Characterization of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

Abstract

Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.

RXLR and CRN Effectors from the Sunflower Downy Mildew Pathogen Plasmopara halstedii Induce Hypersensitive-Like Responses in Resistant Sunflower Lines

“…Thirty-three percent of HaRXLs were exclusively found in the A. thaliana cell nucleus, and 22% showed nucleolar localizations (Caillaud et al, 2012). Nuclear localization of oomycete CRN effectors is even more frequent, for example all CRNs identified in Phytophthora capsici were localized in the N. benthamiana cell nucleus (Stam et al, 2013a,b). Nuclear localization in N. benthamiana was reported as essential for the necrotic function of CRN effectors from Phytophthora infestans and Aphanomyces euteiches (Schornack et al, 2010).…”

Intervention of Phytohormone Pathways by Pathogen Effectors

“…A number of secreted pathogen effectors contain nuclear localization signals indicating that they may interfere with transcription (Canonne and Rivas, 2012;Stam et al, 2013), although direct targets of these effectors remain to be determined. Bacterial effectors that act as plant transcription factors, such as those belonging to the TAL family, activate host target genes, but their precise mode of action remains unclear (Boch and Bonas, 2010).…”

Random mutagenesis screen shows that Phytophthora capsici CRN83_152‐mediated cell death is not required for its virulence function(s)

“…Here, we have attempted to gain new insights into the role(s) of CRN‐mediated cell death in virulence function using PcCRN83_152 as a model. Over‐expression of PcCRN83_152 enhances P. capsici virulence and induces plant cell death (Mafurah et al ., ; Stam et al ., ). Moreover, this CRN has also been shown to mediate host chromatin re‐localization when over‐expressed in N. benthamiana leaves (Stam et al ., ).…”

“…Over‐expression of PcCRN83_152 enhances P. capsici virulence and induces plant cell death (Mafurah et al ., ; Stam et al ., ). Moreover, this CRN has also been shown to mediate host chromatin re‐localization when over‐expressed in N. benthamiana leaves (Stam et al ., ). In order to test whether PcCRN83_152‐mediated cell death is required for its virulence function(s), a polymerase chain reaction (PCR)‐based random mutagenesis screen was performed on the PcCRN83_152 C‐terminal domain (the domain responsible for both PcCRN83_152 virulence and cell death functions) (Mafurah et al ., ; Stam et al ., ).…”