The barley powdery mildew candidate secreted effector protein CSEP0105 inhibits the chaperone activity of a small heat shock protein.

Plant Physiology Preview. Published on March 13, 2015, as DOI:10.1104/pp.15.00278

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Running head: Pathogen Effector Inhibition of Small Hsp

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Hans Thordal-Christensen

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Department of Plant and Environmental Sciences

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University of Copenhagen

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Thorvaldsensvej 40

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DK-1871 Frederiksberg C

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Tel: +45 35 33 34 43

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E-mail: [email protected]

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1 Downloaded from www.plantphysiol.org on April 5, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Copyright 2015 by the American Society of Plant Biologists

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Barley Powdery Mildew Effector Candidate CSEP0105 Inhibits Chaperone Activity of Small

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Heat Shock Protein

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Ali Abdurehim Ahmed, Carsten Pedersen, Torsten Schultz-Larsen, Mark Kwaaitaal2, Hans

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Jørgen Lyngs Jørgensen, and Hans Thordal-Christensen*

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Section for Plant and Soil Science, Department of Plant and Environmental Sciences, Faculty of

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Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

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(A.A.A., C.P., T.S.L., M.K., H.J.L.J., H.T.C.)

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SUMMARY

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Barley powdery mildew effector candidate contributes to fungal aggressiveness, while it targets and

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inhibits host small heat shock proteins, known to be required for defense against pathogens.

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This work was supported by the Danish Strategic Research Council, The Danish Council for

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Independent Research/Technology and Production Sciences as well as Department of Plant and

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Environmental Sciences, University of Copenhagen.

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Present address: Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen

University, Worringer Weg 1, D-52056 Aachen, Germany (M.K.)

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Address correspondence to [email protected]

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ABSTRACT

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Pathogens secrete effector proteins to establish a successful interaction with their host. Here, we

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describe two barley powdery mildew Candidate Secreted Effector Proteins (CSEPs), CSEP0105

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and CSEP0162, which contribute to pathogen success and appear to be required during or after

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haustorial formation. Silencing of either CSEP using host-induced gene silencing significantly

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reduced the fungal haustorial formation rate. Interestingly, both CSEPs interact with the barley

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small heat shock proteins, 16.9 and 17.5, in a yeast two-hybrid assay. Small heat shock proteins are

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known to stabilize several intracellular proteins including defense-related signaling components

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through their chaperone activity. CSEP0105 and CSEP0162 localized to the cytosol and the nucleus

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of barley epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Intriguingly, only those

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specific CSEPs changed localization and became restricted to the cytosol when co-expressed with

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Hsp16.9 and Hsp17.5, confirming the CSEP-sHsp interaction. As predicted, Hsp16.9 showed

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chaperone activity as it could prevent aggregation of Escherichia coli proteins during thermal

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stress. Remarkably, CSEP0105 compromised this activity. These data suggest that CSEP0105

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promotes virulence by interfering with the chaperone activity of barley small heat shock proteins

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essential for defense and stress responses.

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INTRODUCTION

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Powdery mildew fungi are obligate biotrophs, which cause significant economic loss in

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temperate areas by infecting more than 9000 dicot and 650 monocot species including grape,

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tomato, barley and wheat (Takamatsu, 2013). The barley powdery mildew fungus (Blumeria

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graminis f. sp. hordei; Bgh) reproduces and survives on the epidermal cell layer of wild and

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cultivated Hordeum species. Under favorable conditions, the conidia germinate and produce a

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primary and an appressorial germ tube. The appressorial germ tube swells at the end and forms an

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appressorium, from which a penetration peg develops. From here a penetration hypha penetrates the

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plant cell wall and forms a specialized feeding structure, the haustorium, which is vital for the

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obligate biotrophic interaction. The fungus utilizes the haustorium to acquire nutrients from the

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plant cell and it has been suggested as a site of effector biosynthesis and delivery to the plant cell

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(Panstruga, 2003; Stergiopoulos and de Wit, 2009). Pathogens deliver effector molecules to the host

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cell to manipulate cellular function to redirect nutrient transport and to interfere with the host

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defense mechanisms (Kamoun, 2006; Thomma et al., 2011; Ahmed et al., 2014).

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A genomic study supported by proteomics and transcriptomics identified around 500 CSEPs

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from Bgh. Some of them share a Y/F/WxC motif in the N-terminal part of the mature protein.

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(Bindschedler et al., 2009; 2011; Godfrey et al., 2010; Spanu et al., 2010; Pedersen et al., 2012). Of

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these, only a few CSEPs have been studied and shown to play a role in virulence. A method for

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genetic transformation of powdery mildew fungi is not available. Therefore, these results were

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obtained using Host-Induced Gene Silencing (HIGS), an RNA interference method in which hair-

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pin constructs targeting fungal transcripts are expressed in the attacked host cell (Nowara et al.,

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2010; Zhang et al., 2012a; Pliego et al., 2013). For example, CSEP0055 interacts with the barley

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PR17c protein and promotes fungal aggressiveness by suppressing defense (Zhang et al., 2012a).

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Similarly, a ribonuclease-like effector candidate, CSEP0264, suppresses defense by obstructing

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pathogen-induced host cell death (Pliego et al., 2013). In addition, two avirulence (Avr) proteins,

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Avrk1 and Avra10, that lack conventional N-terminal signal sequences, have been identified. These

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Avr proteins trigger host cell death when recognized by their cognate resistance (R) proteins, Mlk1

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and Mla10 (Ridout et al., 2006).

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Plants utilize a wide range of responses to defend themselves against invasion by pathogens and

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parasites. These include the expression of heat shock proteins (Hsps), which allow the cells to adapt


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to and survive severe environmental changes (Didelot et al., 2006; Gupta et al., 2010). Hsps are

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highly conserved molecular chaperones that stabilize intracellular proteins and facilitate the

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refolding of misfolded or denatured proteins during stress, for instance inflicted by microbial attack

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(Didelot et al., 2006). Two well-studied ATP-dependent chaperones, Hsp70 and Hsp90, play

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significant roles in hypersensitive response (HR) induction and non-host resistance in Nicotiana

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benthamiana (Kanzaki et al., 2003; Jelenska et al., 2010). Moreover, Hsp90 stabilizes and is

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required for the function of numerous R proteins such as I-2, RPM1, N and Rx (Hubert et al., 2003;

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Lu et al., 2003; Liu et al., 2004; de la Fuente van Bentem et al., 2005; Kadota and Shirasu, 2012).

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ATP-independent small Hsp (sHsp) chaperones also protect the cell from stress-induced protein

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aggregation and misfolding (McHaourab et al., 2009; Basha et al., 2012). sHsps are characterized

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by a conserved domain of approximately 90 residues forming an α-crystallin domain (ACD),

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flanked by a variable N-terminal sequence and a short C-terminal extension (Basha et al., 2012).

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They assist the Hsp70/Hsp90-mediated refolding process by maintaining the misfolded or denatured

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proteins in a folding-competent state (Lee et al., 1997; Van Ooijen et al., 2010). The monomeric

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size of sHsps ranges between 12 and 42 kDa. However, in most cases, they form large oligomers of

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eight to 32 subunits (Basha et al., 2012). The oligomers are activated to a higher substrate affinity

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state during stress and they bind non-native proteins of similar weight (Basha et al., 2006;

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Tyedmers et al., 2010). While the main function of sHsps is to provide thermo-tolerance, some are

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also involved in defense against pathogens. For instance, the tomato sHsp20, RSI2, is involved in

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Fusarium oxysporum resistance by stabilizing the tomato resistance protein, I-2 (Van Ooijen et al.,

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2010). The Nicotiana tabacum sHsp, Ntshsp17, and its N. benthamiana orthologue, Nbshsp17, are

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required for basal immunity against Ralstonia solanacearum (Maimbo et al., 2007).

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In this study, we are now able to demonstrate small heat shock proteins being targeted by

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pathogen effectors. We found that two barley powdery mildew effector candidates, CSEP0105 and

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CSEP0162, contributed to the Bgh infection success. Two barley small heat shock proteins,

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Hsp16.9 and Hsp17.5, were identified as the host targets for these two CSEPs. Notably, Hsp16.9

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showed in vitro chaperone activity, which was specifically suppressed by CSEP0105.

