PROTEIN DISULFIDE ISOMERASE LIKE 5-1 is asusceptibility factor to plant virusesPing Yanga, Thomas Lüpkenb, Antje Habekussb, Goetz Henselc, Burkhard Steuernageld,1, Benjamin Kiliana,Ruvini Ariyadasaa, Axel Himmelbacha, Jochen Kumlehnc, Uwe Scholzd, Frank Ordonb,2, and Nils Steina,2,3

aGenome Diversity, Department Genebank, Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany; bInstitute forResistance Research and Stress Tolerance, Julius Kuehn Institute, Federal Research Centre for Cultivated Plants, D-06484 Quedlinburg, Germany; cPlantReproductive Biology, Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany;and dBioinformatics and Information Technology, Department of Cytogenetics and Genome Analyses, Leibniz Institute of Plant Genetics and Crop PlantResearch, D-06466 Gatersleben, Germany

Edited by David C. Baulcombe, University of Cambridge, Cambridge, United Kingdom, and approved January 3, 2014 (received for review October 29, 2013)

Protein disulfide isomerases (PDIs) catalyze the correct folding ofproteins and prevent the aggregation of unfolded or partiallyfolded precursors. Whereas suppression of members of the PDIgene family can delay replication of several human and animalviruses (e.g., HIV), their role in interactions with plant viruses islargely unknown. Here, using a positional cloning strategy weidentified variants of PROTEIN DISULFIDE ISOMERASE LIKE 5–1(HvPDIL5-1) as the cause of naturally occurring resistance to mul-tiple strains of Bymoviruses. The role of wild-type HvPDIL5-1 inconferring susceptibility was confirmed by targeting induced locallesions in genomes for induced mutant alleles, transgene-inducedcomplementation, and allelism tests using different natural resis-tance alleles. The geographical distribution of natural genetic var-iants of HvPDIL5-1 revealed the origin of resistance conferringalleles in domesticated barley in Eastern Asia. Higher sequencediversity was correlated with areas with increased pathogen di-versity suggesting adaptive selection for bymovirus resistance.HvPDIL5-1 homologs are highly conserved across species of theplant and animal kingdoms implying that orthologs of HvPDIL5-1or other closely related members of the PDI gene family may bepotential susceptibility factors to viruses in other eukaryotic species.

allele mining | resistance breeding | soil-borne virus disease | chaperone

Infectious diseases caused by plant viruses threaten agriculturalproductivity and reduce globally attainable agricultural pro-

duction by about 3% (1). In specific pathosystems, plant virusescan result in the loss of the entire crop. For example, the dev-astation of cassava production by cassava mosaic geminiviruses(CMGs) in Uganda during the 1990s led to widespread foodshortages and famine-related deaths (2). Unfortunately pro-tecting plants against viruses (especially soil-borne viruses) byusing agrochemicals to control virus vectors is seldom efficientfrom economic or ecological perspectives. Therefore, crop pro-tection based on naturally occurring virus resistance is key tominimizing losses and achieving sustainable crop yields.Positive-strand RNA viruses represent the largest group of

plant viruses (3). They cause a very high proportion of the im-portant infectious virus diseases in agriculture (4, 5). Such plantviruses carry a reduced genome that encodes a limited set offunctional proteins (4–10 viral proteins)—insufficient to com-plete the entire virus replication and proliferation cycle (6). In-stead, over evolutionary time, viruses recruited host factors toperform the infectious life cycle (7). This dependence on hostfactors establishes a possibility that plants can evolve escape, tol-erance, or resistance mechanisms to ameliorate the consequencesof viral infection. The absence of essential host factors could in-terfere with the infection process or restrict proliferation (8) lead-ing to either mono- or polygenic recessive resistance (5). Prominentexamples of such susceptibility factors that are conserved in mul-tiple plant–virus systems are the EUKARYOTIC TRANSLATIONINITIATION FACTORS (EIF)4E, iso4E, 4G, and iso4G (9).Translation initiation factors may interact directly with viral RNAwhere they catalyze the initiation of translation of viral polyproteins

