PMID: 35468894 A fungal extracellular effector inactivates plant polygalacturonase-inhibiting protein

[CuzickA]

Just seen this whilst preparing the latest Batch 112 of articles for MC curation. From the abstract looks like it could be a good first host target paper for curation.

[JonMWilkes]

Any preference who curates this paper? I have just cleared my current set of papers. I've looked over the pdf, it seems quite a 'chunky' paper with a lot of supplementary material.

[CuzickA]

Hi @JonMWilkes, thanks for your message. This paper will probably be for @smvelasquez as it contains a pathogen effector first host target which she is currently training on for the plant community.

[smvelasquez]

https://canto.phi-base.org/curs/010b2ec99d0d70e8

[smvelasquez]

@CuzickA How do you annotate the following combination: Host overexpressing the pathogen's gene which is then infected with the pathogen's wt strain or the deletion mutant of the pathogen's gene?

[CuzickA]

I think this example covers it

Select the condition terms to capture the host expressing the pathogen's gene and then the pathogen WT inoculation.

Hope that helps :-)

[smvelasquez]

I need new PHIPO terms: reduced/absent inhibition of pathogen polygalacturonase by host, normal sclerotia formation, +polygalacturonate acid, +dinitrosalicylic acid

[CuzickA]

Moved to correct issue

[smvelasquez]

@CuzickA Yes, those are the situations I was referring to

[CuzickA]

Checking this session now.

In the first results paragraph there is this text

"The S. sclerotiorum genome contains a single copy of SsPINE1 (Ss1g_08128)"

When I search for 'Ss1g_08128' in UniProt I get A7ES23 https://www.uniprot.org/uniprotkb/A7ES23/entry which is not listed below

Has the correct UniProt Id been used here?

[CuzickA]

It looks like 'sscle_10g074920' 'A0A1D9QD76' has been used for the Pathogen effector 'SsPINE1'. I'm not sure why? This UniProt may need changing but I will continue checking the session for now.

Pathogen phenotype Fig S1c

changed to

(note 'Two SsPINE1 knockout (ΔSsPINE1) mutants (KoSsPINE1-1 and KoSsPINE1-7)' combined as single DeltaSsPINE1 annotation).

Deleted WT control as not required for Pathogen phenotypes and also deleted complementation control as these are not curated.

[smvelasquez]

@CuzickA I used this Uniprot ID A0A1D9QD76. I don't know what happened, but I presume that I couldn't find the Uniprot ID code with just adding the gene's id, and I found this one instead

[CuzickA]

@CuzickA I used this Uniprot ID A0A1D9QD76. I don't know what happened, but I presume that I couldn't find the Uniprot ID code with just adding the gene's id, and I found this one instead

Okay thanks for letting me know. Perhaps UniProt made some changes.

I will try and change the UniProt ID at the end once I've finished checking.

[CuzickA]

Fig 1a PHI Phenotypes

changed to

Removed complementation controls. Changed PHIPO term annotation 'presence of pathogen-associated host defense induced lesions' to 'presence of pathogen-associated host lesions' as no evidence in this expt that lesions were caused by host defense. (see point 4 in 'curation issues' comment in https://github.com/PHI-base/curation/issues/104) Added conditions and AEs

[CuzickA]

Fig 1b PHI phenotype

changed to PHI phenotype

Simplified genotypes. Remove second direction of Y2H I think we only need to annotate one combination.

[CuzickA]

Fig 1c

changed to

Simplified genotypes. Added condition and AE compared to.

[CuzickA]

See https://github.com/pombase/canto/issues/2677

[CuzickA]

Changed to

[CuzickA]

Fig 2

Changed to (Made similar edits as in Fig 1 above).

This was fine

I'm not sure if the correct UniProt id has been used for SsPG1. In this text from the paper, when I search UniProt for Ss1G_10167, I get the below 2 entries. I think the top entry should have been used A7EXV2. Instead an alternative UniProt Q8NKE6 has been used. I'm not sure where this has come from? @smvelasquez, can you remember how you obtained this UniProt please?

[smvelasquez]

@CuzickA I probably couldn't find that Uniprot ID with just that code, but if you look at Fungi DB, they have the same gene number.

[smvelasquez]

@CuzickA and looking at ENA, it says that my ID is mRNA, and the other one is DNA. Maybe that is the difference

[CuzickA]

@smvelasquez, thanks for looking into this. It's a bit confusing as the FungiDB record above seems to have multiple UniProt Ids listed including both A7EXV2 and Q8NKE6.

I'm still inclined to change the UniProt ids to those found using the gene ids in the paper (especially as this is a recent paper 2022).

Therefore, (noted in earlier comment above)For SsPINE1, change 'sscle_10g074920 / A0A1D9QD76' to 'A7ES23' which is found with search for 'Ss1g_08128' from paper. For SsPG1, change 'sspg1d / Q8NKE6' to 'A7EXV2' which is found with search for 'Ss1G_10167' from paper.

