PROTEIN DISULFIDE ISOMERASE LIKE 5-1 Is a Susceptibility Factor to Plant Viruses

PROTEIN DISULFIDE ISOMERASE LIKE 5-1 is a susceptibility factor to plant viruses

Ping 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 for Resistance Research and Stress Tolerance, Julius Kuehn Institute, Federal Research Centre for Cultivated Plants, D-06484 Quedlinburg, Germany; cPlant Reproductive 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 Plant Research, 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 of (10, 11). In addition, to establish replication and assembly com- proteins and prevent the aggregation of unfolded or partially plexes during infection, viruses typically create membrane-bound folded precursors. Whereas suppression of members of the PDI environments, referred to as “virus factories” (12). There, cellular gene family can delay replication of several human and animal chaperones such as HSP70 and DNAJ-like proteins likely contrib- viruses (e.g., HIV), their role in interactions with plant viruses is ute to the correct folding and translocation of substrates (12, 13). largely unknown. Here, using a positional cloning strategy we However, only a few such host factors are known (7, 9, 14). identified variants of PROTEIN DISULFIDE ISOMERASE LIKE 5–1 Barley yellow mosaic virus disease caused by barley yellow (HvPDIL5-1) as the cause of naturally occurring resistance to mul- mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) tiple strains of Bymoviruses. The role of wild-type HvPDIL5-1 in (both belong to Bymoviruses) seriously threatens winter barley conferring susceptibility was confirmed by targeting induced local production in Europe and East Asia (4). Infection leads to yel- lesions in genomes for induced mutant alleles, transgene-induced low discoloration and stunted growth and may result in yield loss complementation, and allelism tests using different natural resis- of up to 50% (15). Soil-borne transmission via the plasmodio- tance alleles. The geographical distribution of natural genetic var- phorid Polymyxa graminis prohibits plant protection by chemical iants of HvPDIL5-1 revealed the origin of resistance conferring measures, and breeding for resistant cultivars is therefore the alleles in domesticated barley in Eastern Asia. Higher sequence only practicable way to prevent yield loss. The naturally occurring recessive resistance locus rym11 confers complete broad- diversity was correlated with areas with increased pathogen di- spectrum resistance to all known European isolates of BaMMV versity suggesting adaptive selection for bymovirus resistance. and BaYMV (16–19). In the present study, we used a positional HvPDIL5-1 homologs are highly conserved across species of the cloning strategy to identify the gene underlying rym11-based re- plant and animal kingdoms implying that orthologs of HvPDIL5-1 PDI sistance. We show that it is a susceptibility factor belonging to or other closely related members of the gene family may be a gene family of PROTEIN DISULFIDE ISOMERASES (PDIs), potential susceptibility factors to viruses in other eukaryotic species. and is highly conserved across eukaryotic species. We observe a strong correlation between natural allelic variation and geographic allele mining | resistance breeding | soil-borne virus disease | chaperone distribution, suggesting that both the origin and subsequent