(10, 11). In addition, to establish replication and assembly com-plexes during infection, viruses typically create membrane-boundenvironments, referred to as “virus factories” (12). There, cellularchaperones such as HSP70 and DNAJ-like proteins likely contrib-ute to the correct folding and translocation of substrates (12, 13).However, only a few such host factors are known (7, 9, 14).Barley yellow mosaic virus disease caused by barley yellow

mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV)(both belong to Bymoviruses) seriously threatens winter barleyproduction in Europe and East Asia (4). Infection leads to yel-low discoloration and stunted growth and may result in yield lossof up to 50% (15). Soil-borne transmission via the plasmodio-phorid Polymyxa graminis prohibits plant protection by chemicalmeasures, and breeding for resistant cultivars is therefore theonly practicable way to prevent yield loss. The naturally occur-ring recessive resistance locus rym11 confers complete broad-spectrum resistance to all known European isolates of BaMMVand BaYMV (16–19). In the present study, we used a positionalcloning strategy to identify the gene underlying rym11-based re-sistance. We show that it is a susceptibility factor belonging toa gene family of PROTEIN DISULFIDE ISOMERASES (PDIs),and is highly conserved across eukaryotic species. We observe astrong correlation between natural allelic variation and geographicdistribution, suggesting that both the origin and subsequent

Significance

This work describes a susceptibility factor to plant viruses thatbelongs to the conserved PROTEIN DISULFIDE ISOMERASE (PDI)gene family. We show that loss-of-function HvPDIL5-1 allelesat the recessive RESISTANCE TO YELLOW MOSAIC DISEASE 11(rym11) resistance locus confer broad-spectrum resistance tomultiple strains of Bymoviruses and could therefore play acentral role in durable virus resistance breeding in barley. Thegeographic distribution of functional alleles of rym11 in EastAsia suggests adaptive selection for resistance in this region.Orthologues of HvPDIL5-1 or related members of the PDI genefamily potentially provide susceptibility factors to viruses acrossanimal and plant kingdoms.

Author contributions: F.O. and N.S. designed research; T.L. mapped and identified the can-didate gene HvPDIL5-1; P.Y., A. Habekuss, G.H., A. Himmelbach, and J.K. performed research;P.Y., T.L., B.S., B.K., R.A., and U.S. analyzed data; and P.Y. and N.S. wrote the paper.

Conflict of interest statement: A patent application has been filed relating to this work.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the EMBLdatabase (accession nos. PRJEB4765 and HG793095).1Present address: The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH,United Kingdom.

2F.O. and N.S. contributed equally to this work.3To whom correspondence should be addressed. E-mail: stein@ipk-gatersleben.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1320362111/-/DCSupplemental.

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adaptive selection for rym11-based resistance in winter barley oc-curred in East Asia.

ResultsMap-Based Cloning of rym11. The resistance gene rym11 was pre-viously located to a 0.074-cM region on chromosome 4HL (18–20). BAC contigs were identified by sequence comparison offlanking markers and BAC library screening. Sequence analysisof the entire BAC contigs revealed an overlap of ∼18 kb betweenBAC clones HVVMRXALLhA0173N06 and HVVMRXALL-hA0172k23 residing on the original finger-printed contigs (FPCs)ctg551 and ctg1996 (SI Appendix, Fig. S1), respectively. Thus,a physical contig harboring rym11 and its flanking markers wasestablished. New markers were then developed and the geneticinterval of rym11 narrowed to 0.0196 cM between markersC5B4C and C2B18S2 (Fig. 1A). This target interval was repre-sented by 12 overlapping BAC clones (SI Appendix, Fig. S1 andFig. 1A) spanning a distance of about 1.25 Mbp (Fig. 1A). An-notation of the nonrepetitive sequences of this interval identifiedfour ORFs (Fig. 1A). This included two genes with sequencehomology to ELONGATION FACTOR 1-α and one homologof β3-GLUCURONYLTRANSFERASE 43. Resequencing ofthese three genes revealed no polymorphisms between suscep-tible (Rym11) and resistant (rym11) genotypes. The fourth ORFencoded a homolog of PROTEIN DISULFIDE ISOMERASELIKE 5–1 (HvPDIL5-1). This gene contained a 1,375-bp de-letion of the putative promoter region and the first three exons inthe resistant genotypes W757/112 and W757/612 (SI Appendix,Fig. S2A). Due to this large deletion, no transcript fromHvPDIL5-1 could be detected (SI Appendix, Fig. S2B). HvPDIL5-1

was therefore a likely candidate for rym11-based resistance. Theallele carrying the 1,375-bp deletion was named rym11-a.

