varying levels of infectivity, I selected the PHIPO term "presence of pathogen-associated host lesions" for both control and altered metagenotype, and added has_severity variable severity as an annotation extension for the altered metagenotype. AC I also added infective ability 'reduced virulence' as shown by 2 of the 3 mutant lines.

[MPiovesana]

Curation completed pending review.

[CuzickA]

Phytophthora sojae is an oomycete, rather than a fungus, so may need flagging up in the table of curated papers.

[CuzickA]

I couldn't find archetype info for B-tubulin P sojae in Nichola's S file or the Mair et al 2016 publication.

[CuzickA]

New terms added, session approved, closing ticket.

[CuzickA]

Try and and look up archetype info in FRAST

[CuzickA]

I could not find a Gene id in the publication.

Is it ok to do this search in Genbank 'B-tubulin Phytophthora sojae' and use this entry (there was only one) https://www.ncbi.nlm.nih.gov/nuccore/829497727 Generated AA FASTA file in notepad and saved. FRAST alignment (22_09_2023)

NOTE (18_10_2023) This residue number alignment is not correct- see comment below after discussion with Nichola. ~Codon~ Residue 239 in the sequence index is 'S' which aligns with with archetype index ~codon~ residue 352 but a different wildtype AA 'A' in the Cyp51A archetype sequence. Ps βTub(aaC239S)[Not assayed] '352S; β-tubulin; ASPEND'

Saved mafft in notepad and session in FRAST

[CuzickA]

Next step Check this with Nichola. If this looks ok then add to the curation session.

[CuzickA]

After discussion with Nichola

Above is incorrect due to a partial genbank seq causing a tricky alignment.

In the above Residue 239 in the sequence index is 'S' which is the mutated AA residue C239S. This does not make sense. Either the input sequence represents the mutant sequence or the alignment is incorrect.

From the publication

If we try and match up AA by eye we can see that the above AE suggestion is incorrect. Instead of looking at the input sequence at 239 we need to look at the archetype sequence at 239. This then corresponds with the input sequence of 126. Note: the residue position in the AE relates to the archetype seq and not the input seq and thus would be 239 and not 126. The sequences align as expected with 'C' being located at 239/126. (Went back to past saved FRAST alignment to make this screenshot.)

Therefore change AE to 'C239S; β-tubulin; ASPEND'

[CuzickA]

Added AE alteration in archetype.

Approved session. Closing ticket.