nfectious diseases caused by plant viruses threaten agricultural Significance Iproductivity and reduce globally attainable agricultural production by about 3% (1). In specific pathosystems, plant viruses This work describes a susceptibility factor to plant viruses that can result in the loss of the entire crop. For example, the dev- belongs to the conserved PROTEIN DISULFIDE ISOMERASE (PDI) astation of cassava production by cassava mosaic geminiviruses gene family. We show that loss-of-function HvPDIL5-1 alleles (CMGs) in Uganda during the 1990s led to widespread food at the recessive RESISTANCE TO YELLOW MOSAIC DISEASE 11 shortages and famine-related deaths (2). Unfortunately pro- (rym11) resistance locus confer broad-spectrum resistance to tecting plants against viruses (especially soil-borne viruses) by multiple strains of Bymoviruses and could therefore play a using agrochemicals to control virus vectors is seldom efficient central role in durable virus resistance breeding in barley. The from economic or ecological perspectives. Therefore, crop pro- geographic distribution of functional alleles of rym11 in East tection based on naturally occurring virus resistance is key to Asia suggests adaptive selection for resistance in this region. minimizing losses and achieving sustainable crop yields. Orthologues of HvPDIL5-1 or related members of the PDI gene Positive-strand RNA viruses represent the largest group of family potentially provide susceptibility factors to viruses across plant viruses (3). They cause a very high proportion of the important infectious virus diseases in agriculture (4, 5). Such plant animal and plant kingdoms. viruses carry a reduced genome that encodes a limited set of – — Author contributions: F.O. and N.S. designed research; T.L. mapped and identified the can- functional proteins (4 10 viral proteins) insufficient to com- didate gene HvPDIL5-1; P.Y., A. Habekuss, G.H., A. Himmelbach, and J.K. performed research; plete the entire virus replication and proliferation cycle (6). In- P.Y., T.L., B.S., B.K., R.A., and U.S. analyzed data; and P.Y. and N.S. wrote the paper. stead, over evolutionary time, viruses recruited host factors to Conflict of interest statement: A patent application has been filed relating to this work. perform the infectious life cycle (7). This dependence on host factors establishes a possibility that plants can evolve escape, tol- This article is a PNAS Direct Submission. erance, or resistance mechanisms to ameliorate the consequences Freely available online through the PNAS open access option. of viral infection. The absence of essential host factors could in- Data deposition: The sequences reported in this paper have been deposited in the EMBL terfere with the infection process or restrict proliferation (8) lead- database (accession nos. PRJEB4765 and HG793095). ing to either mono- or polygenic recessive resistance (5). Prominent 1Present address: The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, examples of such susceptibility factors that are conserved in mul- United Kingdom. tiple plant–virus systems are the EUKARYOTIC TRANSLATION 2F.O. and N.S. contributed equally to this work. INITIATION FACTORS (EIF)4E, iso4E, 4G, and iso4G (9). 3To whom correspondence should be addressed. E-mail: [email protected]. Translation initiation factors may interact directly with viral RNA This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. where they catalyze the initiation of translation of viral polyproteins 1073/pnas.1320362111/-/DCSupplemental.

2104–2109 | PNAS | February 11, 2014 | vol. 111 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1320362111 Downloaded by guest on September 24, 2021 was therefore a likely candidate for rym11-based resistance. The allele carrying the 1,375-bp deletion was named rym11-a.

Functional Validation of HvPDIL5-1 as a Susceptibility Factor for Bymovirus Infection. We used three independent approaches to validate the hypothesis that HvPDIL5-1 is a susceptibility factor for bymovirus infection. First, we screened an EMS-induced mutant population of barley by targeting induced local lesions in genomes (TILLING) (21). Two independent mutants 9699 and 10253 were identified each carrying a premature STOP codon in the coding sequence (CDS) of HvPDIL5-1 (SI Appendix, Table S1 and Fig. 1B). M3 progeny of the originally heterozygous M2 plants segregated for virus resistance. After phenotyping, the plants were genotyped for the presence of the mutation. All homozygous mutant M3 plants were found to be completely resistant. Homozygous wild-type and heterozygous M3 plants were predominantly susceptible with infection rates comparable to the susceptible controls (Table 1 and SI Appendix, Table S2). Re- sistance tests were replicated twice in M4 progeny of a homozygous mutant and a wild-type M3 plant of each of the mutant lines 9699 and 10253, respectively. All homozygous mutant M4 plants were tested resistant. Homozygous wild-type M4 progeny were susceptible, with infection rates between 66% and 100%, similar to susceptible controls (Table 1 and SI Appendix, Table S2 and Fig. S3). Mechanically infected homozygous wild-type HvPDIL5- 1 M4 progenies showed the characteristic yellow discoloration during vegetative growth stage (Fig. 1C), stunted growth at heading stage (Fig. 1D), as well as late maturation (Fig. 1E). Mock inoculated homozygous mutant M4 individuals did not show any detectable difference in overall growth habit at heading and Fig. 1. Map-based cloning of rym11.(A) Genetic and physical mapping of maturation stages compared with control plants (SI Appendix, rym11. The number below each marker indicates the recombination events between rym11 and the adjacent marker. The overlap of black-highlighted Fig. S4). We conclude that HvPDIL5-1 loss-of-function mutants BAC clones was confirmed by PCR amplification and dot-plot analysis together exhibit no deleterious growth effects while acquiring resistance (SI Appendix,Fig.S1). (B) Naturally occurring and induced alleles of rym11 sur- to bymovirus infection. veyed by resequencing and TILLING. The black boxes represent the functional Second, we amplified a 576-bp cDNA containing the 456-bp thioredoxin (TRX)-like domain. The phenotypes at the vegetative growth (C), full-length ORF of wild-type HvPDIL5-1 from barley cv. Naturel,

heading (D), and maturation (E) stages of homozygous M4 mutant (Left)and wild type (Right) of heterozygous M2 mutant 10253 are shown. Table 1. Functional validation of HvPDIL5-1 underlying rym11- based resistance by three independent approaches adaptive selection for rym11-based resistance in winter barley oc- Genotypes Tested Infected Comments curred in East Asia.