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RESULTS

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CSEP0105 and CSEP0162 promote Bgh virulence

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Effectors promote the virulence of pathogens for instance by suppressing the host defense and by

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enhancing nutrient uptake. Thus, we studied the contribution of Bgh CSEPs to the fungal infection

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success using HIGS (Nowara et al., 2010). Among the 500 described CSEPs (Godfrey et al., 2010;

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Pedersen et al., 2012), a number was selected based on their level of expression, the presence or

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absence of nuclear localization signal and to which CSEP family they belong. Our data revealed

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that independent silencing of approximately half of them reduced the Bgh aggressiveness

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significantly. Of these, RNAi of CSEP0105 and CSEP0162 (Supplemental Fig. S1) reduced the Bgh

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haustorial formation rate by approximately 40% relative to empty vector control (Fig. 1). The Mlo

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RNAi positive control reduced the haustorial formation rate by approximately 60% (Fig. 1). Using

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the SI-FI software tool (http://labtools.ipk-gatersleben.de/), the CSEP0162 RNAi construct was

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predicted to generate an off-target silencing effect on CSEP0163 and CSEP0164. These CSEPs are

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highly similar to CSEP0162 and belong to the same CSEP family 4, which has a total of 19

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members (Pedersen et al., 2012). Thus, the reduced haustorial formation rate caused by the

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CSEP0162 RNAi construct may be due to silencing of more members of this family. CSEP0105

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belongs to CSEP family 31, consisting of four members (Pedersen et al., 2012). Despite this, no off-

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target was predicted in the Bgh transcriptome for the CSEP0105 RNAi construct. In addition,

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neither of the RNAi constructs were predicted to have off-target in the barley transcriptome

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(version 12, released on 2011_03_19) as determined by the siRNA scan software

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(http://bioinfo2.noble.org/RNAiScan.htm). Therefore, the reduced infection rates observed after

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silencing indicate that CSEP0105 and CSEP0162 contribute to Bgh infection success, most likely as

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effectors.

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CSEP0105 and CSEP0162 are highly expressed during and after haustorial formation

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Effectors are differentially expressed and assumed to be required at different stages of infection.

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To determine the expression pattern of CSEP0105 and CSEP0162 at different fungal development

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stages, we performed a time-course experiment in a compatible Bgh/barley interaction. The time-

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course corresponds to ungerminated conidia (0 hours post inoculation (hpi)), primary germ tube

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formation (3 hpi), and appressorial germ tube formation (6 hpi), penetration (12 hpi), haustorial

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formation (24 hpi) and secondary penetration (48 hpi). For the first four time points, total RNA


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from the entire infected leaves was analyzed. Since haustorial development is in progress at 24 hpi,

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haustorial and epiphytic expressions were analyzed separately at 24 and 48 hpi. A qPCR expression

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analysis revealed that the CSEP0105 and CSEP0162 transcripts were predominantly expressed in

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haustoria and only poorly in the epiphytic tissue (Fig. 2). Although present at earlier time points,

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both CSEP transcripts were dramatically up-regulated during haustorial formation and secondary

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penetrations at 24 hpi and 48 hpi (Fig. 2).

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CSEP0105 and CSEP0162 interact with the barley Hsp16.9 and Hsp17.5 proteins

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CSEP0105 and CSEP0162 have predicted N-terminal signal peptides and they are expected to be

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secreted from the fungus. Thus, we anticipated that both CSEPs could have barley host targets and

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that interactions with these will promote disease. To identify such targets, we conducted yeast two-

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hybrid (Y2H) screens for both CSEPs. Approximately 1.75x106 clones were screened for each

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CSEP bait construct using a prey cDNA library generated from Bgh-infected barley leaves (Zhang

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et al., 2012a). This led to identification of one prey clone, encountered twice, using CSEP0162 as

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bait (Fig. 3). The clone has a 456 bp cDNA insert, encoding a full-length protein that has 98.7%

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amino acid-identity to a barley class I sHsp with a molecular weight of 16.9 kDa, i.e., HvHsp16.9-

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CI (Gene bank accession number AK362925) (Reddy et al., 2014). The identified cDNA has 14

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nucleotide substitutions compared to HvHsp16.9-CI, where 12 are synonymous and two are non-

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synonymous (Supplemental Fig. S2). With this number of variants, we consider the identified gene

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to be different from HvHsp16.9-CI; thus, we named it HvHsp16.9-CI-H (homologue) (Fig. 4).

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Another prey clone was identified using CSEP0105 as bait. This prey clone carried a 477 bp cDNA

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insert, which encodes the full-length protein of another class I sHsp of 17.5 kDa, namely

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HvHsp17.5-CI (AK250749). HvHsp16.9-CI-H and HvHsp17.5-CI are 71% identical at the amino

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acid level and are hereafter named Hsp16.9 and Hsp17.5. To assess the specificity of these

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interactions, a targeted Y2H assay was performed between CSEP0105, CSEP0162 and two negative

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control CSEPs (CSEP0081 and CSEP0254) as baits, and Hsp16.9 and Hsp17.5 as preys. The Hsps

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were found to interact with both CSEP0105 and CSEP0162, but not with the negative controls,

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indicating that the interactions were specific (Fig. 3). 3-amino-1,2,4-triazole (3-AT) is a competitive

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inhibitor of an enzyme catalyzing the sixth step of histidine biosynthesis. This inhibitor is normally

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added to the selective medium at a concentration of 1 mM to rule out false positives and weak

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interactors (James et al., 1996; Gietz et al., 1997). Yeast cells with Hsp16.9 and Hsp17.5/CSEP0105

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and CSEP0162 Y2H protein combinations grew at 5 mM 3-AT, indicating strong protein


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interactions.

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To confirm these results using another protein–protein interaction method, a bimolecular

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fluorescence complementation assay (BiFC) (Hu et al., 2002) was carried out in N. benthamiana

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and barley plants. These experiments supported that Hsp16.9 and Hsp17.5 interact with both

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CSEP0105 and CSEP0162, but not with CSEP0254 (Supplemental Fig. S3). The interactions were

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observed only for the N-terminal nYFP CSEP fusions combined with the C-terminal cCFP Hsp

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fusions, which appeared to form a stable CSEP-Hsp BiFC aggregate proximal to the nucleus. As a

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positive control, we used dimerization of 14-3-3 (Aitken, 2006). As a negative control, interactions

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of 14-3-3 with CSEP0105 and CSEP0162 were tested (Supplemental Fig. S3). Together, the Y2H

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and BiFC results provide strong evidence that CSEP0105 and CSEP0162 interact with both

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Hsp16.9 and Hsp17.5 proteins in yeast and in planta.

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Using a genome-wide sequence survey, Reddy et al. (2014) discovered 18 full-length sHsps that

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are differentially regulated during drought stress and seed development in barley. We searched and

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identified 19 additional full-length sHsps (Supplemental Table S1) in the recently published barley

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genome (The International Barley Genome Sequencing Consortium, 2012) and the full-length

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barley cDNAs at NCBI (Matsumoto et al., 2011). A phylogenetic tree generated based on the amino

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acid sequences of these 37 barley sHsps, as well as tomato RSI2, NtsHsp17 and NbsHsp17,

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revealed that Hsp16.9 and Hsp17.5 belong to a subgroup of ten highly homologous cytosolic sHsps

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in barley (Fig. 4). Within this, Hsp16.9 belongs to a clade of six sHsps, between which the identities

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are above 90% (Fig. 4), while Hsp17.5 belongs to another clade of four sHsps which have more

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than 78% identities.

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Small Hsps function as pathogen defense components (Maimbo et al., 2007; Van Ooijen et al.,

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2010). Thus, to investigate the role of Hsp16.9 and Hsp17.5 proteins, we generated a single RNAi

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construct, which can target the transcript of both sHsps and performed transient induced gene

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silencing (TIGS) in epidermal cells using particle bombardment. This experiment did not result in

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any susceptibility difference between the sHsp-silenced and empty vector control transformed cells.

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CSEP0105 and CSEP0162 localize to the cytosol and the nucleus, while Hsp16.9 and Hsp17.5

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are cytosolic

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To investigate the subcellular localizations of CSEP0105, CSEP0162 and CSEP0254 in the host

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cell, N-terminal YFP fusions to the mature CSEPs (lacking the signal peptide) were constructed.

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The fusion constructs were expressed in barley epidermal cells using particle bombardment together


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with a free mCherry marker, which localizes to the cytosol and the nucleus. The YFP-CSEP0105,

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YFP-CSEP0162 and YFP-CSEP0254 fusion proteins accumulated both in the cytosol and the

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nucleus as there was a complete overlap between the YFP and mCherry fluorescent signals (Fig. 5,

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A-C). Similar localizations were observed when these CSEPs were expressed as C-terminal YFP

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fusions. Usually, effectors localize together with their host targets in order to execute their virulence

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function. Therefore, to determine in which of these locations the CSEP targets reside, we expressed

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C-terminally mCherry-tagged Hsp fusions in barley epidermal cells. This revealed that both the

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Hsp16.9-mCherry and Hsp17.5-mCherry fusion proteins accumulated exclusively in the cytosol, in

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contrast to the co-expressed yellow fluorescent marker that localized both in the cytosol and the

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nucleus (Fig. 5, D and E). This indicated that Hsp16.9 and Hsp17.5 are cytosolic proteins, as

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predicted by Reddy et al. (2014), and their interactions with CSEP0105 and CSEP0162 likely occur

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in the cytosol.