Functional Validation of HvPDIL5-1 as a Susceptibility Factor forBymovirus Infection. We used three independent approaches tovalidate the hypothesis that HvPDIL5-1 is a susceptibility factorfor bymovirus infection. First, we screened an EMS-inducedmutant population of barley by targeting induced local lesions ingenomes (TILLING) (21). Two independent mutants 9699 and10253 were identified each carrying a premature STOP codon inthe coding sequence (CDS) of HvPDIL5-1 (SI Appendix, TableS1 and Fig. 1B). M3 progeny of the originally heterozygous M2plants segregated for virus resistance. After phenotyping, theplants were genotyped for the presence of the mutation. Allhomozygous mutant M3 plants were found to be completely re-sistant. Homozygous wild-type and heterozygous M3 plants werepredominantly susceptible with infection rates comparable to thesusceptible controls (Table 1 and SI Appendix, Table S2). Re-sistance tests were replicated twice in M4 progeny of a homozy-gous mutant and a wild-type M3 plant of each of the mutant lines9699 and 10253, respectively. All homozygous mutant M4 plantswere tested resistant. Homozygous wild-type M4 progeny weresusceptible, with infection rates between 66% and 100%, similarto susceptible controls (Table 1 and SI Appendix, Table S2 andFig. S3). Mechanically infected homozygous wild-type HvPDIL5-1 M4 progenies showed the characteristic yellow discolorationduring vegetative growth stage (Fig. 1C), stunted growth at headingstage (Fig. 1D), as well as late maturation (Fig. 1E). Mock in-oculated homozygous mutant M4 individuals did not show anydetectable difference in overall growth habit at heading andmaturation stages compared with control plants (SI Appendix,Fig. S4). We conclude that HvPDIL5-1 loss-of-function mutantsexhibit no deleterious growth effects while acquiring resistanceto bymovirus infection.Second, we amplified a 576-bp cDNA containing the 456-bp

full-length ORF of wild-type HvPDIL5-1 from barley cv. Naturel,

Fig. 1. Map-based cloning of rym11. (A) Genetic and physical mapping ofrym11. The number below each marker indicates the recombination eventsbetween rym11 and the adjacent marker. The overlap of black-highlightedBAC clones was confirmed by PCR amplification and dot-plot analysis together(SI Appendix, Fig. S1). (B) Naturally occurring and induced alleles of rym11 sur-veyed by resequencing and TILLING. The black boxes represent the functionalthioredoxin (TRX)-like domain. The phenotypes at the vegetative growth (C),heading (D), and maturation (E) stages of homozygous M4 mutant (Left) andwild type (Right) of heterozygous M2 mutant 10253 are shown.

Table 1. Functional validation of HvPDIL5-1 underlying rym11-based resistance by three independent approaches

Genotypes Tested Infected Comments

M3-9699-MT 3 0 M3, TILLINGM3-9699-HET 10 8 M3, TILLINGM3-9699-WT 6 5 M3, TILLINGM3-10253-MT 15 0 M3, TILLINGM3-10253-HET 21 17 M3, TILLINGM3-10253-WT 11 9 M3, TILLINGM4-9699-MT† 13 0 M4, TILLINGM4-9699-WT† 15 13 M4, TILLINGM4-10253-MT† 13 0 M4, TILLINGM4-10253-WT† 18 13 M4, TILLINGE1-OX* 49 30 T1, transgene analysisE1-rym11** 21 0 T1, transgene analysisE2-OX* 48 45 T1, transgene analysisE2-rym11** 10 0 T1, transgene analysisrym11-a + b 8 0 F1, test for allelismrym11-a + c 11 0 F1, test for allelismrym11-a + d 14 0 F1, test for allelismW757/612‡ 49 0 Resistant controlBarke‡ 45 37 Susceptible controlIgri‡ 26 22 Susceptible controlMaris Otter‡ 58 51 Susceptible control

WT, wild type; HET, heterozygous; MT, mutant; *OX, T1 plants carryingthe wild-type Rym11 transgene; ** T1 null-segregant plants for the trans-gene; Res, resistance; Sus, susceptibility.†Sum of two biological repeats.‡Sum of five independent experiments. For further details, see SI Appendix,Table S2.