M3-9699-MT 3 0 M3, TILLING Results M3-9699-HET 10 8 M3, TILLING Map-Based Cloning of rym11. The resistance gene rym11 was pre- M -9699-WT 6 5 M , TILLING – 3 3 viously located to a 0.074-cM region on chromosome 4HL (18 M3-10253-MT 15 0 M3, TILLING 20). BAC contigs were identified by sequence comparison of M3-10253-HET 21 17 M3, TILLING

flanking markers and BAC library screening. Sequence analysis M3-10253-WT 11 9 M3, TILLING of the entire BAC contigs revealed an overlap of ∼18 kb between † M4-9699-MT 13 0 M4, TILLING BAC clones HVVMRXALLhA0173N06 and HVVMRXALL- † M4-9699-WT 15 13 M4, TILLING hA0172k23 residing on the original finger-printed contigs (FPCs) † M4-10253-MT 13 0 M4, TILLING ctg551 and ctg1996 (SI Appendix, Fig. S1), respectively. Thus, M -10253-WT† 18 13 M , TILLING a physical contig harboring rym11 and its flanking markers was 4 4 E1-OX* 49 30 T1, transgene analysis established. New markers were then developed and the genetic E1-rym11** 21 0 T , transgene analysis interval of rym11 narrowed to 0.0196 cM between markers 1 E2-OX* 48 45 T , transgene analysis C5B4C and C2B18S2 (Fig. 1A). This target interval was repre- 1 E2-rym11** 10 0 T , transgene analysis sented by 12 overlapping BAC clones (SI Appendix, Fig. S1 and 1 rym11-a + b 80F1, test for allelism Fig. 1A) spanning a distance of about 1.25 Mbp (Fig. 1A). An- + notation of the nonrepetitive sequences of this interval identified rym11-a c 11 0 F1, test for allelism rym11-a + d 14 0 F1, test for allelism four ORFs (Fig. 1A). This included two genes with sequence ‡ α W757/612 49 0 Resistant control homology to ELONGATION FACTOR 1- and one homolog ‡ of β3-GLUCURONYLTRANSFERASE 43. Resequencing of Barke 45 37 Susceptible control Igri‡ 26 22 Susceptible control these three genes revealed no polymorphisms between suscep- ‡ tible (Rym11) and resistant (rym11) genotypes. The fourth ORF Maris Otter 58 51 Susceptible control encoded a homolog of PROTEIN DISULFIDE ISOMERASE – WT, wild type; HET, heterozygous; MT, mutant; *OX, T1 plants carrying LIKE 5 1 (HvPDIL5-1). This gene contained a 1,375-bp de- the wild-type Rym11 transgene; ** T1 null-segregant plants for the trans- letion of the putative promoter region and the first three exons in gene; Res, resistance; Sus, susceptibility. † the resistant genotypes W757/112 and W757/612 (SI Appendix, Sum of two biological repeats. SCIENCES ‡ Fig. S2A). Due to this large deletion, no transcript from Sum of five independent experiments. For further details, see SI Appendix, AGRICULTURAL HvPDIL5-1 could be detected (SI Appendix,Fig.S2B). HvPDIL5-1 Table S2.

