Abstract:
Oomycetes of the genus Phytophthora encompass several of the most
successful plant pathogens described to date. The success of infection by Phytophthora
species is attributed to the pathogens’ ability to secrete effector proteins that alter the
host’s physiological processes. Structural analyses of effector proteins mainly from bacterial
and viral pathogens have revealed the presence of intrinsically disordered regions
that host short linear motifs (SLiMs). These motifs play important biological roles by
facilitating protein-protein interactions as well as protein translocation. Nonetheless,
SLiMs in Phytophthora species RxLR effectors have not been investigated previously and
their roles remain unknown. Using a bioinformatics pipeline, we identified 333 candidate
RxLR effectors in the strain INRA 310 of Phytophthora parasitica. Of these, 71 (21%) were
also found to be present in 10 other genomes of P. parasitica, and hence, these were
designated core RxLR effectors (CREs). Within the CRE sequences, the N terminus exhibited
enrichment in intrinsically disordered regions compared to the C terminus, suggesting
a potential role of disorder in effector translocation. Although the disorder content
was reduced in the C-terminal regions, it is important to mention that most SLiMs were
in this terminus. PpRxLR1 is one of the 71 CREs identified in this study, and its genes
encode a 6-amino acid (aa)-long SLiM at the C terminus. We showed that PpRxLR1 interacts
with several host proteins that are implicated in defense. Structural analysis of this
effector using homology modeling revealed the presence of potential ligand-binding
sites. Among key residues that were predicted to be crucial for ligand binding, L102 and
Y106 were of interest since they form part of the 6-aa-long PpRxLR1 SLiM. In silico substitution
of these two residues to alanine was predicted to have a significant effect on
both the function and the structure of PpRxLR1 effector. Molecular docking simulations revealed possible interactions between PpRxLR1 effector and ubiquitin-associated proteins.
The ubiquitin-like SLiM carried in this effector was shown to be a potential mediator
of these interactions. Further studies are required to validate and elucidate the
underlying molecular mechanism of action.
IMPORTANCE The continuous gain and loss of RxLR effectors makes the control of
Phytophthora spp. difficult. Therefore, in this study, we endeavored to identify RxLR
effectors that are highly conserved among species, also known as “core” RxLR effectors
(CREs). We reason that these highly conserved effectors target conserved proteins
or processes; thus, they can be harnessed in breeding for durable resistance in
plants. To further understand the mechanisms of action of CREs, structural dissection
of these proteins is crucial. Intrinsically disordered regions (IDRs) that do not adopt a
fixed, three-dimensional fold carry short linear motifs (SLiMs) that mediate biological
functions of proteins. The presence and potential role of these SLiMs in CREs of
Phytophthora spp. have been overlooked. To our knowledge, we have effectively
identified CREs as well as SLiMs with the potential of promoting effector virulence.
Together, this work has advanced our comprehension of Phytophthora RxLR effector function and may facilitate the development of innovative and effective control
strategies.