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and used this to complement the resistant genotype W757/612 byAgrobacterium-mediated transformation with the intention toinduce susceptibility. W757/612 contains the recessive resistanceallele rym11-a. Two independent transgenic events (T0) wereobtained. T1 plants were mechanically infected, phenotyped, andsubsequently genotyped for the presence of the transgene. RT-PCR amplification revealed a 3:1 segregation (presence/absence)of the transgene in the respective T1 families, indicating thepresence of a single transgene locus (Table 1). Plants carryingthe transgene were observed to be predominantly susceptible toBaMMV infection (Table 1 and SI Appendix, Table S2) whereasT1 null segregants were consistently resistant (Table 1 and SIAppendix, Table S2). Therefore, complementation of rym11-a with wild-type HvPDIL5-1 converted a resistant genotype intoa susceptible one.Third we performed allelism tests between naturally occurring

rym11 alleles. The breeding line Russia57 was previously reportedto carry the recessive monogenic resistance locus rym11 (18).Resequencing HvPDIL5-1 from Russia57 revealed a 17-bp de-letion causing a frameshift (henceforth named rym11-b, Fig. 1B). F1plants obtained by crossing W757/112 (rym11-a) and Russia57(rym11-b) showed complete resistance to mechanical inoculationwith BaMMV (Table 1 and SI Appendix, Table S2). Thus, bothrym11-a and -b confer bymovirus resistance.

Natural Variation of HvPDIL5-1 and the Origin of rym11-BasedResistance. Natural variation of HvPDIL5-1 was surveyed byresequencing the entire ORF in a geographically referencedcollection of 365 wild (Hordeum vulgare ssp. spontaneum) and1,452 domesticated barley accessions (H. vulgare ssp. vulgare)(SI Appendix, Table S3). High-quality sequence was obtainedfrom 1,732 accessions for 1,974 bp of genomic sequence in-cluding the complete ORF. We used the 456 bp of assembledCDS for calling exon-based sequence diversity. Overall, fourdeletions and 25 SNPs defined 28 independent exon hap-lotypes (SI Appendix, Table S4). Relative to haplotype I (i.e.,wild-type HvPDIL5-1 of cv. Naturel), nine haplotypes con-tained exclusively synonymous variants, whereas the remaining18 contained deletions or nonsynonymous variants, sometimes incombination with additional synonymous SNPs (SI Appendix,Table S4). Twenty-three haplotypes were exclusively observed inaccessions originating from the Near East (SI Appendix, Fig. S5).Three haplotypes (I, III, and IV) were shared between wild anddomesticated barleys (Fig. 2). Haplotype I was found in 1,607 ofthe 1,732 accessions (92.8%) (SI Appendix, Table S3) and was

present in all geographical regions (Fig. 3A). Median-joining(MJ) network analysis revealed that HvPDIL5-1 homologs fromBrachypodium distachyon, Secale cereale, Triticum aestivum, andHordeum bulbosum forming the outgroup, were most closelyrelated to haplotype I in the H. vulgare lineage (SI Appendix,Fig. S6). Haplotype I is therefore most closely related to orrepresents the ancestral HvPDIL5-1 haplotype. The remaininghaplotypes in barley, therefore, likely evolved by natural selection(Fig. 2).Among these 1,732 accessions, only a single genotype,

HOR1363, carried the rym11-a resistance allele (haplotypeXXVIII). Twenty-seven accessions carried rym11-b (haplotype II)(SI Appendix, Table S4). All 28 accessions showed resistanceupon natural and artificial virus infection (SI Appendix, Table S5).Two additional haplotypes (VII and VIII) encoded defectivePDIL5-1 proteins. Haplotype VII carried a 1-bp deletion andhaplotype VIII exhibited a nonsynonymous substitution at base-pair position 182 of CDS (SI Appendix, Table S4 and Fig. 1B).Accessions carrying either of these haplotypes conferred re-sistance to BaMMV and BaYMV (SI Appendix, Table S5) andwere named rym11-c and -d, respectively (Fig. 1B). F1 hybridscarrying rym11-a and either rym11-c or -d were resistant toBaMMV (rym11-a + c, rym11-a + d, Table 1). All naturally oc-curring resistance alleles (rym11-a, -b, -c, and -d) were deriveddirectly by single mutation events (deletion or substitution) ofhaplotype I (Fig. 2). Four haplotype-I–containing accessions weretested for resistance by mechanical infection with BaMMV andall were susceptible (SI Appendix, Table S5). Overall haplotype I

Fig. 2. Median-joining (MJ) network analysis of naturally occurring hap-lotypes of the gene HvPDIL5-1. Twenty-eight haplotypes were found in 1,732accessions including four resistance-conferring alleles indicated as rym11-a,-b, -c, and -d, respectively. Circle size corresponds to the frequency of aparticular haplotype. Red, wild barley; light blue, landrace barley; dark blue,barley cultivar; yellow, H. vulgare ssp. agriocrithon. If not otherwise in-dicated (XXVIII, rym11-a; II, rym11-b; V) the distance between haplotypesrepresents one nucleotide substitution or deletion.

