PMID:21933337 Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat

[jingluodatacurator]

Curated by @jingluodatacurator

https://canto.phi-base.org/curs/fa6dafc2380a5876

Notes: Uniprot IDs: G4XX47, G4XX53, G8EI90 ~Strains~ Genes/proteins: SdhB, SdhC, SdhD (see Table 3 and Table 4) Strains: IPO323 and IRE30 Main expt. for curation: see in Results 'Identification of Sdh mutations in carboxin-resistant UV mutants' and 'Relationship between mutant Sdh variants and SDHI sensitivity', and Table 3, Table 4, Figure 4.

Notes on Uniprot IDs: basesd on GenBank accession numbers JF916683-916700 were given, G8EI90 was from JF916683 for SdhB, G4XX47 was from JF916689 for SdhD, G4XX53 was from JF916695 for SdhC.

[CuzickA]

@jingluodatacurator has now added double mutants. Ready for AC to check.

[CuzickA]

Checking currently used uniprots SdhD https://www.uniprot.org/uniprotkb/G4XX47/entry SdhC https://www.uniprot.org/uniprotkb/G4XX53/entry SdhB https://www.uniprot.org/uniprotkb/G8EI90/entry

All these these are unreviewed Uniprot entries.

@jingluodatacurator, please could you note here the method that you used to find these uniprot ids?

(note to self: Search uniprot with 'SDH Zymoseptoria tritici' and the only reviewed entry is for SdhB (Sdh2)

This reviewed entry uniprot was used in #126 for curation of Zt SdhB. We may need to switch uniprot over to this one in this current session. I could not see reviewed entries for Zt SdhD or C).

[CuzickA]

Current annotations

[CuzickA]

Hi @jingluodatacurator,

Looking at the first set of three pathogen phenotypes in Zt strain IPO323 it looks as though you have combined all the mutations within one gene into one genotype eg When I look at Table 3 It looks to me as though these are two separate lines of Zt strain IPO323, one line with a mutation SdhC-L85P and another line with SdhC-N86K. Please could you have another go at these genotypes so that they align with the info in Table 3?

I think there is a similar issue for the Zt strain IRE30 and info in Table 4.

This is a bit tricky and you may have been thinking about the multi-locus genotypes in the next set of annotations.

Happy to talk this through further if required.

[CuzickA]

Hi @jingluodatacurator,

These multi-locus annotations for Table 3 look great. The genotypes are correct. I have made a few edits to the figure, AE and conditions. In the conditions we want to describe the phenotyping 'Phenotyping and genotyping of carboxin-resistant UV mutants and field strains' of the uv mutants not the creation of the uv mutants 'Generation of SDHI-resistant UV mutants' :-).

[CuzickA]

@jingluodatacurator, for the Table 4 multi-locus genotype annotations we currently have

For Please could you change the multi-locus genotype to a single locus genotype containing two mutations within SdhB and the strain needs changing to SRE30.

For Please could you change the strain to IRE30 and edit the conditions and figure as noted above.

[CuzickA]

In summary note: this is a great learning paper for genotype construction :-) 1) we have a uniprot query 2) @jingluodatacurator to make changes to the single locus genotypes. 3) @jingluodatacurator to make changes to the multi-locus genotypes. 4) @jingluodatacurator try and make any relevant PHI phenotype annotations for this section 'In planta fitness studies on SDHI-resistant UV mutants of M. graminicola' and Table 5. 5) AC to check these genotype edits once done. 6) AC to try and add in AE alteration in archetype (will try and do this once 2 and 3 above have been done).

[jingluodatacurator]

In summary note: this is a great learning paper for genotype construction :-)

1. we have a uniprot query
2. @jingluodatacurator to make changes to the single locus genotypes.
3. @jingluodatacurator to make changes to the multi-locus genotypes.
4. @jingluodatacurator try and make any relevant PHI phenotype annotations for this section 'In planta fitness studies on SDHI-resistant UV mutants of M. graminicola' and Table 5.
5. AC to check these genotype edits once done.
6. AC to try and add in AE alteration in archetype (will try and do this once 2 and 3 above have been done).

