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Polyploidization is prevalent in the evolutionary history of many important crop species (Glover et al., 2016). One example is bread wheat (Triticum aestivum L.), a staple crop for human consumption. This allohexaploid species arose from a primary hybridization between two diploid wild progenitors, Triticum urartu (AA genome) and Aegilops speltoides (BB genome), followed by a secondary hybridization with a third wild diploid species, Aegilops tauschii (DD genome) (International Wheat Genome Sequencing Consortium, 2018; Pont et al., 2019). Recent domestication and polyploid speciation have increased the adaptability of wheat to various environments/stresses, as well as its versatility for end-use in food products (Dubcovsky Jones Liu et al., 2021). Plant RNA viruses transmitted by the soil-borne plasmodiophorid Polymyxa graminis severely threaten globally important cereal crops, including wheat and barley (Hordeum vulgare L.) (Kanyuka et al., 2003; Jiang et al., 2020). Polymyxa graminis transmits the bymoviruses barley yellow mosaic virus (BaYMV), barley mild mosaic virus (BaMMV) – which cause barley yellow mosaic disease–and wheat yellow mosaic virus (WYMV) – which causes wheat yellow mosaic disease (Supporting Information Fig. S1). Most Bymovirus resistance loci in diploid barley are recessively inherited, whereas those found in polyploid wheat are generally dominant. In wheat, 46 resistance genes against fungal pathogens have been cloned (Hafeez et al., 2021), but no virus resistance gene has been isolated (Kühne, 2009; Jiang et al., 2020). We hypothesize that polyploidization has resulted in functionally redundant homoeologous genes that compensate for genetic deficiency in any single gene, thus blocking the identification of recessive resistance in hexaploid wheat. To test this hypothesis, we used the widely adopted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology (Gao, 2021) to edit the wheat orthologues of the barley susceptibility factor gene protein disulfide isomerase like 5-1 (HvPDIL5-1) (Methods S1). In barley, HvPDIL5-1 encodes for an endoplasmic reticulum-localized chaperone in the quality check system of correct protein folding, and loss of HvPDIL5-1 confers broad-spectrum resistance to multiple strains of the bymoviruses BaMMV and BaYMV (Yang et al., 2014). Our search for orthologues of HvPDIL5-1 in hexaploid wheat revealed three homoeologous genes: TaPDIL5-1-4A (TraesCS4A02G107900.1), TaPDIL5-1-4B (TraesCS4B02G196700.1), and TaPDIL5-1-4D (TraesCS4D02G197000.1) (Fig. 1a). We used CRISPR/Cas9-based genome editing to target a 20-bp fragment within the conserved region of these genes via Agrobacterium-mediated transformation of immature embryos of the wheat variety ‘Fielder’. This genotype is susceptible to WYMV, as well as to Chinese wheat mosaic virus (CWMV; genus Furovirus), another P. graminis-transmitted RNA virus occurring in China. Out of 84 regenerated T0 plants, 12, 11, and 12 plants showed editing at the target site in subgenomes 4A, 4B, and 4D, respectively, with editing efficiencies of 14.3%, 13.1%, and 14.3%, respectively (Table S1). Overall, editing was detected in 22 (26.2%) plants, and the majority of the editing events were deletions of 1–4 bp (Table S2). After crossing and marker-assisted selection and seed multiplication, we obtained four homozygous triple mutants (aabbdd-1, -2, -3, and -4) with frameshifts in the three homoeoalleles, as well as single and double mutants (Fig. S2a–c; Table S3). To examine whether TaPDIL5-1-edited wheat plants acquired virus resistance, four triple mutants were tested through mechanical inoculation with either WYMV or CWMV-infected leaves (Fig. 1b). The triple mutants stayed green, in contrast to the yellow discoloration of the wild-type plants at 6 wk postinoculation (Fig. 1c). No infection-induced oxidative burst was detected in these triple mutants by diaminobenzidine tetrahydrochloride staining (Fig. 1d). Using reverse transcription (RT)-PCR and quantitative RT-PCR to detect and quantify WYMV replication, respectively, the virus RNA was barely detectable (c. 10−5) in the triple mutants compared with the susceptible wild-type plants (Fig. 1e,f). For CWMV, no significant difference in the virus accumulation was observed between the triple mutants and the wild-type plants, both of which were considered susceptible to this virus (Fig. S3). These results demonstrate that triple editing of the three TaPDIL5-1 homoeoalleles was sufficient to achieve reliable resistance against WYMV in hexaploid wheat. The responses to WYMV inoculation were further tested in single, double, and triple knockout mutants. All the mutants except aabbdd-1 were susceptible to WYMV (Fig. 1g–j). The double mutants exhibited a statistically significant