Fig. 3. Geographic distribution of barley germplasm carrying differentrym11 alleles and haplotypes. Geographic information system (GIS)-basedtopographical maps for the ancestral haplotype I and the combination offour resistance-conferring alleles (rym11-a, -b, -c, and -d) are shown. (A)Geographic distribution of accessions carrying the ancestral haplotype I. (B)Distribution of accessions carrying either one of the resistance-conferringrym11 alleles. The size of filled cycles is proportional to the number ofaccessions found at each particular site. For accessions without georeferenceinformation the capital state of the source country was arbitrarily chosen asthe collecting site, which is explaining circles in regions that do not representbarley cultivation area.

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predominates among accessions of a worldwide diversity collec-tion. We conclude that susceptibility to Bymovirus is the ancestralstate. We propose that recessive rym11 resistance alleles mostlikely arose by selection of mutations in HvPDIL5-1 during themigration of domesticated barleys into new environments wherenaturally occurring Bymoviruses were prevalent. This proposal issupported by our observation that no resistance-conferring alleleswere found in wild barleys (Fig. 2). Furthermore, only a singlerym11-a carrying barley landrace HOR1363 originated fromwithin the accepted natural distribution range of the wild species(Turkey). All 32 accessions carrying recessive resistance alleles(rym11-b, -c, and -d) were collected from Eastern Asian countries(SI Appendix, Fig. S5 and Fig. 3B) where high BaMMV/BaYMVdiversity has previously been observed (4, 22).

PDIL5-1 Is a Highly Conserved Protein in Plant and Animal Species. Inmany plants, recessive resistance to Potyviruses is controlledby natural variation in the highly conserved EUKARYOTICTRANSLATION INITIATION FACTOR 4E (eIF4E) (9). Todetermine the level of PDIL5-1 conservation, we conducteda sequence comparison and phylogenetic analysis among puta-tive functional orthologs from different plant and animal species.HvPDIL5-1 is a specific member of the PDI gene family andencodes 151 amino acids. This particular member carries a singlefunctional thioredoxin (TRX) domain (SI Appendix, Table S6)putatively catalyzing the formation, reduction, and isomerizationof disulfide bonds. It contains the C′-terminal tetrapeptideEDEL, which controls the retention of the protein within theendoplasmic reticulum (ER), as could be demonstrated for thehomologous human protein ERp16 (also called ERp18, ERp19,or hLTP19) (23). Eighteen homologous genes in plants and 30genes in animals were identified (SI Appendix, Table S6). Aphylogenetic analysis for 17 PDI proteins of species from allevolutionary and organizational stages (e.g., unicellulate andmulticellulate organisms, algae/primitive land plants/higher vas-cular plants, etc.) revealed a single rooted tree with a split be-tween the animal and plant branch but otherwise consistentsorting according to the evolutionary level or organization (Fig.4A). Sequence similarity inside the TRX domain separated theplant from the animal branch and diversity in the C′-terminalregion revealed differentiation between monocotyledonous anddicotyledonous plant species (SI Appendix, Figs. S7 and S8).Furthermore, the majority of homologous proteins are predictedto be secreted to the ER (SI Appendix, Table S6). The 3Dstructure of the homologous proteins of a protist (Capsasporaowczarzaki), of a unicellulate algae (Chlamydomonas rein-hardtii) and of barley were simulated in reference to the hu-man protein ERp18 (24). The predicted 3D structure washighly conserved across kingdoms (Fig. 4B). As PDIL5-1homologs share structure, function, and subcellular localiza-tion between organisms, it is tempting to speculate that somemay similarly function as dominant virus susceptibility factorsin organisms other than barley.

DiscussionHvPDIL5-1 Is a Susceptibility Factor to Bymoviruses in Barley and It IsHighly Conserved Between Plants and Animals. We identifieda susceptibility factor to bymovirus disease in barley encodedby HvPDIL5-1, a member of the PROTEIN DISULFIDEISOMERASE (PDI) gene family. The PDI gene family usuallycontains more than 10 members in different eukaryotic species;e.g., there are 20 genes in Homo sapiens and 18 in rice (25–27).The major function of PDIs in humans is to contribute to theformation of disulfide bonds by induction, oxidization, and isom-erization; all catalyzed by the active TRX domain (25, 27). PDIsalso play a role as chaperones in the quality check system forcorrect protein folding (28). Based on its high amino acid sequenceconservation to human ERp16, which encodes for an ER-residentthioldisulfide oxidoreductase fulfilling chaperone activities (23), weconclude that plant homologs have retained this function. Thus,HvPDIL5-1 could have been recruited by Bymoviruses in barley to