@CuzickA, all changes have been made for the single and multi-locus genotypes, please check.

[jingluodatacurator]

In summary note: this is a great learning paper for genotype construction :-)

1. we have a uniprot query
2. @jingluodatacurator to make changes to the single locus genotypes.
3. @jingluodatacurator to make changes to the multi-locus genotypes.
4. @jingluodatacurator try and make any relevant PHI phenotype annotations for this section 'In planta fitness studies on SDHI-resistant UV mutants of M. graminicola' and Table 5.
5. AC to check these genotype edits once done.
6. AC to try and add in AE alteration in archetype (will try and do this once 2 and 3 above have been done).

@CuzickA, re point 4, have tried and made a PHI phenotype annotation for the In planta fitness studies on SDHI-resistant UV mutants of M. graminicola section, but unsure if the host organism and gene added were correct. Please check.

[CuzickA]

feedback on current changes made for point 2)

@jingluodatacurator, looks good. These are just a subset of the mutants listed in Table 3. Please let me know your reasoning for only curating this subset eg were these the main mutations reported on in the authors text? Were they the first mutation listed in the table per gene? I think more of the mutants could be curated :-)

The same query for the Table 4 annotations

[CuzickA]

@jingluodatacurator, for the Table 4 multi-locus genotype annotations we currently have

For Please could you change the multi-locus genotype to a single locus genotype containing two mutations within SdhB and the strain needs changing to SRE30.

For Please could you change the strain to IRE30 and edit the conditions and figure as noted above.

Hi @jingluodatacurator, I don't think these changes have been made yet for point 3) above

[CuzickA]

Point 4, PHI phenotype annotation

Current annotation

[CuzickA]

My notes on the text in the publication relating to what the PHI phenotypes could be

Text from the summary 'Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations.'

Text from the Introduction 'For a selection of mutants, pathogenicity tests were carried out to establish their fitness in planta.'

Text from methods 'In planta fitness studies on Sdh mutants and field strains of M. graminicola. In planta pathogenicity tests were carried out on 2–3-week-old wheat seedlings of cultivar Riband' '18 leaves per strain were inoculated with spore suspensions' 'After 72 h of incubation at 100% relative humidity in polystyrene boxes, inoculated plants were incubated at 16 °C with a 16-h light period at 88% relative humidity for up to 25 days to allow symptoms to develop.' 'Visual assessments, recording the extent of pycnidia formation and chlorosis, were conducted 21 days after inoculation.' 'DNA was also extracted from four 2-cm inoculated leaf segments at several time points (7, 14, 21 and 25 days after inoculation) and the amount of pathogen DNA was quantified' 'Spores were also counted from three inoculated leaves after 21 days.' 'The amount of spores collected was determined microscopically using a haemocytometer.'

Text from results section 'In planta fitness studies on SDHI-resistant UV mutants of M. graminicola A selection of field strains (BC1, BC4 and IRE30) and Sdh mutants derived from IRE30 were further examined with regard to their in planta pathogenicity and the ability to produce spores (Table 5). After 21 days, the inoculated areas were completely necrotic and covered with pycnidia for the three field isolates. All mutants produced lesions with pycnidia, but some areas of green leaf tissue were still observed within the inoculated areas. The largest areas of necrosis were observed for mutants M74 and M79 (data not shown). In comparison with IRE30, where 36 ng of pathogen DNA were detected per 50 ng of total DNA after 25 days, the level of pathogen DNA was lower for the mutants, with measured values between 5 and 30 ng. However, half of the mutants tested produced more pycnidiospores than IRE30 after 21 days. The amount of spores harvested per leaf after 21 days was at least five-fold higher than the initial amount of spores inoculated for all mutants and strains tested. Although some small differences in symptom expression, fungal growth and spore production were measured for individual mutants, these differences were not linked to a particular Sdh variant (e.g. B-S221P, B-H267N, B-H267Y and D-D129E).'