Yang et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2105 Downloaded by guest on September 24, 2021 present in all geographical regions (Fig. 3A). Median-joining (MJ) network analysis revealed that HvPDIL5-1 homologs from Brachypodium distachyon, Secale cereale, Triticum aestivum,and Hordeum bulbosum forming the outgroup, were most closely related to haplotype I in the H. vulgare lineage (SI Appendix, Fig. S6). Haplotype I is therefore most closely related to or represents the ancestral HvPDIL5-1 haplotype. The remaining haplotypes 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 (haplotype XXVIII). Twenty-seven accessions carried rym11-b (haplotype II) (SI Appendix, Table S4). All 28 accessions showed resistance upon natural and artificial virus infection (SI Appendix, Table S5). Two additional haplotypes (VII and VIII) encoded defective PDIL5-1 proteins. Haplotype VII carried a 1-bp deletion and Fig. 2. Median-joining (MJ) network analysis of naturally occurring hap- haplotype VIII exhibited a nonsynonymous substitution at base- lotypes of the gene HvPDIL5-1. Twenty-eight haplotypes were found in 1,732 pair position 182 of CDS (SI Appendix, Table S4 and Fig. 1B). accessions including four resistance-conferring alleles indicated as rym11-a, Accessions carrying either of these haplotypes conferred re- -b, -c, and -d, respectively. Circle size corresponds to the frequency of a sistance to BaMMV and BaYMV (SI Appendix,TableS5)and particular haplotype. Red, wild barley; light blue, landrace barley; dark blue, were named rym11-c and -d, respectively (Fig. 1B). F1 hybrids barley cultivar; yellow, H. vulgare ssp. agriocrithon. If not otherwise in- carrying rym11-a and either rym11-c or -d were resistant to dicated (XXVIII, rym11-a; II, rym11-b; V) the distance between haplotypes BaMMV (rym11-a + c, rym11-a + d, Table 1). All naturally oc- represents one nucleotide substitution or deletion. curring resistance alleles (rym11-a, -b, -c,and-d) were derived directly by single mutation events (deletion or substitution) of haplotype I (Fig. 2). Four haplotype-I–containing accessions were and used this to complement the resistant genotype W757/612 by tested for resistance by mechanical infection with BaMMV and Agrobacterium-mediated transformation with the intention to all were susceptible (SI Appendix, Table S5). Overall haplotype I induce susceptibility. W757/612 contains the recessive resistance allele rym11-a. Two independent transgenic events (T0) were obtained. T1 plants were mechanically infected, phenotyped, and subsequently 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 the presence of a single transgene locus (Table 1). Plants carrying the transgene were observed to be predominantly susceptible to BaMMV infection (Table 1 and SI Appendix, Table S2) whereas T1 null segregants were consistently resistant (Table 1 and SI Appendix, Table S2). Therefore, complementation of rym11- a with wild-type HvPDIL5-1 converted a resistant genotype into a susceptible one. Third we performed allelism tests between naturally occurring rym11 alleles. The breeding line Russia57 was previously reported to carry the recessive monogenic resistance locus rym11 (18). Resequencing HvPDIL5-1 from Russia57 revealed a 17-bp deletion causing a frameshift (henceforth named rym11-b,Fig.1B). F1 plants obtained by crossing W757/112 (rym11-a) and Russia57 (rym11-b) showed complete resistance to mechanical inoculation with BaMMV (Table 1 and SI Appendix,TableS2). Thus, both rym11-a and -b confer bymovirus resistance.

Natural Variation of HvPDIL5-1 and the Origin of rym11-Based Resistance. Natural variation of HvPDIL5-1 was surveyed by resequencing the entire ORF in a geographically referenced collection of 365 wild (Hordeum vulgare ssp. spontaneum)and 1,452 domesticated barley accessions (H. vulgare ssp. vulgare) (SI Appendix,TableS3). High-quality sequence was obtained from 1,732 accessions for 1,974 bp of genomic sequence including the complete ORF. We used the 456 bp of assembled CDS for calling exon-based sequence diversity. Overall, four deletions and 25 SNPs defined 28 independent exon haplotypes (SI Appendix,TableS4). Relative to haplotype I (i.e., Fig. 3. Geographic distribution of barley germplasm carrying different wild-type HvPDIL5-1 of cv. Naturel), nine haplotypes con- rym11 alleles and haplotypes. Geographic information system (GIS)-based topographical maps for the ancestral haplotype I and the combination of tained exclusively synonymous variants, whereas the remaining four resistance-conferring alleles (rym11-a, -b, -c, and -d) are shown. (A) 18 contained deletions or nonsynonymous variants, sometimes in Geographic distribution of accessions carrying the ancestral haplotype I. (B) combination with additional synonymous SNPs (SI Appendix, Distribution of accessions carrying either one of the resistance-conferring Table S4). Twenty-three haplotypes were exclusively observed in rym11 alleles. The size of filled cycles is proportional to the number of accessions originating from the Near East (SI Appendix, Fig. S5). accessions found at each particular site. For accessions without georeference Three haplotypes (I, III, and IV) were shared between wild and information the capital state of the source country was arbitrarily chosen as domesticated barleys (Fig. 2). Haplotype I was found in 1,607 of the collecting site, which is explaining circles in regions that do not represent the 1,732 accessions (92.8%) (SI Appendix,TableS3) and was barley cultivation area.