act as cellular chaperone (protein folding, stabilization, or facili-tating transport) during virus infection. Other cellular chaperoneslike HSP70, HSP90, and DNAJ-like proteins have been shown toperform similar functions in other plant species in the inter-actions with several genera of plant viruses (12). Both functionalmotifs and tertiary structures of HvPDIL5-1 and its homologs inother plants and animals are highly conserved. By identifyingHvPDIL5-1 as susceptibility factor to the potyvirus-related Bymo-viruses in barley, it seems plausible that other members of the PDIgene family may play a similar role in other plant–virus interactions.Given that the highly conserved eukaryotic translation initiationfactor gene family has been repeatedly identified as susceptibilityfactors in numerous pathosystems (9), we consider this a realis-tic possibility.Members of plant PDIs have not been reported so far as sensu

stricto susceptibility factors to any plant viruses and little is knownabout plant PDI/virus interactions. However, virus-inducedgene silencing (VIGS) of two PDI family members in tobacco,NtERp57 and NtP5, partially decreased N-gene mediated re-sistance to tobacco mosaic virus (TMV) (29). Whereas thishinted at an interaction between plant PDIs and viruses, it didnot indicate a role as a susceptibility factor. The situation isdifferent in animals. PDI family members, particularly cell-surface PDI proteins, may participate in the infection processof multiple human and animal viruses, e.g., HIV (HIV) (30–36).Nonspecific inhibition of PDI activity suppressed the PDI-mediatedredox environment of plasma membranes (33) and therefore in-terfered with HIV envelope protein-directed cell fusions (32).Knockdown of specific PDImembers could influence the infectivityof several viruses, e.g., HIV, Newcastle disease virus (NDV) andmouse polyomavirus (31, 32, 35). In human cell cultures, knock-down of the PDI family members, PDI, ERp29, ERp57, and ERp72inhibited the accumulation of viral particles (30, 34, 36). Our datasupport and extend the role of PDIs as important host componentsrequired for the successful infection or replication of viruses inanimals to plants.

Fig. 4. Phylogenetic analysis of HvPDIL5-1. (A) Phylogenetic tree of HvPDIL5-1and 16 homologous proteins. (B) Three dimensional model of the homologousproteins in C. owczarzaki ATCC 30864, C. reinhardtii, and H. vulgare. Simulationof 3D protein structure was achieved based on sequence homology to humanHsERp18 (PDB accession no. 2k8vA).

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Loss-of-Function Alleles Are Prevalent in Domesticated BarleyAccessions from Eastern Asia. Resequencing HvPDIL5-1 in alarge georeferenced collection of wild and domesticated barleysrevealed less haplotype diversity in domesticated (12 haplotypes)compared with wild barley (18 haplotypes) (although almostfourfold more accessions of domesticated barley were surveyed).This is in strong contrast to the four naturally occurring loss-of-function alleles of HvPDIL5-1. All four confer resistance toBymoviruses, and all four were exclusively observed in domesticatedbarleys. Geographically, resistant alleles were overrepresented inwinter-type barley from Eastern Asia (SI Appendix, Fig. S9), sug-gesting selection for resistance during range extension as barleycultivation spread east from its center of origin. This is con-sistent with the higher diversity of Bymoviruses in Eastern Asia(4, 22) where a nonfunctional HvPDIL5-1 may have providedan adaptative advantage.

Resistance Breeding Requires Access to Multiple Sources of NaturallyOccurring Resistance. Barley yellow mosaic virus disease is one ofthe most important diseases in winter barley (15). Its soil-bornetransmission precludes plant protection by use of agrochemicals.In European barley breeding this stimulated the extensive use ofrym4/rym5, a single natural recessive resistance conferred byalleles of the EUKARYOTIC TRANSLATION INITIATIONFACTOR 4E (37). However, rym4/rym5 resistance-breakingisolates have recently been observed (16, 17, 38). rym11 is ef-fective against all European strains of BaMMV/BaYMV (16–18). We have shown here that resistance is conferred by loss-of-function mutations in HvPDIL5-1. HvPDIL5-1 must thereforeprovide a host component that is essential for virus establish-ment and/or replication in planta. To overcome rym11-basedresistance the virus would therefore need to establish an in-teraction with an alternative and functionally redundant hostcomponent. Today, rym11 plays a minor role in European barleybreeding. Its implementation may, however, reduce the potentialfor the emergence of resistance-breaking viral isolates. Pyr-amiding several resistance genes in a single genotype is consid-ered to be a promising strategy to obtain durable virus resistance(39). With the identification of rym11 as loss-of-function alleles ofHvPDIL5-1, it is now possible to combine two recessive re-sistance loci (rym11 and rym4/rym5) on the basis of perfectlylinked and diagnostic molecular markers.