Table 5

[CuzickA]

Possible PHI phenotypes that could be reported on

• necrotic lesions with pycnidia (data not shown) Text suggests present in mutants but to a lesser extent 'All mutants produced lesions with pycnidia, but some areas of green leaf tissue were still observed within the inoculated areas.'
• pathogen DNA in host Text 'the level of pathogen DNA was lower for the mutants'
• pathogen spores in host Text 'half of the mutants tested produced more pycnidiospores than IRE30 after 21 days'

@jingluodatacurator, Please look through my notes above and we can discuss further in the new year :-)

[CuzickA]

Note to self

PHI-ECO term '3 weeks post inoculation' needs exact synonym '21 days post inoculation'

[CuzickA]

In summary note: this is a great learning paper for genotype construction :-)

1. we have a uniprot query
2. @jingluodatacurator to make changes to the single locus genotypes.
3. @jingluodatacurator to make changes to the multi-locus genotypes.
4. @jingluodatacurator try and make any relevant PHI phenotype annotations for this section 'In planta fitness studies on SDHI-resistant UV mutants of M. graminicola' and Table 5.
5. AC to check these genotype edits once done.
6. AC to try and add in AE alteration in archetype (will try and do this once 2 and 3 above have been done).

@CuzickA, all changes have been made for the single and multi-locus genotypes, please check.

After our last discussion it turns out the genotype had been created but the phenotypes had not been annotated. It looks as if the phenotype annotations have now been added and I need to check them.

Therefore current next steps list is

1. we have a uniprot query
2. @jingluodatacurator to make changes to the single locus genotypes. _DONEAC needs to check
3. @jingluodatacurator to make changes to the multi-locus genotypes. DONE_AC needs to check
4. @jingluodatacurator try and make any relevant PHI phenotype annotations for this section 'In planta fitness studies on SDHI-resistant UV mutants of M. graminicola' and Table 5.
5. AC to check all annotations once done.
6. AC to try and add in AE alteration in archetype (will try and do this once 2 and 3 above have been done).
7. AC PHI-ECO term '3 weeks post inoculation' needs exact synonym '21 days post inoculation' _DONE in Protege_16_012024
8. AC add new strain 'IRE30' for Z. tritici to strain list _DONE 16_012024 e9723ab

[CuzickA]

Just checking through the updated Pathogen phenotype annotations -mostly look good now with a few issues to address below please @jingluodatacurator

Table 3 information Missing genotypes/phenotypes for 'IPO323 B-H267Y' and 'IPO323 B-S221P'

Table 4 information Missing genotype/phenotype for 'IRE30 B-H267Y'

These genotypes for Table 4 should be in strain 'IRE30' and not 'IPO323' (used in Table 3)

Also the first of these annotations should be two mutations within the same gene rather than a multi-locus allele. B-P155L, B-H267Y

[CuzickA]

Further thoughts on the PHI phenotypes

As noted above the article states in several places that the mutations do not have a fitness cost. Therefore, the PHI phenotype annotations would probably use AE infective ability 'unaffected pathogenicity' and maybe an AE for severity of the phenotypes between the WT and mutant interactions.

From the Summary text 'Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations.'

From the earlier comment... necrotic lesions with pycnidia (data not shown) Text suggests present in mutants but to a lesser extent 'All mutants produced lesions with pycnidia, but some areas of green leaf tissue were still observed within the inoculated areas.'

It may be more straight forward to curate the WT controls with 'presence of pathogen-associated host lesions' and AE severity 'high' and the mutants also with 'presence of pathogen-associated host lesions' with AE severity 'medium' and AE 'unaffected pathogenicity'.

The data on spore counts for the mutants is a bit of a mixed bag and the authors repeated state that there are no fitness penalties.

Suggested mutant pathogen genotypes within the metagenotype (info from Table 5, although 'data not shown' for lesion phenotype) Strain IRE30 B-S221P B-H267N B-H267Y D-D129E

@jingluodatacurator, please have a read through my comments to see if you agree.