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CSEP0105 and CSEP0162 are exclusively targeted to the cytosol when co-expressed with

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Hsp16.9 and Hsp17.5

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The protein-protein interaction and localization results shown in Figures 3 and 5 encouraged us

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to examine the effect of their interactions on the localizations of the CSEPs and Hsps. Therefore,

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the YFP-CSEP0105 and YFP-CSEP0162 fusion constructs were co-expressed with either the

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Hsp16.9-mCherry or Hsp17.5-mCherry fusion constructs in barley leaf epidermal cells. Our data

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revealed that the accumulation of the two Hsps was unaltered by the presence of the interacting

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CSEPs (Fig. 6, A-F). Intriguingly, YFP-CSEP0105 and YFP-CSEP0162 fusion proteins remained

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in the cytosol and did not translocate to the nucleus in the presence of Hsp16.9-mCherry and

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Hsp17.5-mCherry fusion proteins (Fig. 6, A-D). Meanwhile, the non-interacting CSEP, YFP-

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CSEP0254, kept its nuclear and cytosolic localization when co-expressed with either Hsp16.9-

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mCherry or Hsp17.5-mCherry (Fig. 6, E and F). This suggests that CSEP0105 and CSEP0162 are

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exclusively directed to the compartment where their host targets localize. This observation also

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provides additional evidence for the physical interaction between the two partners, substantiating

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that the sHsp/effector candidate interactions are authentic.

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Hsp16.9 prevents heat-induced protein aggregation in vitro Small heat shock proteins have chaperone activity that prevents protein aggregation during heat


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stress. To investigate whether Hsp16.9 is a bona fide chaperone, we used a thermal stability assay

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of Escherichia coli cellular proteins. Recombinant Hsp16.9 protein fused with a histidine tag was

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expressed in E. coli. After heating the total cellular protein fraction in a cell-free extract from β-

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glucuronidase (GUS, control) expressing E. coli at 40°C for 15 min., approximately 55% of the

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proteins remained soluble. After heating at 90°C, only 7% of the proteins remained soluble (Fig. 7).

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However, in lysates containing Hsp16.9, 75% of the proteins were soluble after the 40°C-treatment,

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and remarkably 31% of the proteins were soluble after the 90°C-treatment (Fig. 7). This strongly

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suggests that Hsp16.9 is effective in preventing aggregation of bacterial cellular proteins. Hsp17.5

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was not tested for its chaperone activity due to insolubility of the His-tagged fusion protein in vitro.

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The CSEP0105 interferes with Hsp16.9 chaperone activity

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Effectors interfere with the functions of their targets. As CSEP0105 and CSEP0162 were found

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to interact with Hsp16.9, we wanted to test their effect on its chaperone activity. For this purpose,

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we performed a similar thermal stability assay of E. coli cellular proteins as described above, with

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and without the CSEPs. We chose to perform this experiment at 60°C. Based on the results in

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Figure 7, we concluded that sHsp-mediated protection from protein aggregation is sufficiently high

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to be able to determine an effect of the CSEPs at this temperature. Furthermore, we expect that

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heating at 60°C will not damage the CSEPs. In this experiment, 20% of the proteins were soluble

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after heating the control extract, whereas this value was 40% for the Hsp16.9-containing extract

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(Fig. 8). This corroborated the chaperone activity of Hsp16.9. When CSEP0105 was added to the

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control lysate and heated, no change in thermal stability was seen. Interestingly, CSEP0105

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essentially prevented Hsp16.9 from improving the thermal stability (Fig. 8). This reveals that

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CSEP0105 has a detrimental effect on the chaperone activity of Hsp16.9. While CSEP0105

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specifically interfered with Hsp16.9-mediated increased protein solubility, CSEP0162 and

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CSEP0254 also reduced the solubility of cellular proteins. However, the effects of these CSEPs

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were smaller than that of CSEP0105 and not associated to Hsp16.9 activity, as they occurred in

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both E. coli protein samples (Fig. 8). This can reflect that CSEP0162 and CSEP0254 interfere with

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endogenous E. coli chaperones or that they form insoluble complexes with E. coli proteins. Such

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effects were not seen for CSEP0105, confirming its specific interaction with Hsp16.9.

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CSEP0105 keeps its cytosolic localization in infected barley cells, whereas CSEP0162

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accumulates in the extrahaustorial matrix


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Since CSEP0105 reduced the chaperone activity of Hsp16.9, we further studied the localization

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of these physically and functionally interacting proteins in infected cells. For this purpose, the YFP-

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CSEP and Hsp-mCherry constructs were expressed in barley epidermal cells as described above.

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One day after the particle bombardment, the leaves were inoculated with Bgh; and 2 to 3 days later,

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the protein localization was determined in cells containing fungal haustoria. To our surprise, we

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could detect free YFP and mCherry in the extrahaustorial matrix (EHMx) between the

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extrahaustorial membrane (EHM) and the fungal haustorium (Fig. 9, A, B, D and E). Hsp16.9-

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mCherry and Hsp17.5-mCherry fusion proteins retained their cytosolic localization and did not

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enter the nucleus, as discussed above for uninfected cells (Fig. 5, D and E), and they did not enter

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the EHMx either (Fig. 9, A-C; Supplemental Fig. S4A and B). However, an interesting difference

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was observed for the two CSEPs. YFP-CSEP0105 was cytosolic and nuclear, while YFP-

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CSEP0162 in addition was observed in the EHMx, where it overlapped with free mCherry (Fig. 9,

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C-E; Supplemental Fig. S4B-D). Exclusion of YFP-CSEP0105 from the EHMx occurred

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independent of sHsp over-expression. Since YFP-CSEP0105 is smaller (38.8 kDa) than YFP-

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CSEP0162 (42.7 kDa), the localization difference cannot be a matter of protein size. Yet, the data

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suggest that CSEP0105 co-localized with the Hsp, which it may inhibit. This is parallel to the co-

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localization in the unstressed cells (Fig. 6).

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DISCUSSION

364

Previously, it has been suggested and supported by BiFC that Bgh CSEP0055 interacts with the

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barley pathogenesis-related protein, PR17 (Zhang et al., 2012a), and that the Bgh effector

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candidates, BEC3 and BEC4, interact with a thiopurine methyltransferase and an E2 ubiquitin-

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conjugating enzyme, respectively (Schmidt et al., 2014). These three effector candidates all

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contributed to Bgh virulence. Here we provide evidence that CSEP0105 and CSEP0162 enhance

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Bgh virulence. Interestingly these CSEPs are unrelated and they belong to CSEP families 31 and 4,

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respectively (Pedersen et al., 2012). The expression of both CSEPs occurred predominantly in the

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haustoria and they were highly up-regulated during and after formation of this fungal structure. This

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result is consistent with previous proteomic and transcriptomic analyses, where both CSEPs were

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exclusively identified in the haustorial proteome, and not in sporulating hyphae (Bindschedler et al.,

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2011; Pedersen et al., 2012). This signifies that the two CSEPs could be required at late infection

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stage, during haustorial development and secondary penetration.

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Using the Y2H system, the barley Hsp16.9 and Hsp17.5 proteins were identified as interactors of

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both CSEP0105 and CSEP0162. The growth of the yeast cells at 5 mM 3-AT indicated strong

378

interactions between these proteins. These results were verified by in planta expression using the

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BiFC method in N. benthamiana and barley plants, which has been proved as a reliable method to

380

show protein interactions in living cells. In this assay, the CSEPs and sHSPs were fused to

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complementing N- and C-terminal halves of a YFP molecule. The interaction between a CSEP and

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a Hsps brought the two halves of the YFP in close contact allowing them to irreversibly reconstitute

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an intact, fluorescent YFP molecule. The observation of YFP fluorescence therefore showed that

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the CSEPs and sHsps formed a complex in planta. A drawback of the BiFC system is that YFP

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reconstitution is permanent and for this reason interaction dynamics cannot be studied (Hu et al.,

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2002; Kerppola, 2008). For this reason we cannot draw conclusions on the timing or strength of

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CSEP-sHsp complex formation. The BiFC-CSEP-sHsp protein complexes localized to large

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aggregates proximal to the nucleus (Supplemental Figure S3). This phenomenon is most likely

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caused by the irreversible formation of BiFC-sHSP-CSEP complexes; while, the co-expression of

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the proteins fused to conventional fluorophores (Supplemental Figure S4) does not result in the

391

formation of these aggregates.

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Meanwhile, more conclusive evidence for these interactions comes from a co-expression study

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showing that the localization of CSEPs is following the sHsps. When expressed alone, the CSEPs

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accumulate both in the cytosol and the nucleus; although they lack predicted nuclear localization


395

signals (Pedersen et al., 2012). However, when co-expressed with the two cytosolic sHsps, the

396

interacting CSEPs no longer localized to the nucleus, while a non-interacting, negative control

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CSEP was not affected. This strongly indicates that these interactions occur in the cytosol where the

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host targets reside. Although the endogenous Hsp16.9 and Hsp17.5 proteins were also expected to

399

interact with the overexpressed CSEPs, they appeared to occur in insufficient quantities to bind and

400

trap all CSEP protein in the cytosol. Collectively, we demonstrate that small heat shock proteins are

401

targets of pathogen effectors. Previously, the Pseudomonas syringae HOPI1 effector has been

402

demonstrated to bind and recruit cytosolic Hsp70 to the chloroplast, where they form large

403

complexes. Such complex formation, combined with the HOPI1-stimulated Hsp70 ATP hydrolysis

404

activity, enabled the pathogen to hijack the plant defense and stress machinery to promote virulence

405

(Jelenska et al., 2010).