2106 | www.pnas.org/cgi/doi/10.1073/pnas.1320362111 Yang et al. Downloaded by guest on September 24, 2021 predominates among accessions of a worldwide diversity collection. We conclude that susceptibility to Bymovirus is the ancestral state. We propose that recessive rym11 resistance alleles most likely arose by selection of mutations in HvPDIL5-1 during the migration of domesticated barleys into new environments where naturally occurring Bymoviruses were prevalent. This proposal is supported by our observation that no resistance-conferring alleles were found in wild barleys (Fig. 2). Furthermore, only a single rym11-a carrying barley landrace HOR1363 originated from within 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.S5and Fig. 3B) where high BaMMV/BaYMV diversity has previously been observed (4, 22).

PDIL5-1 Is a Highly Conserved Protein in Plant and Animal Species. In many plants, recessive resistance to Potyviruses is controlled by natural variation in the highly conserved EUKARYOTIC TRANSLATION INITIATION FACTOR 4E (eIF4E) (9). To determine the level of PDIL5-1 conservation, we conducted a sequence comparison and phylogenetic analysis among putative functional orthologs from different plant and animal species. HvPDIL5-1 is a specific member of the PDI gene family and encodes 151 amino acids. This particular member carries a single functional thioredoxin (TRX) domain (SI Appendix, Table S6) putatively catalyzing the formation, reduction, and isomerization of disulfide bonds. It contains the C′-terminal tetrapeptide Fig. 4. Phylogenetic analysis of HvPDIL5-1.(A) Phylogenetic tree of HvPDIL5-1 EDEL, which controls the retention of the protein within the and 16 homologous proteins. (B) Three dimensional model of the homologous endoplasmic reticulum (ER), as could be demonstrated for the proteins in C. owczarzaki ATCC 30864, C. reinhardtii,andH. vulgare.Simulation of 3D protein structure was achieved based on sequence homology to human homologous human protein ERp16 (also called ERp18, ERp19, HsERp18 (PDB accession no. 2k8vA). or hLTP19) (23). Eighteen homologous genes in plants and 30 genes in animals were identified (SI Appendix, Table S6). A phylogenetic analysis for 17 PDI proteins of species from all act as cellular chaperone (protein folding, stabilization, or facili- evolutionary and organizational stages (e.g., unicellulate and tating transport) during virus infection. Other cellular chaperones multicellulate organisms, algae/primitive land plants/higher vas- cular plants, etc.) revealed a single rooted tree with a split be- like HSP70, HSP90, and DNAJ-like proteins have been shown to tween the animal and plant branch but otherwise consistent perform similar functions in other plant species in the inter- sorting according to the evolutionary level or organization (Fig. actions with several genera of plant viruses (12). Both functional 4A). Sequence similarity inside the TRX domain separated the motifs and tertiary structures of HvPDIL5-1 and its homologs in plant from the animal branch and diversity in the C′-terminal other plants and animals are highly conserved. By identifying region revealed differentiation between monocotyledonous and HvPDIL5-1 as susceptibility factor to the potyvirus-related Bymo- dicotyledonous plant species (SI Appendix, Figs. S7 and S8). viruses in barley, it seems plausible that other members of the PDI Furthermore, the majority of homologous proteins are predicted gene family may play a similar role in other plant–virus interactions. to be secreted to the ER (SI Appendix,TableS6). The 3D Given that the highly conserved eukaryotic translation initiation structure of the homologous proteins of a protist (Capsaspora factor gene family has been repeatedly identified as susceptibility owczarzaki), of a unicellulate algae (Chlamydomonas rein- factors in numerous pathosystems (9), we consider this a realis- hardtii ) and of barley were simulated in reference to the hu- tic possibility. man protein ERp18 (24). The predicted 3D structure was Members of plant PDIs have not been reported so far as sensu highly conserved across kingdoms (Fig. 4B). As PDIL5-1 homologs share structure, function, and subcellular localiza- stricto susceptibility factors to any plant viruses and little is known tion between organisms, it is tempting to speculate that some about plant PDI/virus interactions. However, virus-induced may similarly function as dominant virus susceptibility factors gene silencing (VIGS) of two PDI family members in tobacco, in organisms other than barley. NtERp57 and NtP5, partially decreased N-gene mediated resistance to tobacco mosaic virus (TMV) (29). Whereas this Discussion hinted at an interaction between plant PDIs and viruses, it did HvPDIL5-1 Is a Susceptibility Factor to Bymoviruses in Barley and It Is not indicate a role as a susceptibility factor. The situation is Highly Conserved Between Plants and Animals. We identified different in animals. PDI family members, particularly cell- a susceptibility factor to bymovirus disease in barley encoded surface PDI proteins, may participate in the infection process by HvPDIL5-1, a member of the PROTEIN DISULFIDE of multiple human and animal viruses, e.g., HIV (HIV) (30–36). ISOMERASE (PDI) gene family. The PDI gene family usually Nonspecific inhibition of PDI activity suppressed the PDI-mediated contains more than 10 members in different eukaryotic species; redox environment of plasma membranes (33) and therefore in- – e.g., there are 20 genes in Homo sapiens and 18 in rice (25 27). terfered with HIV envelope protein-directed cell fusions (32). The major function of PDIs in humans is to contribute to the Knockdown of specific PDI members could influence the infectivity formation of disulfide bonds by induction, oxidization, and isom- of several viruses, e.g., HIV, Newcastle disease virus (NDV) and erization; all catalyzed by the active TRX domain (25, 27). PDIs also play a role as chaperones in the quality check system for mouse polyomavirus (31, 32, 35). In human cell cultures, knock- correct protein folding (28). Based on its high amino acid sequence down of the PDI family members, PDI, ERp29, ERp57, and ERp72 conservation to human ERp16, which encodes for an ER-resident inhibited the accumulation of viral particles (30, 34, 36). Our data