[CuzickA]

AE alteration in archetype

Nichola's Table S4

SdhB(aaC137R)[Not assayed] 'C147R; SdhB; PYRNTE'

SdhB(aaS221T)[Not assayed] 'S231T; SdhB; PYRNTE'

SdhB(aaR265P)[Not assayed] 'R275P; SdhB; PYRNTE'

SdhB(aaH267F)[Not assayed] 'H277F; SdhB; PYRNTE'

SdhB(aaH267L)[Not assayed] 'H277L; SdhB; PYRNTE'

SdhB(aaH267N)[Not assayed] 'H277N; SdhB; PYRNTE'

SdhB(aaI269V)[Not assayed] 'H279V; SdhB; PYRNTE'

SdhB(aaS221P)[Not assayed] 'S231P; SdhB; PYRNTE'

SdhB(aaH267Y)[Not assayed] 'H277Y; SdhB; PYRNTE'

SdhB(aaS221P)[Not assayed] 'S231P; SdhB; PYRNTE'

SdhB(aaP155L, H267Y)[Not assayed] Info from above for SdhB(aaH267Y)[Not assayed] 'H277Y; SdhB; PYRNTE'

Cannot find info for 'SdhB(aaP155L' In Nicola's S file or Mair et al 2016. Might need to use FRAST.

~AE not added~

Update 17_01_2024 FRAST done below. SdhB aaP155L (IRE30) 'P165L; SdhB; PYRNTE' AE now added SdhB(aaP155L, H267Y)[Not assayed] 'P165L, H277Y; SdhB; PYRNTE'

[CuzickA]

SdhC(aaL85P)[Not assayed] 'L74P; SdhC; PYRNTE'

(assume typo in Table 'N75A' same entry as row above) SdhC(aaN86K)[Not assayed] 'N75K; SdhC; PYRNTE'

[jingluodatacurator]

Just checking through the updated Pathogen phenotype annotations -mostly look good now with a few issues to address below please @jingluodatacurator

Table 3 information Missing genotypes/phenotypes for 'IPO323 B-H267Y' and 'IPO323 B-S221P'

Table 4 information Missing genotype/phenotype for 'IRE30 B-H267Y'

These genotypes for Table 4 should be in strain 'IRE30' and not 'IPO323' (used in Table 3)

Also the first of these annotations should be two mutations within the same gene rather than a multi-locus allele. B-P155L, B-H267Y

Hi @CuzickA, missing genotype/phenotype annotations have been added, please check thanks.

[CuzickA]

This previously missing entries now look good thanks @jingluodatacurator

Thanks for changing the strain in these multi-locus gneotypes

One more genotype change needs to be made please for This should not be a multi-locus genotype as there are two mutations within the SdhB gene

[CuzickA]

Hi @jingluodatacurator,

These multi-locus annotations for Table 3 look great. The genotypes are correct. I have made a few edits to the figure, AE and conditions. In the conditions we want to describe the phenotyping 'Phenotyping and genotyping of carboxin-resistant UV mutants and field strains' of the uv mutants not the creation of the uv mutants 'Generation of SDHI-resistant UV mutants' :-).

Hi @jingluodatacurator, I have also just noticed that the edits I made to annotations in this comment here have not yet been applied to the rest of the annotations. Please could you go ahead and do this? Let me know if you have any questions :-)

[jingluodatacurator]

This previously missing entries now look good thanks @jingluodatacurator

Thanks for changing the strain in these multi-locus gneotypes

One more genotype change needs to be made please for This should not be a multi-locus genotype as there are two mutations within the SdhB gene

Hi @CuzickA, I don't quite understand why 'C-L85P, D-V96A' is multi-locus genotype but 'B-P155L, B-H267Y' is not?

[CuzickA]

SdhD(aaI127V)[Not assayed] 'I143V; SdhD; PYRNTE'

SdhD(aaD129E)[Not assayed] 'D145E; SdhD; PYRNTE'

SdhD(aaD129T)[Not assayed] 'D145T; SdhD; PYRNTE'

[CuzickA]

This previously missing entries now look good thanks @jingluodatacurator Thanks for changing the strain in these multi-locus gneotypes One more genotype change needs to be made please for This should not be a multi-locus genotype as there are two mutations within the SdhB gene

Hi @CuzickA, I don't quite understand why 'C-L85P, D-V96A' is multi-locus genotype but 'B-P155L, B-H267Y' is not?