406

The extrahaustorial matrix is a separate compartment that exists between the host cell and

407

haustoria-forming microbes. Effectors synthesized from such microbes are secreted into the

408

extrahaustorial matrix, where some of them may execute their virulence function. Others will be

409

transferred over the extrahaustorial membrane (EHM) to the host cytosol to execute their mission

410

there. The localization of CSEP0162 to the extrahaustorial matrix suggests that this CSEP perhaps

411

has a virulence role in the matrix, in addition to the cytosol where we have shown it to target sHsps.

412

If Bgh-expressed CSEP0105 is transferred over the EHM to the plant cell, our data would suggest it

413

only to interact with its target in the cytosol.

414

The phylogenetic tree illustrated that Hsp16.9 and Hsp17.5 belong to a subgroup of cytosolic

415

sHsps in barley that consists of ten members with high similarity (Fig. 4). Therefore, other members

416

of this subgroup might interact with the two CSEPs. In contrast, CSEP0105 and CSEP00162 are

417

distinct and belong to different CSEP families. It is well established that effectors from the same or

418

different pathogens target a common cellular hub to gain efficient suppression of host defense and

419

facilitate pathogen fitness (Mukhtar et al., 2011; Weßling et al., 2014). For example, six

420

Pseudomonas syringae effectors (AvrB, AvrRpm1, AvrRpt2, AvrPto, AvrPtoB, and HopF2) all

421

target Arabidopsis thaliana RIN4 (Deslandes and Rivas, 2012). Thus, our finding that two unrelated

422

Bgh effectors target identical proteins of the stress machinery agrees with accumulating data that

423

effectors converge on common host hubs that are important for defense.

424

Generally, misfolded proteins expose hydrophobic residues, which otherwise are buried in the

425

protein structure. Such hydrophobic surfaces have a strong tendency to induce protein aggregation

426

(Tyedmers et al., 2010). Small Hsps recognize and bind such hydrophobic surfaces to refold the


427

protein either alone or with the help of other ATP-dependent chaperones (Basha et al., 2012; Fu,

428

2014). Thus, in order to investigate whether the interactions we found resemble binding of sHsps

429

with their substrate, we predicted the hydrophobic surface areas for the two CSEPs that interact

430

with sHsps and two non-interacting CSEPs (CSEP0081 and CSEP0254). Protein structure models

431

were created for the four CSEPs using the IntFOLD server (Roche et al., 2011). These models were

432

analyzed with the Swiss-PdbViewer 4.1.0 (Guex and Peitsch, 1997) to detect potential hydrophobic

433

patches on the surfaces of the proteins. This predicted that CSEP0081, CSEP0105, CSEP0162 and

434

CSEP0254 have more or less similar surface hydrophobicity of 22%, 23%, 19% and 15%,

435

respectively. As the exposed hydrophobic amino acids occur singly or in groups of a few, and only

436

make up small patches of hydrophobicity (less than 200 Å2), hydrophobic effects cannot explain the

437

CSEPs-Hsps interaction. Hence, we consider that the CSEPs bind the Hsps according to other

438

principles.

439

Similar to the well-studied high-molecular-weight Hsps, small Hsps are also involved in basal

440

defense response and HR-mediated immunity in plants (Maimbo et al., 2007; Van Ooijen et al.,

441

2010). Silencing of the N. benthamiana shsp17 (Nbshsp17) significantly reduced the expression of

442

defense-related genes, such as PR1, PR4 and the ET-responsive element-binding protein (EREBP),

443

which are marker genes for SA, MeJA and ET signaling, respectively (Maimbo et al., 2007). In

444

addition, silencing of Nbshsp17 increased the growth of both virulent and avirulent Ralstonia

445

solanacearum isolates and accelerated wilt symptoms. However, HR induced by avirulent R.

446

solanacearum, Pseudomonas cichorii, and INF1 was not affected in Nbshsp17 silenced plants.

447

Accordingly, Nbshsp17 is involved in HR-independent basal defense responses in N. benthamiana

448

plants (Maimbo et al., 2007). In contrast, the tomato small Hsp20 (RSI2) is required for HR-

449

dependent defense responses (Van Ooijen et al., 2010). Silencing of RSI2 compromises the HR

450

triggered by an auto-active version of the I-2 R gene. This phenotype is correlated with reduced I-2

451

protein accumulation, which led the authors to conclude that RSI2 is required for stabilization of I-2

452

and/or other signaling components involved in the execution of HR (Van Ooijen et al., 2010). The

453

phylogenetic tree in Fig. 4 shows that Nbshsp17 is distantly related to RSI2 and the two CSEP-

454

interacting barley sHsps. Plants possess a large number of highly diverse sHsps with various

455

subcellular localizations (Fig. 4; Jiang et al., 2009; Basha et al., 2010). Hsp16.9, Hsp17.5 and RSI2

456

are cytosolic class I proteins, whereas Nbshsp17 is clustered with potentially non-cytosolic sHsps

457

(Fig. 4). Therefore, RSI2 and Nbshsp17 could have a specialized role in R-gene mediated immunity

458

and basal defense, respectively. However, the role of RSI2 as a component of basal defense has not


459

been investigated and cannot be excluded.

460

The barley Hsp16.9 and Hsp17.5 have 68% amino acid-identity to the full-length RSI2; and they

461

have 81 and 83% identities to the α-crystallin domain of RSI2, respectively. In general, sHsps

462

diverge more in the N-terminal region, upstream of the α-crystallin domain (Basha et al., 2012).

463

Thus, as they share a high sequence similarity, localization, and for Hsp16.9 experimentally verified

464

chaperone activity, we expect that Hsp16.9 and Hsp17.5 proteins could have similar biochemical

465

functions as RSI2 in immunity. However, there was no observable change in the susceptibility of

466

barley after introduction of the Hsp16.9 and Hsp17.5 RNAi construct. This is likely due to sHsp

467

redundancy caused by other small Hsps (Fig. 4) not targeted by the RNAi construct, which would

468

mask the effect. This notion is consistent with the observation that CSEP0105 and CSEP0162 each

469

interact with at least two sHsps. In fact, a recent study in wheat implicates Mds-1, a close

470

homologue of barley Hsp16.9 and Hsp17.5 (Fig. 4), in resistance to powdery mildew (Liu et al.,

471

2013). Here knock-down of Msd-1 made wheat resistant to powdery mildew. This observation is

472

not necessarily in conflict with small Hsps playing a positive role in defense and being effector

473

targets. Knocking out positive defense genes often activates defense (eg. Nishimura et al., 2003;

474

Stein et al., 2006; Zhang et al., 2008), which can be mediated by R-proteins detecting the absence

475

of effector targets (Mackey et al., 2002; Jones and Dangl, 2006; Zhang et al., 2012b).

476

We found that CSEP0105 hampered the chaperone activity of Hsp16.9 in vitro. By hydrophobic

477

interactions, the melittin peptide binds and outcompetes the hydrophobic binding site of an eye lens

478

α-crystallin protein thereby diminishing its chaperone activity (Sharma et al., 1998; Sharma et al.,

479

2000; Santhoshkumar and Sharma, 2001). Our predictions indicate that CSEP0105 is not

480

particularly rich in exposed hydrophobic amino acids, therefore it remains to be seen how this

481

protein interferes with the chaperone activity of Hsp16.9. Yet, we speculate that it can interfere with

482

the function of multiple sHsps. On the other hand, CSEP0162 did not have a specific effect on the

483

chaperone activity of Hsp16.9, potentially because its sHsp inhibition is not revealed in our assay.

484

To summarize, we found that CSEP0105 and CSEP0162 promoted Bgh virulence in barley,

485

suggesting them to be effector proteins. In addition, both CSEPs were demonstrated to interact with

486

the barley Hsp16.9 and Hsp17.5 proteins. CSEP0105 compromised the chaperone activity of

487

Hsp16.9, which we assume to be the mechanism by which it suppresses defense.

488 489

MATERIALS AND METHODS

490

Plant and Fungal Materials


491

One-week-old barley seedlings (Hordeum vulgare cultivar Golden Promise) were used for the

492

HIGS, BiFC, localization and expression profiling studies, in combination with the virulent isolate,

493

DH14, of Blumeria graminis f.sp. hordei. This fungal isolate was maintained on cv. Golden

494

Promise by weekly inoculum transfers. Barley seedlings were grown at 16 h light (20°C, 150 µEs-

495 496

1

m-2)/8 h darkness (15°C). Two to four-weeks-old Nicotiana benthamiana plants were used for

BiFC studies.