thioldisulfide oxidoreductase fulfilling chaperone activities (23), we support and extend the role of PDIs as important host components SCIENCES

conclude that plant homologs have retained this function. Thus, required for the successful infection or replication of viruses in AGRICULTURAL HvPDIL5-1 could have been recruited by Bymoviruses in barley to animals to plants.

Yang et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2107 Downloaded by guest on September 24, 2021 Loss-of-Function Alleles Are Prevalent in Domesticated Barley Putative ORFs of potential candidate genes were amplified. The amplicons Accessions from Eastern Asia. Resequencing HvPDIL5-1 in a were purified by using NucleoFast 96 PCR kit (Macherey-Nagel) and then large georeferenced collection of wild and domesticated barleys sequenced by using ABI-3730xl technology (Applied Biosystems). revealed less haplotype diversity in domesticated (12 haplotypes) compared with wild barley (18 haplotypes) (although almost Expression Analysis. RNA extraction, first-strand cDNA synthesis, and RT-PCR fourfold more accessions of domesticated barley were surveyed). were performed as described previously (51). The young leaves of seedlings This is in strong contrast to the four naturally occurring loss-of- at three-leaf stage or BaMMV-inoculated leaves 5 wk postinoculation were function alleles of HvPDIL5-1. All four confer resistance to sampled for total RNA extraction. The barley UBIQUITIN CARRIER gene (NCBI Bymoviruses, and all four were exclusively observed in domesticated accession no. AK361071, HvUBC) was used as endogenous constitutively barleys. Geographically, resistant alleles were overrepresented in expressed control (51). winter-type barley from Eastern Asia (SI Appendix,Fig.S9), sug- TILLING for Chemically Induced Mutants. A large ethyl methanesulfonate gesting selection for resistance during range extension as barley (EMS)-induced mutant population of BaMMV-susceptible cv. Barke, com- cultivation spread east from its center of origin. This is con- prising 10,279 M2 families (21), was screened for induced mutations in the sistent with the higher diversity of Bymoviruses in Eastern Asia gene HvPDIL5-1 by TILLING (21). Two amplicons separately covered the first (4, 22) where a nonfunctional HvPDIL5-1 may have provided three and the last exon of the gene (SI Appendix, Table S7), respectively. To an adaptative advantage. identify potential mutants, heteroduplex analysis of PCR amplicons was performed applying the Mutation Discovery kit and gel-dsDNA reagent kit, Resistance Breeding Requires Access to Multiple Sources of Naturally and samples were subsequently run on an Advance FS96 system (Advanced Occurring Resistance. Barley yellow mosaic virus disease is one of Analytical). To verify the identified mutations, the gene was subsequently