Hi @jingluodatacurator, good question. 'C-L85P, D-V96A' is a multi-locus genotype because the L85P mutation takes place in the SdhC gene and the V96A mutation in the SdhD gene. Two different genes and thus multi-locus. For 'B-P155L, B-H267Y', both mutations P155L and H267Y take place in the same gene SdhB so this a single locus genotype rather than a multi-locus genotype. Hope that helps.

[jingluodatacurator]

This previously missing entries now look good thanks @jingluodatacurator Thanks for changing the strain in these multi-locus gneotypes One more genotype change needs to be made please for This should not be a multi-locus genotype as there are two mutations within the SdhB gene

Hi @CuzickA, I don't quite understand why 'C-L85P, D-V96A' is multi-locus genotype but 'B-P155L, B-H267Y' is not?

Hi @jingluodatacurator, good question. 'C-L85P, D-V96A' is a multi-locus genotype because the L85P mutation takes place in the SdhC gene and the V96A mutation in the SdhD gene. Two different genes and thus multi-locus. For 'B-P155L, B-H267Y', both mutations P155L and H267Y take place in the same gene SdhB so this a single locus genotype rather than a multi-locus genotype. Hope that helps.

Thanks @CuzickA, could you please point me the direction where this information is in the text?

[CuzickA]

SdhB(aaS221P)[Not assayed] SdhC(aaR54G)[Not assayed]

This previously missing entries now look good thanks @jingluodatacurator Thanks for changing the strain in these multi-locus gneotypes One more genotype change needs to be made please for This should not be a multi-locus genotype as there are two mutations within the SdhB gene

Hi @CuzickA, I don't quite understand why 'C-L85P, D-V96A' is multi-locus genotype but 'B-P155L, B-H267Y' is not?

Hi @jingluodatacurator, good question. 'C-L85P, D-V96A' is a multi-locus genotype because the L85P mutation takes place in the SdhC gene and the V96A mutation in the SdhD gene. Two different genes and thus multi-locus. For 'B-P155L, B-H267Y', both mutations P155L and H267Y take place in the same gene SdhB so this a single locus genotype rather than a multi-locus genotype. Hope that helps.

Thanks @CuzickA, could you please point me the direction where this information is in the text?

We are annotating three genes SdhB, SdhC and SdhD

In the Tables the mutant name prefix corresponds to the Sdh gene that is mutated eg B-P155L, B-H267Y refers to gene SdhB.

[CuzickA]

SdhB(aaS221P)[Not assayed] SdhC(aaR54G)[Not assayed]

Two AES, one for each locus 'S231P; SdhB; PYRNTE' (from above)

SdhC(aaR54G) Not listed in Nichola's S file or Mair et al 2016

AE not added

Update 17_01_2024 Did FRAST search below - not clear alignment -not sure what to do here yet.

[jingluodatacurator]

SdhB(aaS221P)[Not assayed] SdhC(aaR54G)[Not assayed]

This previously missing entries now look good thanks @jingluodatacurator Thanks for changing the strain in these multi-locus gneotypes One more genotype change needs to be made please for This should not be a multi-locus genotype as there are two mutations within the SdhB gene

Hi @CuzickA, I don't quite understand why 'C-L85P, D-V96A' is multi-locus genotype but 'B-P155L, B-H267Y' is not?

Hi @jingluodatacurator, good question. 'C-L85P, D-V96A' is a multi-locus genotype because the L85P mutation takes place in the SdhC gene and the V96A mutation in the SdhD gene. Two different genes and thus multi-locus. For 'B-P155L, B-H267Y', both mutations P155L and H267Y take place in the same gene SdhB so this a single locus genotype rather than a multi-locus genotype. Hope that helps.

Thanks @CuzickA, could you please point me the direction where this information is in the text?

We are annotating three genes SdhB, SdhC and SdhD

In the Tables the mutant name prefix corresponds to the Sdh gene that is mutated eg B-P155L, B-H267Y refers to gene SdhB.

Thanks @CuzickA, now I fully understand it with prefix and multi-locus genotypes/double mutations.