497 498

Construction of Gateway entry clones

499

Gateway entry clones were produced for the coding sequences of effector candidates CSEP0081

500

(BGHDH14_bgh03006), CSEP0105 (BGHDH14_bgh03459), CSEP0162 (BGHDH14_bgh03874)

501

and CSEP0254 (BGHDH14_bgh05751) with and without signal peptides, as available in the

502

EnsemblFungi database (http://fungi.ensembl.org/index.html), and for Hsp16.9-CI-H and Hsp17.5

503

(AK250749), all with and without stop codons. The sequences were Polymerase Chain Reaction

504

(PCR) amplified on fungal and plant cDNA, using the primer pairs described in Supplemental Table

505

S2. The PCR products were TOPO-cloned either into the pCR8/GW/TOPO or the pENTR/D-TOPO

506

entry vectors. From here the inserts were transferred to destination vectors using Gateway LR

507

cloning reactions (Invitrogen). All entry and destination clones were confirmed by sequencing.

508 509

HIGS and TIGS

510

Gene silencing in Bgh and barley was conducted using the particle bombardment technique as

511

described by Nowara et al. (2010) and Douchkov et al. (2005), respectively. The RNAi constructs

512

were generated in the 35S promoter-driven hairpin destination vector, pIPKTA30N (Douchkov et

513

al., 2005). They were co-transformed with a β-glucuronidase (GUS) reporter gene construct into

514

barley epidermal cells. Seven leaves were transformed for each construct using particle

515

bombardment. Two days later, the leaves were inoculated with Bgh and three days after that, they

516

were stained for GUS activity. Transformed (blue) cells were assessed microscopically for presence

517

of haustoria as an indication of fungal aggressiveness. Haustorial formation rates were calculated as

518

the number of blue cells with haustoria divided by the total number of blue cells. The empty vector

519

pIPKTA30N and the Mlo-RNAi (pIPKTA36) constructs were used as negative and positive

520

controls, respectively. Relative haustorial formation rates were calculated as the haustorial

521

formation rate for each construct divided by the haustorial formation rate for the empty vector


522

control treatment. The data were analyzed using the software package SAS (version 9.4, SAS

523

Institute, Cary, NC) by logistic regression (PROC GENMOD, corrected for overdispersion), using a

524

generalized linear model and assuming a binomial distribution. The probability of Bgh haustorial

525

formation was modeled as a linear function of an intercept parameter, a CSEP effect and an

526

experiment effect. The significance of differences between each construct and the empty vector

527

RNAi were analyzed using Pearson's Chi-Square test.

528 529

Yeast Two-Hybrid

530

A pAD-GAL4-2.1-based prey cDNA library, which was generated from Bgh-infected barley

531

leaves (Zhang et al., 2012a), was used for the Y2H screens. The CSEP bait constructs were made in

532

the pDEST-AS2-1 destination vector producing fusions to the C-terminus of the DNA-binding

533

domain of the GAL4 transcription factor (Robertson, 2004).

534

The J69-4a yeast strain, which contains HIS3 and ADE2 reporter genes, was used for the Y2H

535

screen (James et al., 1996). The CSEP0105 and CSEP0162 bait constructs were transformed

536

independently into J69-4a and selected on dropout (DO) medium lacking tryptophan (W).

537

Subsequently, the clones with the bait plasmid were re-transformed with the prey cDNA library.

538

Positive clones were selected on DO medium lacking W, leucine (L), histidine (H) and adenine (A)

539

and supplemented with 2 mM 3-amino-1,2,4-triazole (3-AT). Positive clones were subjected to 2, 3

540

and 5 mM 3-AT to select strong interactors. Prey plasmids from the positive clones that grew at 5

541

mM 3-AT were extracted and re-transformed to bait-containing clones to confirm their interactions.

542

Expression of another reporter gene, Lac Z, was examined using the Y190 strain as described in Bai

543

(1997). The yeast clones grown in DO-WL liquid medium were transferred to filter paper and

544

incubated with an X-gal solution at 30°C for 10-12 h to test the synthesis of β-galactosidase. The

545

prey plasmids from the double-confirmed clones were sequenced and analyzed by Blast screens in

546

NCBI. The interaction of SNF1 (bait) and SNF4 (prey) was used as a positive control (Durfee et al.,

547

1993). CSEP0081 and CSEP0254 were used as negative bait controls. The DO medium is

548

composed of glucose (20 g L-1), yeast nitrogen base (6.7 g L-1), DO mix minus W, L, H and A (2 g

549

L-1), supplemental amino acids, agar (15 g L-1) and water.

550 551 552

BiFC Coding sequences for full-length barley Hsp and fungal CSEP, lacking signal peptides, with and


553

without stop codons were transferred to 35S promoter-driven BiFC binary Ti destination vectors

554

(Liu et al., 2009) as N- and C-terminal fusions of nYFP and cCFP. nYFP denotes amino acids 1-

555

172 of YFP and cCFP denotes amino acids 154-239 of CFP. Constructs were transiently

556

transformed to N. benthamiana using Agrobacterium-mediated transformation. All eight possible

557

combinations were evaluated for each CSEP/Hsp combinations. Fluorescent signal was evaluated

558

using a Leica SP5 confocal laser scanning microscope at 488 nm excitation; and the emission was

559

detected between 518 and 540 nm, according to Hu and Kerppola (2003). Construct combinations

560

that gave positive results from the N. benthamiana BiFC assay were co-transformed into barley leaf

561

epidermal tissues together with an mCherry transformation marker using particle bombardment, and

562

evaluated for protein interaction.

563 564

Localization

565

The CSEP coding sequences with and without stop codons were transferred to a ubiquitin

566

promoter-driven over-expression destination vector, to mediate fusion to the N- and C-terminus of

567

YFP. The coding sequences for Hsp16.9 and Hsp17.5 were transferred to a ubiquitin promoter-

568

driven over-expression destination vector, to mediate fusion to the N-terminus of mCherry. Particle

569

bombardment was used to transform the constructs into barley epidermal cells. Fluorescent signal

570

was monitored using a Leica SP5 confocal laser scanning microscope. Lasers of 514 and 543 nm

571

were used to excite YFP and mCherry, respectively. The YFP and mCherry emissions were

572

detected between 524 to 539 nm and 590 to 640 nm, respectively. Free YFP and mCherry markers,

573

which localize to the cytosol and the nucleus, were used as controls.

574 575

Identification of sHsps in the barley genome and clustering analysis

576

To identify small Hsps in barley, we used the 19 barley sHsps described in Reddy et al. (2014)

577

and rice sHsps obtained from the Phytozome-server (http://www.phytozome.net/) as queries in

578

BLASTp searches of the high-confidence barley genome sequence predicted by the International

579

Barley

580

muenchen.de/plant/barley/download/index.jsp) and of barley ESTs at NCBI, HarvEST (Close et al.,

581

2004) and PLEXdb (Dash et al., 2012). An amino acid-based alignment and a phylogenetic tree

582

were made for the identified sHsps, as well as the tomato RSI2 (Van Ooijen et al., 2010), Ntshsp17

583

and Nbshsp17 (Maimbo et al., 2007) using the CLC main workbench (CLC Bio). Protein

Sequencing

Consortium

(2012)

(http://mips.helmholtz-


584

localizations were predicted using TargetP (Emanuelsson et al., 2007), WoLF PSORT (Horton et

585

al., 2006) and PlantLoc (http://cal.tongji.edu.cn/PlantLoc/index.jsp). Only sHsps that were

586

predicted consistently by the three softwares are presented here.

587 588

RNA isolation, RT-PCR and quantitative PCR

589

Total RNA was isolated from Bgh-infected barley leaves at 0, 3, 6, 12, 24 and 48 hpi using the

590

polyvinylpolypyrrolidone method (Chen et al., 2000). RNA was isolated from total infected leaves

591

for the first four time points, and from the epiphytic fungal material and the separated leaves

592

containing the haustoria for the 24 and 48 hpi time points. This separation was done by dipping the

593

Bgh-infected leaves into 10% cellulose acetate (in acetone). The leaves were dried for 10 min and

594

cellulose acetate strips containing the epiphytic fungal material were detached from the leaves.

595

cDNA was synthesized using the SMARTTM MMLV RT kit (Clontech) following the

596

manufacturer’s instructions with gene specific primers described in Supplemental Table S2.

597

Transcript quantification was performed on a Stratagene MX3000P real-time PCR detection system

598

using the FIREPol® EvaGreen® qPCR-kit (SOLIS BIODYNE). Each reaction consisted of 20 µL

599

containing 4 µL 5 x HOT FIREPol® EvaGreen® qPCR Mix Plus, 2 µL cDNA, 1 µL of 10 mM

600

solution of each gene specific primer (Supplemental Table S2) and 12 µL water. Reactions for each

601

CSEP and GAPDH (reference gene) were combined in one 96-well plate. PCRs were carried out

602

using the following thermocycle: 15 min at 95°C, followed by 40 cycles of 15 s at 95°C, 20 s at

603

58°C, and 20 s at 72°C and the dissociation curves of the PCR products were recorded between

604

55°C and 95°C degrees. Amplification efficiencies of reference gene and genes of interest were

605

between 90 and 100%. Results were analyzed using Stratagene MX3000 qPCR analysis software.