Just to note UniProt ref proteome

I don't plan on changing the UniProts until I've finished checking the session. At this point I will also flag up any UniProt entries that need their name updating using the regular feedback protocol.

[CuzickA]

Fig 3. SsPINE1 outcompetes SsPG1 in binding with AtPGIP1.

with tidied up genotypes

Hi @ValWood, I'm not sure how best to curate these protein binding affinity assays or whether it is even possible. The PHIPO term 'presence of pathogen host protein-protein interaction' doesn't seem adequate.

Paper text for Fig 3a

Fig 3b is a dissociation expt. This can be looked at after finishing Fig 3a.

[ValWood]

You are right we don't have a good way to capture this.

[CuzickA]

Thanks @ValWood. For now I will delete the annotations for Fig 3a and b. I have added a note to the FAQ doc under 'Information not to curate' and also in https://github.com/PHI-base/canto-docs/issues/10

[CuzickA]

Fig 4 a and b

note to self for Fig 4a,b i) SsPG1 shows PG activity in vitro expt ii) add AtPGIP1 to SsPG1 -> inhibition of SsPG1 PG activity iii) add SsPINE1 to AtPGIP1 and SsPG1 -> suppression of inhibitory affect of AtPGIP1 on SsPG1. Therefore restoring PG activity of SsPG1

I think these should just be GO annotations as WT proteins, rather than phenotype annotations.

[CuzickA]

Delete PHI Phenotype annotations for Fig4 a and b in favour of GO annotations.

@ValWood is it ok to have the AE 'has input SsPG1' here? I remember in a past ticket (#103) we discussed that for GO the species much match in the primary annotation and the AE

Also how best to capture 'iii) add SsPINE1 to AtPGIP1 and SsPG1 -> suppression of inhibitory affect of AtPGIP1 on SsPG1. Therefore restoring PG activity of SsPG1'? _(I have now deleted this annotation 30_012023). We currently have this with a new GO term suggestion. sscle_10g074920 is SsPINE1.

[ValWood]

I think it is OK to have the host species as an extension, especially if annotating the effects of an effector on a host protein. I have a feeling that wen we discussed this it was some non-biological mixing of species in some assay system (which would be incorrect).

[CuzickA]

Ok thanks. Yes the other example #103 had a protein from an additional (second) host species. So really the rule should be that AEs in GO annotations must match either the primary pathogen or host species when a PHI (making biological sense).

[CuzickA]

Fig 4c, d expt tricky Pea leaf infected with either WT SsPINE1 inoculum mixed with purified WT AtPGIP1 protein WT SsPINE1 inoculum mixed with purified mutant AtPGIP1 protein mutant SsPINE1delta inoculum mixed with purified WT AtPGIP1 protein mutant SsPINE1delta inoculum mixed with purified mutant AtPGIP1 protein

Pathogen = Ss Host = pea How to record adding the purified WT or mutant Arabidopsis AtPGIP1 protein?

[CuzickA]

Fig 4 c, d Tricky to curate these types of expts. The problem is that we need to capture information from 2 hosts - WT pea and then the purified WT or mutant PGIP1 protein from Arabidopsis.

I think we could add '+ polygalacturonase inhibitor' as a 'condition' to represent the WT AtPGIP1. We probably don't need to record the mutant AtPGIP1 as this is really used as a control, demonstrating that functional WT AtPGIP1 is required for SsPG1 inhibition.

I think it would also we useful to record the presence of the WT SsPG1 within the pathogen genotype. These would therefore become multiallele genotypes.

I think the annotations would then be for SsPINE1+, SsPG1+ on Pea ->lesions present /pathogen growth present SsPINE1+, SsPG1+ on Pea + condition '+ polygalacturonase inhibitor' ->lesions reduced/pathogen growth reduced SsPINE1delta, SsPG1+ on Pea ->lesions present /pathogen growth present SsPINE1delta, SsPG1+ on Pea + condition '+ polygalacturonase inhibitor' ->lesions absent/pathogen growth absent

[CuzickA]

A record of the current Fig 4c, d annotations before I make changes

[CuzickA]

Note: Fig 1 and 2 pathogen inoculation expt may also benefit from multiallele genotypes containing both SsPINE1 and SsPG1.

[CuzickA]

I now have the following I have compared metagenotypes with and without the condition '+ polygalacturonase inhibitor' rather than between differences in the metagenotype. It's a tricky call to know which comparisons to make. @smvelasquez please could you have a look at the changes and see if you think they captures Fig 4c,d?

[CuzickA]

Correcting GO MF annotation AE happens during for host AtPGIP1

I think 'happens_during induction by symbiont of host immune response' would be used for a pathogen gene not a host gene.

changed to below

[CuzickA]

Adding new GO MF annotation for SsPINE1 protein binding to AtPGIP1 (modelled on https://github.com/PHI-base/curation/issues/66) Current GO MF annotations

Current Fig 4 GO BP annotations

changed to

[CuzickA]

Fig 5a PHI phenotypes changed to

Unable to read text in S fig 8 reporting exact aa changes with the P1, P2, P3 mutants of At PGIP1. In each case multiple aa substitutions within LRR regions.