[CuzickA]

SdhB(aaH267Y)[Not assayed] SdhC(aaN86S)[Not assayed]

Two AES, one for each locus 'H277Y; SdhB; PYRNTE' (from above) 'N75S; SdhC; PYRNTE'

[CuzickA]

SdhB(aaD166G)[Not assayed] SdhD(aaD129G)[Not assayed]

Two AES, one for each locus SdhD(aaD129G)[Not assayed] 'D145G; SdhD; PYRNTE' ' From Nichola's S file Table S4

SdhB(aaD166G) Not listed in Nichola's S file or Mair et al 2016

~AE not added~

Update 17_01_2024 FRAST search done below SdhB aaD166G (IPO323) 'D176G; SdhB; PYRNTE'

SdhB(aaD166G)[Not assayed] SdhD(aaD129G)[Not assayed] Two AES, one for each locus 'D176G; SdhB; PYRNTE' (from FRAST below) 'D145G; SdhD; PYRNTE' (from above) now added

[CuzickA]

SdhB(aaI13V)[Not assayed] SdhD(aaD129E, V154A)[Not assayed]

Two AES, one for each locus SdhD(aaD129E, V154A) 'D145E, XXX; SdhD; PYRNTE'

SdhD(aaV154A) Not listed in Nichola's S file or Mair et al 2016

SdhB(aaI13V) Not listed in Nichola's S file or Mair et al 2016

AE not added

Update 17_01_2024 Did FRAST search below - not clear alignment -not sure what to do here yet.

[CuzickA]

SdhC(aaL85P)[Not assayed] SdhD(aaV96A)[Not assayed]

Two AES, one for each locus 'L74P; SdhC; PYRNTE' (from above)

SdhD(aaV96A) Not listed in Nichola's S file or Mair et al 2016

[jingluodatacurator]

Hi @jingluodatacurator, These multi-locus annotations for Table 3 look great. The genotypes are correct. I have made a few edits to the figure, AE and conditions. In the conditions we want to describe the phenotyping 'Phenotyping and genotyping of carboxin-resistant UV mutants and field strains' of the uv mutants not the creation of the uv mutants 'Generation of SDHI-resistant UV mutants' :-).

Hi @jingluodatacurator, I have also just noticed that the edits I made to annotations in this comment here have not yet been applied to the rest of the annotations. Please could you go ahead and do this? Let me know if you have any questions :-)

Hi @CuzickA, I'm not quite sure what to do here, please could you give me a pointer.

[CuzickA]

Notes to self on AE alteration in archetype 1) I have successfully added AE to all single locus genotypes (still waiting for SdhB double mutant to be created). 2) I have tried to add AE to multi-locus genotypes. This has only been possible in one case. In the other cases I have not been able to find the information in Nichola's S file or Mair et al 2016 paper.

Next steps for AE alteration in archetype 1) Add to SdhB double mutant once created. _Tried to do this but will need to use FRAST 17_012024 2) Maybe use FRAST to look up missing information for multi-allele genotypes. 3) Once I have completed 1 and 2, ask Nichola to check the AEs.

[CuzickA]

Hi @CuzickA, I'm not quite sure what to do here, please could you give me a pointer.

Hi @jingluodatacurator, no problem.

In my comment here

I have made a few edits to the figure, AE and conditions. In the conditions we want to describe the phenotyping 'Phenotyping and genotyping of carboxin-resistant UV mutants and field strains' of the uv mutants not the creation of the uv mutants 'Generation of SDHI-resistant UV mutants' :-).

I am saying that the 'figure' and 'conditions' should reflect the phenotype expt rather than the creation of the mutants by uv. In this example I have changed the 'figure' and 'conditions' to reflect the 'phenotype expt'

In the other annotations where the changes have not been made there is unnecessary information like 'uv' etc Figure 4 refers more to the mutant genotypes and not the actual expt to look at the phenotypes which are reported in the Table 4 (or Table 3 for the IPO323 strain mutants).

Please could you carefully go through the annotations and make the edits to correct the 'figure' and 'conditions'. In one case it looks as though I had also added the AE severity to indicate that the increase in carboxin resistance is very high >200 mg/mL. You could also add this AE severity to the other two genotypes in the table with >200 mg/mL B-H267L and D-D129T

[CuzickA]

@jingluodatacurator I see this new genotype/phenotype annotation has been added thanks

Is this for B-P155L, B-H267Y? If so then the second mutation also needs adding :-). Sorry if I've jumped the gun here and you are currently working on it ;-).