Materials and MethodsPlant Material. High-resolution genetic mapping of rym11 was conducted in5,102 F2 individuals of the cross cv. Naturel (Rym11/Rym11) × resistant ge-notype W757/112 and W757/612 (both rym11/rym11) (20). Eighteenrecombinants of this population were used for further genetic and physicaldelimitation of rym11. For allele mining, 1,816 georeferenced accessionsfrom 70 countries including 365 wild and 1,451 domesticated barleys wereselected (SI Appendix, Table S3). F1 hybrids were obtained by crossing theresistant genotypes W757/112 and/or W757/612 with the newly identifiedgene haplotypes.

Physical Mapping. BAC contigs were identified either by sequence comparisonof flanking markers (20) to sequence resources integrated to the physicalmap (40) or by PCR-based screening of cv. Morex BAC library (41). Presenceof previously unobserved overlaps was surveyed by (i) reassembly of all BACsbelonging to the identified contigs with software FPC (42) at Sulston cutoffe-10 as well as (ii) sequencing a minimum tiling path (MTP) of BACs of therespective physical contigs. BAC sequencing was performed on Roche/454 GSFLX Titanium (Roche Applied Science) and 454-shotgun reads were assem-bled using the software MIRA version 3.2.1 essentially as described previously(40). Sequence overlaps between BACs were confirmed by PCR amplificationand inspected by sequence comparison (BLASTn) (43) and dot-plot analysis(Dotter) (44).

Candidate Gene Identification. Repetitive elements of BAC clone sequenceswere masked by applying K-mer statistics (45). Nonmasked sequences werecompared by BLASTn analysis to barley high-confidence genes (40), barleyfull-length cDNAs (46), HarvEST_U35 (47), as well as annotated gene sets ofthe sequenced grass species rice (48), Brachypodium (49) and sorghum (50).

Putative ORFs of potential candidate genes were amplified. The ampliconswere purified by using NucleoFast 96 PCR kit (Macherey-Nagel) and thensequenced by using ABI-3730xl technology (Applied Biosystems).

Expression Analysis. RNA extraction, first-strand cDNA synthesis, and RT-PCRwere performed as described previously (51). The young leaves of seedlingsat three-leaf stage or BaMMV-inoculated leaves 5 wk postinoculation weresampled for total RNA extraction. The barley UBIQUITIN CARRIER gene (NCBIaccession no. AK361071, HvUBC) was used as endogenous constitutivelyexpressed control (51).

TILLING for Chemically Induced Mutants. A large ethyl methanesulfonate(EMS)-induced mutant population of BaMMV-susceptible cv. Barke, com-prising 10,279 M2 families (21), was screened for induced mutations in thegene HvPDIL5-1 by TILLING (21). Two amplicons separately covered the firstthree and the last exon of the gene (SI Appendix, Table S7), respectively. Toidentify potential mutants, heteroduplex analysis of PCR amplicons wasperformed applying the Mutation Discovery kit and gel-dsDNA reagent kit,and samples were subsequently run on an Advance FS96 system (AdvancedAnalytical). To verify the identified mutations, the gene was subsequentlyamplified from identified M2 individuals and PCR amplicons were Sangersequenced. All M2 individuals with nonsynonymous mutations were propa-gated (SI Appendix, Table S1) for resistance tests.

Allele Mining. For the identification of naturally occurring allelic diversity,the full-length coding sequence of HvPDIL5-1 was analyzed by PCR andamplicon sequencing. Four primer pairs were designed to cover the en-tire length of 1,974-bp of HvPDIL5-1 genomic sequence (SI Appendix,Table S7). PCR products were amplified, purified, and sequenced as de-scribed above. Sequence alignment and assembly was performed withSequencher 4.7 (Gene Codes), and allelic haplotypes and polymorphicloci were defined by DNASP 5.10.01 (52). A MJ network was constructedwith DNA Alignment 1.3.1.1, Network 4.6.1.1, and Network Publisher1.3.0.0 software (Fluxus Technology).