[CuzickA]

Fig 5b

changed to

[CuzickA]

Fig 5c,d

I think the control WT metagenotype here would require 'NTR: inhibition of pathogen polygalacturonase by host present' following

I don't think we need 'dinitrosalicylic acid' as a condition for the DNSA method used in Fig 5d as the method text states 'DNSA solution was added to stop the reaction'.

[CuzickA]

S Fig 9 Host phenotypes

Change expression from not assayed to overexpression as 35::AtPGIP is being expressed at higher level in Arabidopsis than the WT Arabidopsis PGIP.

Delete second row annotation 'increased RNA level' as this is really just a control 'overexpression of AtPGIP1-3xFlag was confirmed by Western blot and RT-qPCR (Supplementary Fig. 9c, d). '

Add PHI phenotype for 'Stable 35S::SsPINE1-GFP Arabidopsis (Col-0) overexpressing line' modelled on #104, #42 and #89

Also added GO CC

[CuzickA]

Fig 6 current annotations (next comment will show changes made)

[CuzickA]

Fig 6 text - this is a tricky expt to curate 'The three genotypes (Col-0, 35S::SsPINE1 and 35S::AtPGIP1) of A. thaliana plants were tested for their reactions to infection by three genotypes of S. sclerotiorum (wild-type, ΔSsPINE1 and ΔSsPG1) strains.'

Mock ups (to be continued and checked) +WMA1 (WT) SsPINE1+, PG1+ (WT level) and AtPGIP1+ (WT level) (or just At WT) = lesions present (Fig 6a,b) SsPINE1+(overexpression) and AtPGIP1+ (WT level)(or just At WT), delivery mechanism: pathogen gene expressed by transgenic host, and + wild type pathogen inoculation (including wt Pine1 and pg1)= increased lesions (Fig 6a,b) SsPINE1+, PG1+ (WT level) and AtPGIP1+ (overexpression)= lesions present (Fig 6a,b)

+SsPINE1delta SsPINE1delta, PG1+ (WT level) and AtPGIP1+(WT level) (or just At WT) = lesions present (less than WT SsPINE1+ on WT At)(Fig 6a,b) CANNOT CURATE SsPINE1+(overexpression) and AtPGIP1+ (WT level) (or just At WT), delivery mechanism: pathogen gene expressed by transgenic host, and + wild type pathogen inoculation BUT pathogen inoculation is SsPINE1delta increased lesions (Fig 6a,b) SsPINE1delta, PG1+ (WT level) and AtPGIP1+ (overexpression) = decreased lesions / lesions absent (Fig 6a,b)

+SsPG1delta SsPG1delta, PINE1+ (WT level) and AtPGIP1+(WT level) (or just At WT) = lesions absent (close enough to absent, compared to WT SsPG1+ on WT At )(Fig 6a,b) CANNOT CURATE SsPINE1+(overexpression) and AtPGIP1+ (WT level) (or just At WT), delivery mechanism: pathogen gene expressed by transgenic host, and + wild type pathogen inoculation BUT pathogen inoculation is SsPG1delta lesions present (Fig 6a,b) SsPINE1delta, PG1+ (WT level) and AtPGIP1+ (overexpression) = absent lesions (Fig 6a,b)

[CuzickA]

Not all of the information can be curated for Expt 6 We cannot curate pathogen genotype - host genotype + mutant pathogen inoculation

Need to decide whether it is worth curating part of Fig 6 or none of it. Partial curation may not be very informative.

I think I will leave this for now and come back to it after checking the other annotations.

[CuzickA]

Fig 7 'BcPINE1 (BC1G_04506) of Botrytis cinerea wildtype strain B05.10' I couldn't find a UniProt for 'BC1G_04506' Google indicates locus tag name has been updated to 'BCIN_04g02570' UniProt search for 'BCIN_04g02570' gives 'A0A384JF20', which is the UniProt id that has been used in this session.

[CuzickA]

Fig 7a, b

changed to

I have deleted below as cannot curate info on 2 pathogen species (Bc and Ss) and 1 host (At) BcPINE1+ inoculated onto AtPGIP1+ transgenically expressing SsPINE1 BcPINE1Delta inoculated onto AtPGIP1+ transgenically expressing SsPINE1

[CuzickA]

Fig 7c

changed to

Again deleted as not possible to curate

[CuzickA]

Fig 7d PHI phenotype Physical interaction GO BP

edits to

Added GO MF

Deleted GO BP

I don't think there is enough data to annotate BcPINE1 as an effector.

[CuzickA]

Fig S2

edits to

removed annotation for AT2G35790 following note in text 'Since AT2G35790 is a trans-membrane protein in the mitochondria with no known functions and SsPINE1 is in the apoplast, its interaction with SsPINE1 is likely an artifact of yeast 2-hybrid assay.'

GO MF for At PGIP2

[CuzickA]

Note: This session still needs curation/re-curation/checking.