Deliverable D1.1: List of candidate cell wall target enzymes - M6 (P1 and P2) (ICPSR doi:10.13130/RD_UNIMI/KKCY2I)

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Document Description

Citation

Title:

Deliverable D1.1: List of candidate cell wall target enzymes - M6 (P1 and P2)

Identification Number:

doi:10.13130/RD_UNIMI/KKCY2I

Distributor:

UNIMI Dataverse

Date of Distribution:

2020-01-04

Version:

1

Bibliographic Citation:

Pesaresi, Paolo, 2020, "Deliverable D1.1: List of candidate cell wall target enzymes - M6 (P1 and P2)", https://doi.org/10.13130/RD_UNIMI/KKCY2I, UNIMI Dataverse, V1

Study Description

Citation

Title:

Deliverable D1.1: List of candidate cell wall target enzymes - M6 (P1 and P2)

Identification Number:

doi:10.13130/RD_UNIMI/KKCY2I

Authoring Entity:

Pesaresi, Paolo

Grant Number:

828940

Distributor:

UNIMI Dataverse

Access Authority:

Pesaresi, Paolo

Depositor:

Pesaresi, Paolo

Date of Deposit:

2020-01-03

Study Scope

Keywords:

Agricultural Sciences, Target Enzymes

Abstract:

List of candidate cell wall target enzymes to be used for the Yeast two-hybrid screening, as indicated in Deliverable D1.1 due at Month 6.

Notes:

WP1. Deciphering and targeting cell wall biosynthesis in oomycetes D1.1 List of candidate cell wall target enzymes - M6 (P1 and P2) The genomes of Plasmopara viticola, Phytophthora infestans, Phytophthora capsici, Phytophthora ramorum and Pythium ultimum have been mined for predicted proteins. Among them, annotated Carbohydrate-Active Enzymes (CAZy) have been identified using the dbCAN2 meta server. The dataset has been further filtered for enzymes belonging to glycosyltransferases family 2 (GT2), glycosyl hydrolases family 72 (GH72) and potentially transglycosylases. Furthermore, the selected subset of enzymes has been subjected to functional annotation, protein topology analysis and signal peptide identification using the Blat2GO software and TOPCONS web server. In total, 72 candidates have been identified by P2 (KTH) and the list was shared with P1 (UMIL) on March 2019. The 72 potential targets were analyzed by P1 through CD-HIT, a clustering algorithm that groups proteins based on their sequence identity, a 70% threshold was set for the analysis. In total, 28 clusters were identified. The 10 most populated clusters were assumed to contain the most conserved proteins. The priority in the selection was given to proteins without transmembrane domains (TMDs) presence, predicted with TOPCONS, to raise the reliability of the Yeast two-Hybrid technique (it works with soluble proteins). Almost all GT2 proteins display TMDs presence. However, since GT2 proteins have a known relevance in pathogenesis and cell wall formation (Grenville-Briggs et al., 2008; Guerriero et al., 2010; Rzeszutek et al., in preparation by V. Bulone group), some representative members were selected anyway, such as different cellulose synthases and chitin synthases. To grant the best representation of the 10 most populated clusters, a proportional number of sequences to cluster dimension were selected. At the end of the analysis, P1 indicated 15 interesting CAZy targets for the Yeast two-Hybrid screenings, 6 from P. infestans, 3 from P. viticola, 3 from P. capsici, 2 from P. ramorum and 2 from P. ultimum, and a preliminary list of 20 targets was defined including the previously selected P. infestans proteins: AVR3a, PE1, PexRD54 and EPIC2B. During the SKYPE call between P1 and P2 occurred on 7th June 2019, it was decided to discard the proteins classified as “hypothetical”, to delete mannosyltransferases from the list and to reduce the number of target of P. viticola from 3 to 2 to deserve the possibility to select more specific targets after its cell wall characterization. To face the discussed list modifications, a P. ramorum glycoside hydrolase protein (jgi|Phyra1_1|94466|C_scaffold_18000008) was selected, even though it clusters at lower levels of sequence identity (60%). Some of the selected target proteins, present in the final list, could be changed in future due to emerging needs and/or observations.

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D1.1 List of candidate cell wall target enzymes.xps

Text:

List of target proteins from different oomycetes to be used for the Y2H screening

Notes:

application/vnd.ms-xpsdocument