the most important diseases in winter barley (15). Its soil-borne amplified from identified M2 individuals and PCR amplicons were Sanger transmission precludes plant protection by use of agrochemicals. sequenced. All M2 individuals with nonsynonymous mutations were propa- In European barley breeding this stimulated the extensive use of gated (SI Appendix, Table S1) for resistance tests. rym4/rym5, a single natural recessive resistance conferred by alleles of the EUKARYOTIC TRANSLATION INITIATION Allele Mining. For the identification of naturally occurring allelic diversity, FACTOR 4E (37). However, rym4/rym5 resistance-breaking the full-length coding sequence of HvPDIL5-1 was analyzed by PCR and isolates have recently been observed (16, 17, 38). rym11 is ef- amplicon sequencing. Four primer pairs were designed to cover the en- fective against all European strains of BaMMV/BaYMV (16– tire length of 1,974-bp of HvPDIL5-1 genomic sequence (SI Appendix, 18). We have shown here that resistance is conferred by loss-of- Table S7). PCR products were amplified, purified, and sequenced as de- function mutations in HvPDIL5-1. HvPDIL5-1 must therefore scribed above. Sequence alignment and assembly was performed with provide a host component that is essential for virus establish- Sequencher 4.7 (Gene Codes), and allelic haplotypes and polymorphic ment and/or replication in planta. To overcome rym11-based loci were defined by DNASP 5.10.01 (52). A MJ network was constructed resistance the virus would therefore need to establish an in- with DNA Alignment 1.3.1.1, Network 4.6.1.1, and Network Publisher 1.3.0.0 software (Fluxus Technology). teraction with an alternative and functionally redundant host component. Today, rym11 plays a minor role in European barley Agrobacterium-Mediated Transformation. A 576-bp cDNA fragment containing breeding. Its implementation may, however, reduce the potential the entire ORF of wild-type HvPDIL5-1 amplified from cv. Naturel was cloned into for the emergence of resistance-breaking viral isolates. Pyr- the binary vector pIPKb002 (53) to generate ZmUbi_PDIL5-1_ox. The obtained amiding several resistance genes in a single genotype is consid- destination plasmid was transformed into Agrobacterium tumefaciens strain ered to be a promising strategy to obtain durable virus resistance AGL-1 (54). Isolation and culture of immature embryos, Agrobacterium-medi- (39). With the identification of rym11 as loss-of-function alleles of ated transformation and regeneration of transgenic barley plants were con- HvPDIL5-1, it is now possible to combine two recessive re- ducted on the resistance parent W757/612 as previously described (54), with sistance loci (rym11 and rym4/rym5)onthebasisofperfectly minor modifications. Prior to inoculation with Agrobacterium immature em- linked and diagnostic molecular markers. bryos were cultured on BCIM with dicamba at a final concentration of 5 mg/L for five days at 24 °C in the dark. Coculture of pre-treated immature embryos took Materials and Methods place on stacks on moistened (300 μL BCCM) filter paper in 5.5 cm diameter petri Plant Material. High-resolution genetic mapping of rym11 was conducted in dishes. Due to absence of transcript of the endogenous copy of HvPDIL5-1 in

5,102 F2 individuals of the cross cv. Naturel (Rym11/Rym11) × resistant ge- W757/612, transcription of the transgene could be monitored by RT-PCR. notype W757/112 and W757/612 (both rym11/rym11) (20). Eighteen recombinants of this population were used for further genetic and physical Phylogenetic Analysis of PDIL5-1. Homology search of HvPDIL5-1 in other delimitation of rym11. For allele mining, 1,816 georeferenced accessions species was performed by BLASTx and tBLASTx according to the protein from 70 countries including 365 wild and 1,451 domesticated barleys were similarity. All homologs were requested to contain the conserved functional