606

Relative expression was determined compared to time point 0 hpi (ungerminated conidia),

607

arbitrarily set to 1. Three biological and two technical replications were included for each time

608

point.

609 610

Chaperone activity of recombinant barley Hsp16.9 on E. coli proteins

611

A thermal aggregation assay of E. coli cellular proteins was performed according to Yu et al.

612

(2005). The coding sequence for the full-length Hsp16.9 was transferred to a pDest17 (Histidine

613

tag) expression destination vector. The construct was transformed into the E. coli strain Rosetta and

614

expression was induced by adding 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside). GUS


615

expression was used as a negative control, where the entry clone was obtained from the Gateway

616

LR clonase kit (Invitrogen). The cells were harvested and re-suspended in Buffer A (50 mM Tris-

617

HCl, 100 mM NaCl, 10% glycerol) with 1x Protease inhibitor (Roche). A cell-free E. coli protein

618

extract was generated from the cell suspension by sonication for 10 x 15 sec at setting 5 of 10,

619

followed by centrifugation at 12,500 g for 10 min at 4°C to remove cell debris. The cell-free

620

cellular extract (500 µl of 250 µg mL-1 total protein) was subjected to a chaperone activity assay

621

using different heat-treatments (40, 50, 60, 70, 80 and 90°C) for 15 min. Heat-treated proteins were

622

centrifuged at 12,500 g for 10 min to separate the denatured (pelleted) proteins from the non-

623

denatured (supernatant). The protein content of the supernatant was measured using the Quick Start

624

Bradford dye reagent (Bio-Rad). Three replicates were included for each heat-treatment and three

625

measurements were taken for each replicate. The data were analyzed using a general linear model

626

(PROC GLM) using the software package SAS (version 9.4, SAS Institute, Cary, NC). Means of

627

GUS and Hsp16.9 containing extracts at each heat-treatment were separated by Duncan’s multiple

628

range test.

629

To study the effect of the CSEPs on Hsp16.9 chaperone activity, purified CSEPs were mixed

630

with the cell-free cellular lysates (250 µg mL-1 total protein) containing Hsp16.9 or GUS proteins.

631

Since Hsp16.9 expression was high in E. coli, we diluted the Hsp16.9-containing extract with a

632

GUS expression E. coli lysate in a 1:1 total protein ratio. Subsequently, 5 µl purified CSEP in 40

633

mM glutathione was added to 50 µl of cellular lysate, making a 1:2 protein molar ratio with

634

Hsp16.9. As a negative control, 5 µl 40 mM glutathione was added. The mixture was incubated for

635

1 h at 4°C with gentle agitation and centrifuged at 12,500 g for 10 min. Subsequently, a chaperone

636

activity assay was performed as above using 60°C as the heating temperature. Three replicates were

637

included for each treatment and two measurements were taken for each replicate. The data were

638

analyzed using the SAS software as described above. All means were separated using Duncan’s

639

multiple range test.

640 641

Protein Purification

642

The coding sequences for CSEPs without signal peptides were transferred to the pDest15 over-

643

expression destination vector to fuse a GST-tag to their N-termini. The constructs were transformed

644

into the Rosetta E. coli strain. Cultures of 500 mL were prepared from 5 mL overnight cultures and

645

grown for 4 to 5 h at 37°C at 150 rpm. Protein expression was induced by adding 0.5 mM IPTG and

646

cultures were grown for 18 h at 18°C at 150 rpm. The cells were harvested and suspended in 25 mL


647

buffer, containing Buffer A with 0.5% Triton X100 and 1x Protease inhibitor (Roche). Cells were

648

sonicated for 10 x 15 sec at setting 5 of 10. Cellular debris was pelleted by centrifugation at 13,000

649

g for 10 min at 4°C. The supernatant was diluted by adding 25 mL Buffer A, containing 1x protease

650

inhibitor. Subsequently, 100 µL GST-beads slurry (Protino® Glutathione Agarose 4B, Macherey-

651

Nagel) was added to the protein extract, which was incubated by rolling for 2 h at 4°C. The beads

652

were pelleted and washed twice with buffer A containing 0.2% Triton X100. Protein bound to the

653

beads was eluted in 100 µl 40 mM glutathione, pH 8.

654 655

Supplemental Data

656

Supplemental Figure S1. Nucleotide and amino acid sequences of CSEP0105 and CSEP0162.

657

Supplemental Figure S2. Nucleotide and amino acid alignments of HvHsp16.9-CI and

658

HvHsp16.9-CI-H.

659

Supplemental Figure S3. Interaction of CSEP0105 and CSEP0162 with the barley Hsp16.9 and

660

Hsp17.5 proteins in a BiFC assay.

661

Supplemental Figure S4. Localization of CSEP0105, CSEP0162, and Hsp16.9 proteins in Bgh-

662

infected barley cells.

663

Supplemental Table S1. List of small heat shock proteins identified in barley.

664

Supplemental Table S2. Primer sequences for plasmid constructions and gene expression assay.

665 666

ACKNOWLEDGMENTS

667

We thank Dr. Patrick Schweizer, IPK, Gatersleben, Germany, for providing the RNAi vector and

668

the Mlo-RNAi construct; Dr. Masumi Robertson, CSIRO Plant Industry, Canberra, Australia for the

669

Gateway yeast two-hybrid vectors (pDEST-ACT2 and pDEST-AS2-1), Dr. Anja Fuglsang,

670

Copenhagen University, Denmark, for the BiFC binary Ti destination vectors and Prof. Dr. Paul

671

Schulze-Lefert, MPI, Cologne, Germany, for the YFP-and mCherry-fusion Gateway vectors.

672

Imaging data were collected at the Center for Advanced Bioimaging (CAB), University of

673

Copenhagen, Denmark.

674 675 676

Figure legends

677

Figure 1. Silencing of CSEP0105 and CSEP0162 by HIGS reduce Bgh haustorial formation rate.


678

One-week-old barley leaves were bombarded with an RNAi construct and a GUS reporter

679

construct. Two days later, leaves were infected with Bgh, and at 3 dpi they were stained with an X-

680

Gluc solution and scored for fungal haustorial formation rate, which was calculated as the number

681

of haustoria formed GUS expressing cells divided by the total GUS expressing cells. Relative

682

haustorial formation rate of each construct was obtained by comparing to the empty vector control

683

of each experiment, which was set to 100%. Data represent the mean values of five independent

684

experiments, except for Mlo RNAi (n=3). A total of 1821, 2102, 1779 and 617 cells were assessed

685

for the empty vector, CSEP0105, CSEP0162 and Mlo RNAi, respectively. All values are presented

686

± SE. Bars marked with different letters are significantly different at P < 0.01.

687 688

Figure 2. The expression patterns of CSEP0105 and CSEP0162 at different stages of Bgh

689

development. Total RNA was isolated from Bgh-infected (isolate DH14) barley leaves (cultivar

690

Golden Promise) at 0, 3, 6, 12, 24 and 48 h post inoculation. H and E denote haustorial and

691

epiphytic expression. Expression of Bgh GAPDH was used to normalize the CSEP expression in

692

each sample. Relative expression was determined compared to time point 0 hpi, arbitrarily set to 1.

693

Three biological and two technical replications were included for each time point. Data shown are

694

the means of three independent biological replications. All values are presented ± SE.

695 696

Figure 3. CSEP0105 and CSEP0162 interact with the barley Hsp16.9 and Hsp17.5 proteins in a

697

yeast two-hybrid assay. Yeast was transformed with bait and prey constructs. Interactions were

698

selected on dropout (DO) media lacking leucine (L), histidine (H), adenine (A) and tryptophan (W),

699

supplemented with 5 mM 3-amino-1,2,4-triazole (DO-LHAW, 5 mM 3-AT). The β-galactosidase

700

assay (X-gal) was performed on a filter paper print of DO-LW grown yeast. SNF4/SNF1 interaction

701

used as a positive control. CSEP0081 and CSEP0254 used as negative controls.

702 703

Figure 4. Amino acid-based phylogenetic tree of all 37 identified full-length barley sHsps, tomato

704

RSI2, Ntshsp17 and Nbshsp17. The lower left branches are mainly containing cytosolic (black)

705

sHsps with high similarity to each other. Those with non-cytosolic and unknown localizations are

706

shown in purple and green, respectively. Letters following the molecular weight indicate their

707

localizations, C-cytosolic, ER-endoplasmic reticulum, MI-mitochondria, P-chloroplast and Px-

708

peroxisome. The two sHsps identified in the Y2H screen, RSI2, Nthsp17, Nbhsp17 and Mds1 are

709

indicated by arrows. The naming follows Reddy et al. (2014) for those starting with “HvHsp”. The


710

MLOC-names are from the annotation of the barley genome. There are four sequences solely based

711

on cDNA-clones (gene bank accession numbers given).

