Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process

Abstract

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by co-immunoprecipitation and liquid chromatography tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and co-localize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome data set will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network

“…It is possible that AVRcap1b is co-opting NbTOL9a to hijack a similar immunomodulatory trafficking pathway in the plant cell to counteract NRC-mediated HR cell death and suppress immunity. This model would be consistent with the observation that vesicle trafficking is massively reprogrammed by P. infestans effectors during infection [49,[73][74][75].…”

“…This model would be consistent with the observation that vesicle trafficking is massively reprogrammed by P . infestans effectors during infection [ 49 , 73 – 75 ].…”

“…Given that AVRcap1b did not interact with NRCs, we reasoned that this effector targets another host protein that is involved in NLR immunity. To narrow down the list of 13 candidate interactors obtained from the Y2H screen, we subjected AVRcap1b to in planta coIP coupled with tandem mass spectrometry (IP-MS) using methods that are well established in our laboratory [48][49][50]. IP-MS experiments with GFP::AVRcap1b resulted in the identification of 8 unique AVRcap1b interactors that were not recovered with the control GFP::PexRD54, another P. infestans effector of a similar fold and size (S11 Fig, S4 Table).…”

“…Mass spectrometry data processing. As previously described [48][49][50]97,100], peak lists were extracted from raw data using MS Convert [101] and peptides identified on Mascot server 2.4.1 using Mascot Daemon (MatrixAU : PerPLOSstyle; donotuseLtd:; etc:exceptasappropriatein Science). The peak lists were searched against the N. benthamiana genome database called Nicotiana_Benthamiana_Nbv6trPAplusSGNU-niq_20170808 (398,682 sequences; 137,880,484 residues), supplemented with common contaminants.…”

Uncovering the Role of Metabolism in Oomycete–Host Interactions Using Genome-Scale Metabolic Models

“…In one study that was based on yeast-two-hybrid screenings, a protein–protein interaction network of Arabidopsis thaliana and pathogens of three kingdoms uncovered that effectors from different pathogens convergently target the same host proteins ( Weßling et al, 2014 ). In a more recent study based on mass spectrometry analyses of immunoprecipitated effector–host target protein complexes in Nicotiana benthamiana , the deduced protein–protein interaction network revealed the cellular vesicle trafficking machinery as a major effector-targeted process ( Petre et al, 2021 ). In other studies, GEMs have been reconstructed for the bacterial plant pathogens Ralstonia solanacearum , Xanthomonas oryzae , and Pectobacterium parmentieri ( Peyraud et al, 2016 ; Zoledowska et al, 2018 ; Koduru et al, 2020 ), and for the fungus Sclerotinia sclerotiorum ( Peyraud et al, 2019 ).…”