PMID:30686204 Molecular and Biochemical Characterization of Laboratory and Field Mutants of Botrytis cinerea Resistant to Fludioxonil

[MPiovesana]

Curated by @MPiovesana https://canto.phi-base.org/curs/0603f5b854b64f55

[MPiovesana]

This study reports the generation of laboratory mutants of Botrytis cinerea which display resistance to the fungicide fludioxonil. The mutants are phenotypically characterised (mycelial growth, sporulation, virulence, etc) and the sequence of the Bos1 gene revealed point mutations, duplication and premature stop codons in the four highly-resistant mutants isolated by the authors.

[MPiovesana]

Uniprot ID: in the paper authors indicate gene ID BC1G_00374 for the Bos1 gene of Botrytis cinerea (Broad Institute B. cinerea Genome Database). Searching NCBI with this gene ID, we retrieve this page (https://www.ncbi.nlm.nih.gov/gene/?term=BC1G_00374), the locus tag BCIN_01g06260 is provided. Searching Uniprot with this locus tag, two Uniprot IDs are retrieved:

In the paper, authors mention that the predicted Bos1 protein has 1310 amino acids, but neither of these entries correspond to this length (one is 1315 and the other is 1311 aa long). I selected the first ID for now to allow the next steps of curation, but I believe this needs to be checked as I am not sure this is the best ID for this session.

AC (04_12_2023): Session https://github.com/PHI-base/curation/issues/144 also used A0A384J5Y1. We will stick with this for now.

[MPiovesana]

Strains: wild type strains from which resistant mutants were derived were manually added to the session.

Host: as pathogenicity assays were performed, host species strawberry was added. No information on cultivar is provided by the authors, so 'Unknown strain' was selected. Pathogenicity assays are also performed in tomato, with similar results obtained. As this curation session has 4 altered pathogen genotypes, considering the 4 respective wild type genotypes, this results in 8 metagenotypes per host species. So I decided to only record the results obtained in strawberry as representative of the virulence phenotype of these strains.

[MPiovesana]

Genotype creation: genotypes were created according to the mutation type harboured by each mutant line, as described below:

Bos1-Q846stop = allele type nonsense mutation for the replacement of Q846 with a stop codon in strain Nj5-10.

Bos1-E253D = allele type amino acid substitution for the E253D mutation in strain Bt3-4.

Bos1-(55bp duplication + 838stop) = allele type amino acid insertion and substitution for the duplication of 55 bp resulting in a stop codon at position 838 in strain Bt6-4.

Bos1-G415D = allele type amino acid substitution for the G415D mutation in strain Yc-6.

[MPiovesana]

Genotype annotations: the following PHIPO terms were used: resistance to fludioxonil resistance to procymidone (suggested) resistance to iprodione decreased number of asexual spores

[MPiovesana]

Metagenotype creation and annotation: WT (control) and mutant metagenotypes were created with host species strawberry. The WT metagenotypes were annotated with PHIPO term "presence of pathogen-associated host lesions", and altered metagenotypes were assigned the term "decreased extent of pathogen-associated host lesions". Annotation extensions (AE) were used to describe the presence of the disease, the reduced virulence of the mutant genotypes, and comparisons between metagenotypes.

[MPiovesana]

NOTE: authors also report on the sensitivity to osmotic stress displayed by the resistant genotypes compared to the parental lines. However, as several conditions are testes (NaCl, KCl, glucose, sorbitol, CaCl, MgCl2), this would result in a large number of annotations (considering all four mutant genotypes). I did not curate these so far, but they could be included in the future (results shown in Figure 1).

[MPiovesana]

Curation completed pending review.

[CuzickA]

Need to add new Botrytis cinerea strains to PHI-Canto strain list. These are the wild type S field strains which are parents to the spontaneous R lab mutants Nj5-10 Bt3-4 Bt6-4 Yc-6

[CuzickA]

Pathogen phenotypes checked and look good.

Next 1) try to add AE alteration in archetype _AC 06_122023 added for 2/4 genotypes. needs checking by Nichola. 2) check PHI phenotypes _AC 06_12_2023DONE

[CuzickA]

AE alteration in archetype Bc is the archetype species for Bos1/Os-1

Bos1(Q846stop)[Not assayed] Not listed in Nichola's file or Mair et al 2016 - TO ADD 'Q846stop; Bos1 (OS-1); BOTRCI' Bos1(aaE253D)[Not assayed] Listed in Nichola's file ' E253D; Bos1 (OS-1); BOTRCI' Bos1(aa55bp duplication + 838stop)[Not assayed] Not listed in Nichola's file or Mair et al 2016 - duplication causing frameshift and stop (disruption) TO ADD 'frameshift indel 835, 838stop Bos1 (OS-1); BOTRCI' Bos1(aaG415D)[Not assayed] Listed in Nichola's file 'G415D; Bos1 (OS-1); BOTRCI'

[CuzickA]

PHI Phenotypes look fine.

[CuzickA]

Next steps 1) Add new strains to PHI-Canto Botrytis cinerea Nj5-10 Bt3-4 Bt6-4 Yc-6

_Done 11_122023 strains added fe050f8

2) Check AE alteration in archetype with Nichola. 3) Then session is ready for approval

[CuzickA]

Checked AEs with Nichola

Bos1(Q846stop)[Not assayed] 'Q846stop; Bos1 (OS-1); BOTRCI'

Added

[CuzickA]

From paper

Changing genotype from Bos1(aa55bp duplication + 838stop)[Not assayed] to Bos1(aa18.3 (55bp) duplication 835 + 838stop)[Not assayed]

We are reporting on a mix of bp and aa. PHI-Canto allele type selected is referring to aa, and automatically adds 'aa' before the '55bp'. This was unclear in the original genotype. Therefore 55/3 = 18.3 aa.

[CuzickA]

Bos1(aa18.3 (55bp) duplication 835 + 838stop)[Not assayed]

Added AE 'frameshift indel 835, 838stop Bos1 (OS-1); BOTRCI'

[CuzickA]

Session now approved, closing ticket.

[CuzickA]

Made edit from 'frameshift' to 'Frameshift' and reapproved