712 713

Figure 5. CSEP0105, CSEP0162 and CSEP0254 have cytosolic and nuclear localization in barley

714

epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Ubiquitin promoter-driven expression

715

constructs were generated for all. (A-C) Constructs encoding signal peptide-lacking CSEPs fused to

716

the C-terminus of YFP were co-transformed with a free mCherry-construct into barley leaf

717

epidermal cells using particle bombardment. Two to four days later, cells were analyzed using a

718

Leica SP5 confocal laser scanning microscope. All the YFP-CSEP fusion signals are in the cytosol

719

and the nucleus. (D-E) Constructs encoding full-length Hsps fused to the N-terminus of mCherry

720

were co-transformed with a free YFP-construct. Both Hsps-mCherry signals are in the cytosol. The

721

free mCherry and YFP markers localize to the cytosol and nucleus. C, cytosol. N, nucleus. Scale

722

bar, 20 µm.

723 724

Figure 6. CSEP0105 and CSEP0162 exclusively accumulate in the cytosol when co-expressed with

725

Hsp16.9 and Hsp17.5. (A-F) The same YFP-CSEP and Hsp-mCherry fusion constructs were used

726

as described in Fig. 5. The YFP-CSEP constructs were co-transformed with each of the Hsp-

727

mCherry construct into barley leaf epidermal cells using particle bombardment. Two to four days

728

later, individual cells were visualized using a Leica SP5 confocal laser scanning microscope.

729

CSEP0254 was included as a negative control. The YFP-CSEP0105, YFP-CSEP0162, Hsp16.9-

730

mCherry and Hsp17.5-mCherry signals are in the cytosol, whereas YFP-CSEP0254 is in the cytosol

731

and the nucleus. C, cytosol. N, nucleus. Scale bar, 20 µm.

732 733

Figure 7. Hsp16.9 protects heat denaturation of E. coli proteins in vitro. Thermo-stability of total

734

protein from E. coli expressing GUS or Hsp16.9. Cell-free total protein extracts (250 µg mL-1) were

735

heated for 15 min. Heat-denatured proteins were removed by centrifugation and protein content of

736

the supernatant fractions was determined. Values are relative to the unheated controls. Data shown

737

are the means of three replicates ± SE. Asterisks show significant difference (P < 0.05) between

738

GUS control and Hsp16.9 at each heat-treatment analysed by Duncan’s multiple range test. The

739

experiment was repeated once with a similar result.

740


741

Figure 8. CSEP0105 inhibits the chaperone activity of Hsp16.9. Thermo-stability at 60°C of total

742

protein from E. coli expressing GUS or Hsp16.9 with and without CSEPs. Purified CSEPs were

743

added in a 1:2 protein molar ratio with Hsp16.9 in the cellular lysate (250 µg mL-1). CSEP0254 was

744

added as a negative control. Values are relative to the unheated controls. Data shown are the means

745

of three replicates ± SE. Means with different letters are significantly different (P < 0.05). Duncan’s

746

multiple range test was used to compare all the means. The experiment was repeated once with a

747

similar result.

748 749

Figure 9. Localization of CSEP0105, CSEP0162, Hsp16.9 and Hsp17.5 proteins in Bgh-infected

750

barley cells. (A-E) Confocal images of cells transiently co-transformed by particle bombardment

751

with the respective constructs as described in Fig. 5. The Hsp16.9-mCherry and Hsp17.5-mCherry

752

fluorescent signals are in the cytosol. YFP-CSEP0105 fluorescent signal is in the cytosol when co-

753

expressed with Hsp16.9-mCherry, and in the cytosol and nucleus when co-expressed with the free

754

mCherry construct. YFP-CSEP0162, as well as the free YFP and mCherry marker proteins

755

localized to the cytosol, nucleus and extrahaustorial matrix. The fluorescent signals labeled EHMx

756

also include the cytosol surrounding the haustoria. Since the nucleus and haustoria were not found

757

in a single confocal plane, images showing nuclei of the same cells are presented in Supplemental

758

Fig. 3. C, cytosol. N, nucleus. EHMx, extrahaustorial matrix. H, haustorium. Scale bar, 20 µm.

759 760


Figure 1.

Downloaded from www.plantphysiol.org on April 5, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Figure 2.


Figure 3.


100

MLOC_67109.1 MLOC_66935.1 AK363601 MLOC_16644.1 MLOC_6726.1 100 HvHsp21.2

56 100

13 97 21

Nbshsp17 Ntshsp17

15 36

MLOC_50285.1 HvHsp17.1-CV 16

MLOC_47822.1-P MLOC_7795.1-P

42 31

100

99 25

28

14

100

85

21

79

AK372933-P HvHsp21.3-MI MLOC_75175.1 HvHsp26.8-P

HvHsp15.1-Px MLOC_62670.1 HvHsp19.0-CIII HvHsp17.77-CII 46 AK375791-C 38 MLOC_32229.1-C 100 HvHsp17.3-CII HvHsp17.7-CII MLOC_52605.1

9 28

57

100

100 95 43

30 26 52 43 33

57

90

95

HvHsp16.86-CI MLOC_45029.1 MLOC_44536.1-C HvHsp16.9-CI HvHsp16.9-CI-H Mds-1 HvHsp16.88-CI

HvHsp16.7-Cl HvHsp17.7-CI MLOC_6787.1 HvHsp17.5-CI RSI2

Figure 4.

MLOC_10834.2 HvHsp21.9-ER HvHsp19.2-CX AK373460 HvHsp17.76-CIX

0.400

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

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SUPPLEMENTAL INFORMATION

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C A A T C T G T C A GC G A A A T A GC T A T C T C GG A A T T A A GGGC T GG A G T T A C A T GGC A C A C A T C C

T T A T C C GG T A T C A T A T A T A G A GG A A A T C T A T G T GG T T T A C C A A A C C C G A C C T A T C G T T GG

T T C A T A C A A C A G T C GG A A G A T G A G A A T T C GG A GG T GG T G A T G A A A T A C T T T T T A C T GG T C

T C GGC C A G T T GC G A A G T GC A GGGGG T C A T A A GC A C A GGC T GG A GG A A G A A A C T T GG A G A A

G A T G A C A C C T GG T G T A C C G A GGC A T A A

A T GC GC T T T T C T C G T A T GGC C A T C A T C T T T C A T A GC GC A A GC T T C T G T A C A A C A A GC C T T

GC C GC C C A A T A T T C T A G A C A T A T T A A T G A A G A A T T G A A GG T A T T T C A T T G T A A T A A T G A T

A T C C A T C A A G A GG A A T A T T C A C GG A C A C C A C A T T A T A A A A T A C A A G A T C C A G A T A C C A T T

C A GGG A T T G A A C G A GG A GC T C A G T T A T T T A G T T GC A G A C A C T T A T G A T A C G A GG A C T G T T

C A G T T C T A T G A C C A A G A C A A C GG T G A C T A C G A G T A T T T C A A T C T T T C A G A A A T A T G T G A T

A G A T A T C A A G A GC A A G A T GG T A C T G T T G T T A T T G A A C A C A T A C T C G T C A A C G A C C GGC A A

GG T C G T GC A T G T GC C A T G A T G A T G A T T A A G A C T G T A A T A C C A T T C A C C A A T T GGG A C GG A

C A A A G T C C A C A A A G A T A T T A T A GC T T G T GC GC A G T T GGC T C GGG A T A A

Figure S1. Nucleotide and amino acid sequences of CSEP0105 and CSEP0162. Shaded sequences encode for predicted signal peptide. Sequences used for the RNAi constructs are shown underlined.

Figure S2. (A) Nucleotide and (B) amino acid alignments of HvHsp16.9-CI and HvHsp16.9-CI-H. There are 14 nucleotide differences, where 12 are synonymous and two are non-synonymous. Dots indicate identical nucleotides and amino acids.

Figure S3. CSEP0105 and CSEP0162 interact with the barley Hsp16.9 and Hsp17.5 proteins in Nicotiana benthamiana and barley in a BiFC assay. The CSEPs were fused N-terminally by nYFP and the Hsps were fused C-terminally by cCFP. A) Constructs were co-expressed into N. benthamiana leaves using Agrobacterium mediated transformation. Interaction of two 14-3-3 proteins was used as a positive control. CSEP0254 and 14-3-3 proteins were used as negative controls. B) The nYFPCSEP and Hsp-cCFP fusion constructs were co-transformed with the mCherry transformation marker, which localizes to the cytosol and the nucleus, using particle bombardment into barley leaf epidermal cells. C, cytosol. N, nucleus. Scale bar, 20 µm.

Figure S4. Localization of CSEP0105, CSEP0162, and Hsp16.9 proteins in Bghinfected barley cells. (A-D) Confocal images showing the nuclei of the same cells presented in Fig. 9. C, cytosol. N, nucleus. Scale bar, 20 µm.