selected (SI Appendix, Table S3). F1 hybrids were obtained by crossing the domain (TRX domain) and active center (Cys-x-x-Cys) by Conserved Domain resistant genotypes W757/112 and/or W757/612 with the newly identified Database (55). Multiple sequence alignments of all homologous proteins in gene haplotypes. monocots, dicots, and mammals were performed by the online software tool ClustalW2 (56). A neighbor-joining phylogenetic tree was produced based Physical Mapping. BAC contigs were identified either by sequence comparison on deduced amino acid sequences by MEGA 5.05 (57). of flanking markers (20) to sequence resources integrated to the physical map (40) or by PCR-based screening of cv. Morex BAC library (41). Presence Three-dimensional Structure Simulation and Conserved Sequence Motif Analysis. of previously unobserved overlaps was surveyed by (i) reassembly of all BACs Sequence conservation of the active center and the C′-terminal tetrapep- belonging to the identified contigs with software FPC (42) at Sulston cutoff tide of the homologous proteins were graphically represented by WebLogo e-10 as well as (ii) sequencing a minimum tiling path (MTP) of BACs of the (58). A 3D model of HvPDIL5-1 and the homologous proteins was simulated respective physical contigs. BAC sequencing was performed on Roche/454 GS on SWISS-MODEL workspace (59) by using the 3D structure of human ERp18 FLX Titanium (Roche Applied Science) and 454-shotgun reads were assem- (PDB accession no. 2k8vA) (24) as a reference. bled using the software MIRA version 3.2.1 essentially as described previously (40). Sequence overlaps between BACs were confirmed by PCR amplification Resistance Test. Tests for resistance to BaMMV and BaYMV were performed and inspected by sequence comparison (BLASTn) (43) and dot-plot analysis under controlled growth chamber and field conditions as described pre-

(Dotter) (44). viously (17, 20). Tilling mutants (M3 families, M4 progeny of homozygous wild-type and mutant M3 plants), transgenic T1 plants, and F1 hybrids were Candidate Gene Identification. Repetitive elements of BAC clone sequences only tested under growth chamber conditions. In brief, plants at three- were masked by applying K-mer statistics (45). Nonmasked sequences were leaves stage were mechanically inoculated twice with isolate BaMMV-ASL1 compared by BLASTn analysis to barley high-confidence genes (40), barley (60) at an interval of 5–7 d. Five weeks after first inoculation, infected plants full-length cDNAs (46), HarvEST_U35 (47), as well as annotated gene sets of were scored for the presence of mosaic symptoms and virus particles were the sequenced grass species rice (48), Brachypodium (49) and sorghum (50). detected by DAS-ELISA with BaMMV-specific antibodies. Genotype ‘Maris

2108 | www.pnas.org/cgi/doi/10.1073/pnas.1320362111 Yang et al. Downloaded by guest on September 24, 2021 Otter’ was used as susceptible control to monitor the infection rate in each ACKNOWLEDGMENTS. We gratefully acknowledge J. Perovic, M. Ziems, J. Pohl, experiment and showed infection rates between 70% and higher than 90%, E. Heike, S. König, I. Walde, C. Bollmann, K. Wolf (Leibniz Institute of Plant Genetics and Crop Plant Research, IPK), and D. Grau (Julius Kuehn Institute, as reported previously (17). Accessions from the diversity panel showing JKI) for excellent technical support; Dr. F. Rabenstein (JKI) for providing anti- nonfunctional HvPDIL5-1 alleles were analyzed by mechanical inoculation of bodies of BaYMV and BaMMV; Dr. A. Walther (University of Gothenburg) for BaMMV. In parallel, these accessions (30 plants each) were cultivated in assistance with generating GIS-based topographical maps; D. Stengel for se- Quedlinburg, Germany, under field conditions providing natural infection quence submission; Drs. A. Graner, P. Schweizer, H. Knüpffer (IPK), and with BaMMV and BaYMV. The pathological responses were subsequently T. Komatsuda (National Institute of Agricultural Sciences) for helpful discussions; Dr. R. Waugh (James Hutton Institute) for critical reading and commenting on determined by scoring of symptoms. Furthermore, leaf samples pooled from this manuscript. The work was financially supported by grants from the German 15 plants per accession were tested by DAS-ELISA diagnostics with BaMMV Ministry of Education and Research (BMBF) (to N.S. and F.O.) (Projects “GABI- and BaYMV-specific antibodies. BARLEX” FKZ 0314000 and “Plant KBBE II-ViReCrop” FKZ 0315708).

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