It would look like this SdhB(aaP155L, H267Y)[Not assayed]

[CuzickA]

Updated next steps list as this ticket is getting long

1) we have a uniprot query 2) @jingluodatacurator to make changes to the single locus genotype SdhB(aaP155L, H267Y)[Not assayed] _DONE 16_012024 3) @jingluodatacurator try and make any relevant PHI phenotype annotations (see comments above). 4) @jingluodatacurator to make edits to 'figure', 'conditions' and add AE severity where required (comments above). 5) AC to check all annotations once done. 6) AC to try and add in additional AE alteration in archetype to SdhB(aaP155L, H267Y)[Not assayed] and maybe use FRAST to look up info for multi-locus genotypes. _Tried to add AE to SdhB(aaP155L, H267Y) but need to use FRAST for aaP155L. 17_012024 Have now tried FRAST for all queries - some unclear entries to follow up with Nichola. 7) AC to check AEs with Nichola.

[jingluodatacurator]

@jingluodatacurator I see this new genotype/phenotype annotation has been added thanks

Is this for B-P155L, B-H267Y? If so then the second mutation also needs adding :-). Sorry if I've jumped the gun here and you are currently working on it ;-).

It would look like this SdhB(aaP155L, H267Y)[Not assayed]

Hi @CuzickA, I have amended it thanks for spotting it.

[CuzickA]

Need to use FRAST to look up Zt SdhB aaI13V (IPO323) unclear?? SdhB aaP155L (IRE30) Done and AE added SdhB aaD166G (IPO323) Done and AE added SdhC aaR54G (IPO323) unclear?? SdhD aaV96A (IRE30) unclear?? SdhD aaV154A (IPO323) unclear??

Genbank accession numbers from article - note: these are for strain IRE30 (other numbers within the range may cover seqs from other strains IRE30, BC1, BC4, R35-3, R39-1, V212-2 BUT COULD NOT FIND IPO323) JF916683 for SdhB https://www.ncbi.nlm.nih.gov/nuccore/JF916683 JF916695 for SdhC https://www.ncbi.nlm.nih.gov/nuccore/JF916695 JF916689 for SdhD https://www.ncbi.nlm.nih.gov/nuccore/JF916689

Generated FASTA files for FRAST

[CuzickA]

FRAST Zt SdhB strain IRE30 sequence input

SdhB aaI13V (IPO323) -Not sure what to do here?? Ask Nichola maybe '20V; SdhB; PYRNTE'

SdhB aaP155L (IRE30) 'P165L; SdhB; PYRNTE'

SdhB aaD166G (IPO323) 'D176G; SdhB; PYRNTE'

[CuzickA]

FRAST Zt SdhC strain IRE30 sequence input SdhC aaR54G (IPO323) -Not sure what to do here?? Ask Nichola maybe '44G; SdhC; PYRNTE'

Could maybe be... SdhB(aaS221P)[Not assayed] SdhC(aaR54G)[Not assayed]

Two AES, one for each locus 'S231P; SdhB; PYRNTE' (from above) '44G; SdhC; PYRNTE' (FRAST)

AE not added

[CuzickA]

FRAST Zt SdhC strain IRE30 sequence input (note same strain as below)

SdhD aaV96A (IRE30) '112A; SdhD; PYRNTE' Check with Nichola if this is correct??

SdhC(aaL85P)[Not assayed] SdhD(aaV96A)[Not assayed] Two AES, one for each locus 'L74P; SdhC; PYRNTE' (from above) '112A; SdhD; PYRNTE' (FRAST)

AE not added

SdhD aaV154A (IPO323) '170A; SdhD; PYRNTE' Check with Nichola if this is correct??