reduction of WYMV accumulation compared with that of the wild-type (Fig. 1j), implying that TaPDIL5-1 had subtle dosage effects on WYMV accumulation. The susceptibility of the single and double mutants to WYMV, compared with the resistance of the triple mutant, demonstrates the functional redundancy of the TaPDIL5-1 homoeologues and their ability to compensate for each other in their compatibility with virus infection. This explains why recessive resistance against bymoviruses had often been detected in diploid barley but not in polyploid wheat. Under natural conditions, several species in the genera Triticum and Aegilops were found to vary in the level of WYMV accumulation (Fig. S4), suggesting the occurrence of WYMV resistance in wheat progenitors and relatives. We further investigated the agronomic performance of the TaPDIL5-1-edited lines in garden experiments to determine whether editing TaPDIL5-1 homoeoalleles would cause pleiotropic effects. Nine traits were scored throughout the wheat life cycle. Two edited lines (aabbDD and AAbbDD) showed decreases in plant height, spike length, spike number per plant, and grain number per spike, along with a delayed heading date and an increase in spike density (Fig. 2a,b). The remaining lines, including the four triple edits and the remaining single and double knockouts, did not differ from the wild-type plants in any of the traits investigated. Since loss of the TaPDIL5-1 homoeoalleles did not affect agronomic performance, the simultaneous knockout of all three copies of this susceptibility factor gene can be used to improve virus resistance in wheat. Antiviral resistance mediated by clustered regularly interspaced short palindromic repeats can be achieved either by inducing targeted virus degradation (Aman et al., 2018) or by modifying viral compatibility by editing host susceptibility factors (Pyott et al., 2020). Both approaches have drawbacks: targeting specific sequences in the virus genome might drive the evolution of viruses that overcome resistance (Mehta et al., 2019), and editing susceptibility factors, such as members of the conserved eukaryotic initiation factor (eIF) family, can cause pleiotropic growth effects or lethality in plants (Macovei et al., 2018). This work achieved reliable WYMV resistance in wheat with no negative effects, through knocking out a dispensable host factor gene independent of the eIF complex. Furthermore, this study demonstrates a strategy to recover recessive resistance genes against viruses in an allopolyploid species by identifying the susceptibility genes in its diploid progenitors (e.g. T. urartu, A. speltoides, and A. tauschii) or relatives (e.g. Triticum monococcum and H. vulgare), followed by manipulation of their homoeologues in transformable varieties via genome editing. We would like to thank the Federal Ex situ Gene Bank (IPK Gatersleben) for providing the seeds of wild relatives, Dr Xingguo Ye and Dr Ke Wang (Chinese Academy of Agricultural Sciences) for assistance on wheat transformation, Zhentian He and Shiqiang Chen (Agricultural Sciences Institute in Jiangsu Lixiahe Area) for help on field trials of wheat relatives, and Simon G. Krattinger (King Abdullah University of Science and Technology) for critical comments. This work was funded by the National Key R JK, YC, CC and YJ carried out the experiments; JK, PY, YC and CJ analyzed the results; PY and JK wrote the manuscript with inputs from all authors. All authors read and approved the final manuscript. JK and YC contributed equally to this work. The authors declare that all the data involved have been provided in the main text or in the Supporting Information. The sequences of wheat cultivar ‘Chinese spring’ TaPDIL5-1 (TaPDIL5-1-4A, TraesCS4A02G107900.1; TaPDIL5-1-4B, TraesCS4B02G196700.1; TaPDIL5-1-4D, TraesCS4D02G197000.1) were publicly released in WheatOmics 1.0 (http://wheatomics.sdau.edu.cn/). The sequences of PCR primers used in this study are given in Table S3, and the methods are given in Methods S1. Fig. S1 Unrooted phylogenetic tree of the bymoviruses and the furoviruses infecting wheat and/or barley. Fig. S2 The generation and identification of single, double, and triple mutants of TaPDIL5-1. Fig. S3 Editing of all three TaPDIL5-1 homoeoalleles did not hamper Chinese wheat mosaic virus (CWMV) proliferation. Fig. S4 Reverse transcription PCR (RT-PCR) detection of WYMV accumulation in wheat progenitors and relatives. Methods S1 Materials and methods. Table S1 The detected editing events in 84 regenerated T0 plants by Sanger sequencing of plasmids of PCR products. Table S2 Summary of CRISPR/Cas9-guided genome editing in 84 regenerated T0 plants. Table S3 PCR primers used in this study. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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