The Barley Powdery Mildew Candidate Secreted Effector Protein CSEP0105 Inhibits the Chaperone Activity of a Small Heat Shock Protein  

Abstract

Pathogens secrete effector proteins to establish a successful interaction with their host. Here, we describe two barley (Hordeum vulgare) powdery mildew candidate secreted effector proteins, CSEP0105 and CSEP0162, which contribute to pathogen success and appear to be required during or after haustorial formation. Silencing of either CSEP using host-induced gene silencing significantly reduced the fungal haustorial formation rate. Interestingly, both CSEPs interact with the barley small heat shock proteins, Hsp16.9 and Hsp17.5, in a yeast two-hybrid assay. Small heat shock proteins are known to stabilize several intracellular proteins, including defense-related signaling components, through their chaperone activity. CSEP0105 and CSEP0162 localized to the cytosol and the nucleus of barley epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Intriguingly, only those specific CSEPs changed localization and became restricted to the cytosol when coexpressed with Hsp16.9 and Hsp17.5, confirming the CSEP-small heat shock protein interaction. As predicted, Hsp16.9 showed chaperone activity, as it could prevent the aggregation of Escherichia coli proteins during thermal stress. Remarkably, CSEP0105 compromised this activity. These data suggest that CSEP0105 promotes virulence by interfering with the chaperone activity of a barley small heat shock protein essential for defense and stress responses.

The isoelectric point of proteins influences their translocation to the extrahaustorial matrix of the barley powdery mildew fungus

“…The present study was initially meant to analyse the subcellular localisation of CSEPs in barley leaf epidermal cells. Serendipitously, however, our observations instead confirmed our previous finding (Ahmed et al, ) that proteins often are translocated from the host cytosol, across the EHM, into the EHMx. Being an entirely unstudied phenomenon, we looked further into it and found interesting details on protein properties influencing uptake into the EHMx.…”

“…When expressing free mYFP in the plant cytosol, ROI1/ROI2 equals 2.86 (mean of 53 cells; SE, 0.218). Although this indicated that intact proteins are able to cross the EHM, our previous study also demonstrated that certain proteins did not localise to the EHMx, as found for mYFP‐CSEP0105 (Ahmed et al, ). Here, we expanded the analysis and examined mYFP fusions of 11 Bgh CSEPs without signal peptides, thus forcing their targeting to the cytosol.…”

“…We have previously observed that free monomeric (m)Cherry, free monomeric Yellow Fluorescent Protein (mYFP), and mYFP‐CSEP0162 (without signal peptide for secretion), expressed in the cytosol of barley epidermal cells with Bgh haustoria, were not only present in the plant cell cytosol but strikingly also entered the EHMx (Ahmed et al, ). This compartment is distinct in bright field images obtained by confocal microscopy (Figures and S1).…”

“…In order to manipulate plant immunity, effector molecules are likely to be transported from the fungus to the plant cell (Lu et al, ). The Bgh genome encodes approximately 500 so‐called candidate secreted effector proteins (CSEPs; Pedersen et al, ), for some of which there is evidence that they contribute positively to the success of the fungus (e.g., Aguilar, Pedersen, & Thordal‐Christensen, ; Ahmed et al, ; Pennington et al, ).…”

Broadly Conserved Fungal Effector BEC1019 Suppresses Host Cell Death and Enhances Pathogen Virulence in Powdery Mildew of Barley (Hordeum vulgare L.) (Retracted)

“…Genomic and proteomic studies have identified about 540 Blumeria effector candidates (BEC) or candidate secreted effector proteins (CSEP) (Bindschedler et al 2011;Kusch et al 2014;Pedersen et al 2012;Spanu et al 2010). A small number of these have been characterized (Ahmed et al 2015;Pliego et al 2013;Schmidt et al 2014;Zhang et al 2012), but the molecular mechanisms by which effectors manipulate host processes to promote colonization are still poorly understood.…”