SdhB(aaI13V)[Not assayed] SdhD(aaD129E, V154A)[Not assayed] Two AES, one for each locus '20V; SdhB; PYRNTE' (FRAST) query this will Nichola 'D145E, 170A; SdhD; PYRNTE' (FRAST)

AE not added

[CuzickA]

Updated next steps list

1) we have a uniprot query 2) @jingluodatacurator try and make any relevant PHI phenotype annotations (see comments above). 3) @jingluodatacurator to make edits to 'figure', 'conditions' and add AE severity where required (comments above). _DONE 17_012024 4) AC to check all annotations once done. 5) AC to check AEs and queries with Nichola.

[jingluodatacurator]

Further thoughts on the PHI phenotypes

As noted above the article states in several places that the mutations do not have a fitness cost. Therefore, the PHI phenotype annotations would probably use AE infective ability 'unaffected pathogenicity' and maybe an AE for severity of the phenotypes between the WT and mutant interactions.

From the Summary text 'Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations.'

From the earlier comment... necrotic lesions with pycnidia (data not shown) Text suggests present in mutants but to a lesser extent 'All mutants produced lesions with pycnidia, but some areas of green leaf tissue were still observed within the inoculated areas.'

It may be more straight forward to curate the WT controls with 'presence of pathogen-associated host lesions' and AE severity 'high' and the mutants also with 'presence of pathogen-associated host lesions' with AE severity 'medium' and AE 'unaffected pathogenicity'.

The data on spore counts for the mutants is a bit of a mixed bag and the authors repeated state that there are no fitness penalties.

Suggested mutant pathogen genotypes within the metagenotype (info from Table 5, although 'data not shown' for lesion phenotype) Strain IRE30 B-S221P B-H267N B-H267Y D-D129E

@jingluodatacurator, please have a read through my comments to see if you agree.

Hi @CuzickA, I'm not sure if I understood the data in Table 5 completely, but I gave another go at creating the PHI phenotypes and their AEs. Please check and let me know any issues thanks.

[jingluodatacurator]

Hi @CuzickA, I'm not quite sure what to do here, please could you give me a pointer.

Hi @jingluodatacurator, no problem.

In my comment here

I have made a few edits to the figure, AE and conditions. In the conditions we want to describe the phenotyping 'Phenotyping and genotyping of carboxin-resistant UV mutants and field strains' of the uv mutants not the creation of the uv mutants 'Generation of SDHI-resistant UV mutants' :-).

I am saying that the 'figure' and 'conditions' should reflect the phenotype expt rather than the creation of the mutants by uv. In this example I have changed the 'figure' and 'conditions' to reflect the 'phenotype expt'

In the other annotations where the changes have not been made there is unnecessary information like 'uv' etc Figure 4 refers more to the mutant genotypes and not the actual expt to look at the phenotypes which are reported in the Table 4 (or Table 3 for the IPO323 strain mutants).

Please could you carefully go through the annotations and make the edits to correct the 'figure' and 'conditions'. In one case it looks as though I had also added the AE severity to indicate that the increase in carboxin resistance is very high >200 mg/mL. You could also add this AE severity to the other two genotypes in the table with >200 mg/mL B-H267L and D-D129T

Hi @CuzickA, I have amended figures and conditions, and also added in high severity in two >200 cases. Please check and let me know any issues thanks.

[CuzickA]

Hi @CuzickA, I have amended figures and conditions, and also added in high severity in two >200 cases. Please check and let me know any issues thanks.

Hi @jingluodatacurator, Thanks for making the changes to the 'conditions' and adding in the two AEs for high severity.

Please could you also edit the 'Figure' information. The text 'Figure 4' needs deleting. We only want the Table information as this explains the phenotype expt rather than the information about the mutations and how they align in other species shown in Figure 4.

[CuzickA]

Copy of newly made PHI annotations by @jingluodatacurator

[jingluodatacurator]

Hi @CuzickA, I have amended figures and conditions, and also added in high severity in two >200 cases. Please check and let me know any issues thanks.

Hi @jingluodatacurator, Thanks for making the changes to the 'conditions' and adding in the two AEs for high severity.

Please could you also edit the 'Figure' information. The text 'Figure 4' needs deleting. We only want the Table information as this explains the phenotype expt rather than the information about the mutations and how they align in other species shown in Figure 4.

Hi @CuzickA, I have understood it now and have deleted Figure 4 annotations, thanks.