Agrobacterium-Mediated Transformation. A 576-bp cDNA fragment containingthe entire ORF of wild-typeHvPDIL5-1 amplified from cv. Naturel was cloned intothe binary vector pIPKb002 (53) to generate ZmUbi_PDIL5-1_ox. The obtaineddestination plasmid was transformed into Agrobacterium tumefaciens strainAGL-1 (54). Isolation and culture of immature embryos, Agrobacterium-medi-ated transformation and regeneration of transgenic barley plants were con-ducted on the resistance parent W757/612 as previously described (54), withminor modifications. Prior to inoculation with Agrobacterium immature em-bryos were cultured on BCIMwith dicamba at a final concentration of 5 mg/L forfive days at 24 °C in the dark. Coculture of pre-treated immature embryos tookplace on stacks on moistened (300 μL BCCM) filter paper in 5.5 cm diameter petridishes. Due to absence of transcript of the endogenous copy of HvPDIL5-1 inW757/612, transcription of the transgene could be monitored by RT-PCR.

Phylogenetic Analysis of PDIL5-1. Homology search of HvPDIL5-1 in otherspecies was performed by BLASTx and tBLASTx according to the proteinsimilarity. All homologs were requested to contain the conserved functionaldomain (TRX domain) and active center (Cys-x-x-Cys) by Conserved DomainDatabase (55). Multiple sequence alignments of all homologous proteins inmonocots, dicots, and mammals were performed by the online software toolClustalW2 (56). A neighbor-joining phylogenetic tree was produced basedon deduced amino acid sequences by MEGA 5.05 (57).

Three-dimensional Structure Simulation and Conserved Sequence Motif Analysis.Sequence conservation of the active center and the C′-terminal tetrapep-tide of the homologous proteins were graphically represented by WebLogo(58). A 3D model of HvPDIL5-1 and the homologous proteins was simulatedon SWISS-MODEL workspace (59) by using the 3D structure of human ERp18(PDB accession no. 2k8vA) (24) as a reference.

Resistance Test. Tests for resistance to BaMMV and BaYMV were performedunder controlled growth chamber and field conditions as described pre-viously (17, 20). Tilling mutants (M3 families, M4 progeny of homozygouswild-type and mutant M3 plants), transgenic T1 plants, and F1 hybrids wereonly tested under growth chamber conditions. In brief, plants at three-leaves stage were mechanically inoculated twice with isolate BaMMV-ASL1(60) at an interval of 5–7 d. Five weeks after first inoculation, infected plantswere scored for the presence of mosaic symptoms and virus particles weredetected by DAS-ELISA with BaMMV-specific antibodies. Genotype ‘Maris

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Otter’ was used as susceptible control to monitor the infection rate in eachexperiment and showed infection rates between 70% and higher than 90%,as reported previously (17). Accessions from the diversity panel showingnonfunctional HvPDIL5-1 alleles were analyzed by mechanical inoculation ofBaMMV. In parallel, these accessions (30 plants each) were cultivated inQuedlinburg, Germany, under field conditions providing natural infectionwith BaMMV and BaYMV. The pathological responses were subsequentlydetermined by scoring of symptoms. Furthermore, leaf samples pooled from15 plants per accession were tested by DAS-ELISA diagnostics with BaMMVand BaYMV-specific antibodies.

ACKNOWLEDGMENTS. We gratefully acknowledge J. Perovic, M. Ziems, J. Pohl,E. Heike, S. König, I. Walde, C. Bollmann, K. Wolf (Leibniz Institute of PlantGenetics and Crop Plant Research, IPK), and D. Grau (Julius Kuehn Institute,JKI) for excellent technical support; Dr. F. Rabenstein (JKI) for providing anti-bodies of BaYMV and BaMMV; Dr. A. Walther (University of Gothenburg) forassistance with generating GIS-based topographical maps; D. Stengel for se-quence submission; Drs. A. Graner, P. Schweizer, H. Knüpffer (IPK), andT. Komatsuda (National Institute of Agricultural Sciences) for helpful discussions;Dr. R. Waugh (James Hutton Institute) for critical reading and commenting onthis manuscript. The work was financially supported by grants from the GermanMinistry of Education and Research (BMBF) (to N.S. and F.O.) (Projects “GABI-BARLEX” FKZ 0314000 and “Plant KBBE II-ViReCrop” FKZ 0315708).

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