Table S1. List of small heat shock proteins identified in barley Gene expression ressources 4) Genome ressources 5) cDNA ressources 1) 2) 3) Name MW (kD) bp AA HarvEST ID Fl cDNA ID Probe Set ID Morex_contig Chromosome MLOC_62670.1 15.09 417 138 none 46836 7H cM=66 Hv Hsp15.1-Px 15.14 417 138 AK365443 none none identical Hv Hsp16.7-CI 16.76 453 150 35_14864 AK252765 Contig2010_at 120794 4H cM=55 MLOC_45029.1 16.79 459 152 none 276057 2H cM=138 MLOC_44536.1 16.84 453 150 Contig2007_s_at 274906 3H cM=15 Hv Hsp16.86-CI 16.86 456 151 AK376179 EBro08_SQ011_I03_at 54783 (8 SNPs) 3H cM=15 Hv Hsp16.88-CI 16.88 453 150 35_14863 AK355146 Contig2008_s_at none identical 3 4) Hv Hsp16.9-CI 16.93 456 151 35_14860 AK362925 Contig2004_s_at 66925 3HS Hv Hsp16.9-CI-H 16.93 456 151 none none identical Hv Hsp17.1-CV 17.14 468 155 35_5795 AK241741 Contig13073_at 49195 2H cM=58 Hv Hsp17.3-CII 17.34 480 159 35_15451 AK375970 Contig3284_x_at 135539 3H cM=46 MLOC_32229.1 17.41 480 159 Contig3286_s_at 226559 3H cM=45 MLOC_6787.1 17.54 477 158 Contig2006_at 138095 4H cM=56 Hv Hsp17.5-CI 17.57 477 158 35_14859 AK250749 HB18H23r_s_at 64954 4H cM=51 Hv Hsp17.7-CII 17.61 489 162 35_15454 BF263847 Contig3287_x_at none identical 3 4) AK373460 17.63 498 165 none 70124 3H cM=14 Hv Hsp17.76-CIX 17.65 498 165 35_5001 AK370196 Contig11961_at 46949 3HS AK375791 17.67 489 162 Contig3285_at 81807 3H cM=46 Hv Hsp-17.77CII 17.78 489 162 35_15456 AK355146 Contig3289_at 69124 3H cM=46 Hv Hsp17.7-CI 17.80 486 161 35_14866 AK368988 Contig2012_s_at 51731 4H cM=51 Hv Hsp19.0-CIII 19.05 537 178 35_18565 AK360636 Contig10029_at 52273 6H cM=91 Hv Hsp19.2-CX 19.26 534 177 35_5077 AK370937 Contig15445_at 368996 6H cM=49 MLOC_52605.1 20.64 561 186 none 37653 1H cM=66 Hv Hsp21.2 21.25 585 194 35_23844 BF623486 Contig21040_at 77164 4H cM=73 Hv Hsp21.3-MI 21.36 582 193 35_16753 AK370932 Contig6559_at none identical 7 4) Hv Hsp21.9-ER 21.98 618 205 AK374148 none 51617 4H cM=51 AK372933 22.58 621 206 Contig6557 none identical MLOC_75175.1 23.42 651 216 none 67285 6H cM=75 MLOC_10834.2 23.49 651 216 none 1559883 2H cM=58 MLOC_6726.1 24.17 666 221 none 138014 4HL MLOC_47822.1 25.16 711 236 none 312759 5H cM=51 MLOC_50285.1 25.32 681 226 none 358032 1H MLOC_7795.1 26.44 750 249 none 140185 5H cM=50 Hv Hsp26.8-P 26.80 735 244 35_21394 AK376976 EBem05_SQ003_L06_at 78169 4H cM=57 MLOC_16644.1 28.33 780 259 none 1574043 3H cM=52 MLOC_67109.1 29.11 807 268 none 52587 1H AK363601 36.17 1029 342 none none identical MLOC_66935.1 38.96 1104 367 none 52302 1H cM=132

Localization 6) Localization peroxisome cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm mitochondria endoplasmic reticulum chloroplast

chloroplast chloroplast chloroplast

Foot notes: 1) The naming follows Reddy et al. (2014) for those starting with Hv Hsp, the MLOC-names are from the barley genome annotation and there are four sequences solely based on cDNA-clones and their genbank-accession numbers are given. 2) HarvEST ID according to the HarvEST-database: http://harvest.ucr.edu/. 3) Full length cDNA accession numbers in NCBI: http://www.ncbi.nlm.nih.gov/. 4) PLEXdb (http://www.plexdb.org/index.php). The Affymetrix probe set Ids indicated for those present on the array. Chromosomal location based on transcript profiling of wheat-barley ditelosomic chromosome addition lines. 5) The contig IDs and chromosomal localization from the genome ressource at IPK Gatersleben (http://webblast.ipk-gatersleben.de/barley/index.php). 6) Predicted by Reddy et al. 2014 and us using the same softwares. The two identified sHsps are shaded with dark grey

Table S2. Primer sequences for plasmid constructions and gene expression assay (5´ to 3´) Primers for RNAi CSEP0105_RNAi_F

ATCTATCATAGGTCTGGTGCAGTG

CSEP0105_RNAi_R

GGTGTCATCTTCTCCAAGTTTCTT

CSEP0162_RNAi_F

GTATGGCCATCATCTTTCATAGC

CSEP0162_RNAi_R

CAACAGTACCATCTTGCTCTTGAT

Hsp16.9_RNAi_F

CACCACTCAACCGAGTGCCAAGC

Hsp16.9_RNAi_R

TGACCTCCTCCTTCTTCACG

Primers for the bait constructs in Y2H, BiFC, localization and protein expression CSEP0081_F

GGAAATGCAAAGTACAATTG

CSEP0081_R_with stop

TTACATGGCAGTACATGGTAAATAG

CSEP0105_F

ATTACAGAATATTACTGTGGCAAC


TTATGCCTCGGTACACCA

CSEP0105_R_no stop

AAATGCCTCGGTACACCAGGT

CSEP0162_F

GCCCAATATTCTAGACATATTAATGA


TTATCCCGAGCCAACTGC

CSEP0162_R_no stop

AAATCCCGAGCCAACTGCGCA

CSEP0254_F

CACCAACCAACATTACAAATGTGATGA


CTAACTAATATCTTGCAGAGAACATAC

CSEP0254_R_no stop

AAAACTAATATCTTGCAGAGAACATACTAT

Hsp16.9_F

CACCATGTCGATCGTGAGGCGTA

Hsp16.9_R_with stop

TCAGCCGGAGATCTCGAT

Hsp16.9_R_no stop

AAAGCCGGAGATCTCGATGGC

Hsp17.5_F

CACCATGTCGCTGATCCGTCGC

Hsp17.5_R_with stop

CTAGCCGGAGATCTGGATG

Hsp17.5_R_no stop

AAAGCCGGAGATCTGGATGGAC

Primers for qPCR CSEP0105_ qPCR_F

GAGAATTCGGAGGTGGTGAT

CSEP0105_ qPCR_R

GTTTCTTCCTCCAGCCTGTG

CSEP0162_ qPCR_F

GTGATAGATATCAAGAGCAAGATGG

CSEP0162_ qPCR_R

TCTTAATCATCATCATGGCACA

BghGAPDH_qPCR_F

ATGAACTACAAGGCATCCTGTCA

BghGAPDH_ qPCR_R

TACCATGCGACTAGCTTAACAAAG

Small Molecule Inhibitors to Disrupt Protein-protein Interactions of Heat Shock Protein 90 Chaperone Machinery.

The Barley Powdery Mildew Effector Candidates CSEP0081 and CSEP0254 Promote Fungal Infection Success.

The Chaperone Activity of the Developmental Small Heat Shock Protein Sip1 Is Regulated by pH-Dependent Conformational Changes.

A barley SKP1-like protein controls abundance of the susceptibility factor RACB and influences the interaction of barley with the barley powdery mildew fungus.

A barley Engulfment and Motility domain containing protein modulates Rho GTPase activating protein HvMAGAP1 function in the barley powdery mildew interaction.

Bacterial Heat Shock Protein Activity.

De novo Analysis of the Epiphytic Transcriptome of the Cucurbit Powdery Mildew Fungus Podosphaera xanthii and Identification of Candidate Secreted Effector Proteins.

Over-expression of the small heat-shock protein, hsp25, inhibits growth of Ehrlich ascites tumor cells.

The juvenile hormone analogue, methoprene, inhibits ecdysterone induction of small heat shock protein gene expression.

The cardioprotective role of small heat-shock protein 20.

Sequence of the Chinese hamster small heat shock protein HSP27.

Molecular marker analyses of powdery mildew resistance in barley.

Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei.

CUEDC2 interacts with heat shock protein 70 and negatively regulates its chaperone activity.

Multiple oligomeric structures of a bacterial small heat shock protein.

Alpha B-crystallin is a small heat shock protein.

Chaperone function of two small heat shock proteins from maize.

Heat Shock Protein HSP101 Affects the Release of Ribosomal Protein mRNAs for Recovery after Heat Shock.

Small heat shock protein degradation could be an indicator of the extent of myofibrillar protein degradation.

A storage-protein marker associated with the suppressor of Pm8 for powdery mildew resistance in wheat.

grp94 is a pro-oncogenic chaperone, not a tumor suppressor.

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley.

Suppression of the powdery mildew pathogen by chitinase microinjected into barley coleoptile epidermal cells.

Heterogeneity of Powdery Mildew Resistance Revealed in Accessions of the ICARDA Wild Barley Collection.