PMID: 30874562 A phosphorylated transcription factor regulates sterol biosynthesis in Fusarium graminearum

[MPiovesana]

Curated by @MPiovesana https://canto.phi-base.org/curs/286abaf17e490111

[MPiovesana]

Extensive paper with generation of several mutant genotypes and numerous experiments performed. In this paper authors report the identification of the transcription factor FgSR of Fusarium graminearum which regulates sterol biosynthesis. As such, deletion and mutations of FgSR affect the susceptibility of F. graminearum to sterol biosynthesis inhibitor (SBI) fungicides, such as tebuconazole. In this study, authors generate a deletion line of FgSR (FgSRΔ), as well as of interacting partners and other proteins which participate in the same signalling cascade as FgSR (Ssk2, Pbs2, Hog1, Arp9). Authors also generate phospho-inactive and phosphomimic mutants of FgSR, as well as lines where the zinc finger (ZF) and middle linker (ML) domains of FgSR have been deleted. The susceptibility of all mutant lines to fungicides is tested. Finally, the virulence and DON production of FgSRΔ infecting wheat plants are also assessed.

[MPiovesana]

Uniprot IDs: FgSR: the gene code FGSG_01176 provided in the paper was used to locate the Uniprot ID (I1RC73) corresponding to FgSR.

FgSsk2: the ref proteome of F. graminearum (UP000070720) was searched with the gene name Ssk2, returning ID I1RA81.

FgPbs2: the ref proteome was searched with the gene name Pbs2, returning ID I1RWL6.

FgHog1: the ref proteome was searched with the gene name Hog1, returning ID P0C431.

FgArp9: the ref proteome was searched with the gene name Arp9, returning ID A0A098DZN5.

[MPiovesana]

Strains: F. graminearum: PH-1 Wheat: cv Jimai22

[MPiovesana]

Genotype creation: Deletion alleles were created for all genes, generating the following genotypes: FgSRΔ FgSSK2Δ FgHOG1Δ FgPBS2Δ FgARP9Δ

The phospho-inactive and phospho-mimic genotypes, where five phosphorylation sites were replaced with alanine or aspartic acid residues, respectively, were created by combining the FgSRΔ and amino acid substitution alleles (multi locus genotypes), as the mutant alleles were introduced in the FgSRΔ mutant background: FgSRΔ + FgSR-C-5A (aaT82A S92A S102A T217A S305A) = phospho-inactive FgSRΔ + FgSR-C-5D (aaT82D S92D S102D T217D S305D) = phosphomimic

Similarly, to create the partially deleted alleles where the zinc finger domain and the middle linker region of the FgSR protein were deleted, partial deletion, amino acid alleles were combined with FgSRΔ: FgSRΔ + FgSRΔML(53-130) = middle linker deletion FgSRΔ + FgSRΔZF(9-52) = zinc finger deletion

Expression level = ectopic was selected for all multi-locus genotypes.

[MPiovesana]

Genotype annotations: several annotations were made to curate this publication. These include the antifungal sensitivity phenotype of the deletion and mutant lines to tebuconazole, as well as the sensitivity of FgSRΔ to a number of other fungicides. The following PHIPO terms were used: sensitive to tebuconazole normal growth on tebuconazole (suggested) sensitive to triadimefon normal growth on iprodione (suggested) normal growth on fludioxonil (suggested) sensitive to naftifine sensitive to terbinafine sensitive to fluvastatin (suggested) sensitive to lovastatin (suggested) sensitive to spiroxamine sensitive to tridemorph sensitive to prochloraz

Metagenotype annotations included: presence of pathogen-associated host lesions (control and altered metagenotypes) pathogen deoxynivalenol within host present (control) decreased level of deoxynivalenol within host (altered)

[MPiovesana]

I believe the most important genotypes and phenotypes were recorded in this curation session. Additional experiments mentioned in the Supplemental material could be included, but are not as relevant to the main focus of the paper.

Curation completed pending review.

[CuzickA]

PHI phenotypes look fine

Added disease annotation

[CuzickA]

Added GO CC annotation

[CuzickA]

Also added GO MF

[CuzickA]

The phospho-inactive and phospho-mimic genotypes, where five phosphorylation sites were replaced with alanine or aspartic acid residues, respectively, were created by combining the FgSRΔ and amino acid substitution alleles (multi locus genotypes), as the mutant alleles were introduced in the FgSRΔ mutant background: FgSRΔ + FgSR-C-5A (aaT82A S92A S102A T217A S305A) = phospho-inactive FgSRΔ + FgSR-C-5D (aaT82D S92D S102D T217D S305D) = phosphomimic

I think that the FgSRΔ should just be recorded in the background rather than as part of a multi-locus allele.

change to

change to

[CuzickA]

Similarly, to create the partially deleted alleles where the zinc finger domain and the middle linker region of the FgSR protein were deleted, partial deletion, amino acid alleles were combined with FgSRΔ: FgSRΔ + FgSRΔML(53-130) = middle linker deletion FgSRΔ + FgSRΔZF(9-52) = zinc finger deletion

Also moved SRdelta into background for these genotypes

changed to

[CuzickA]

I am removing these annotations as the chemicals are statins.

[CuzickA]

Added protein modification annotation

[CuzickA]

Added reviewed UniProt entry 'I1RJR2' for Fg CYP51A

[CuzickA]

In summary,

I have annotated Protein modification 'SR' is phosphorylated by 'Hog1' GO BP CYP51A is involved in sterol biosynthesis GO MF SR is a TF and binds to cis element in CYP51A (and other) promotor

Main pathogen phenotypes SRdelta (results in reduced transcription of CYP51A) readout increased sensitivity to anti-fungal SBI chemical tebuconazole

Hi @ValWood, I'm a bit rusty on GO annotations. Please could you do a sanity check on these?

[CuzickA]

Next steps 1) add new PHIPO term 2) see if GO annotations seem ok

No alterations are made to the chemical target sites of the CYP51 genes in this session so it is not relevant to add in the AE alteration in archetype.

After 1 and 2 above have been done then this session will be ready to approve.

[ValWood]

This looks good. Could CYP51A be to the child "ergosterol biosynthesis" ?

I think you could also add "regulation of ergosterol biosynthesis" to the transcription factor.

[CuzickA]

Thanks @ValWood, how about this?

[ValWood]

Great but the CYP51A is part of the pathway, so that doesn't need to be "regulation of" that would be just to "ergosterol biosynthetic process"

[CuzickA]

Thanks @ValWood

Now looks like this

[CuzickA]

Session just waiting for new PHIPO term and then it will be ready to approve.

[ValWood]

yes, looks good!

[CuzickA]

PHIPO term added. Session approved. Closing ticket.