The barley leaf rust resistance gene Rph3 encodes a putative executor protein

Host resistance is considered the most effective means to control plant diseases; however, individually deployed resistance genes are often rapidly overcome by pathogen adaptation. Combining multiple effective resistance genes is the optimal approach to durable resistance, but the lack of functional markers for resistance genes has hampered implementation. Leaf rust, caused by Puccinia hordei , is an economically significant disease of barley, 36 but only a few major Resistance genes to P. hordei ( Rph ) have been cloned. In this study, gene Rph3 was isolated 37 by positional cloning and confirmed by mutational analysis and transgenic complementation. The Rph3 gene, 38 which originated from wild barley and was first introgressed into cultivated Egyptian germplasm, encodes a 39 unique transmembrane resistance protein that differs from all known plant disease resistance proteins at the 40 amino acid sequence level. Genetic profiles of diverse accessions indicated limited genetic diversity in Rph3 in 41 domesticated germplasm, and higher diversity in wild barley from the Eastern Mediterranean region. Expression 42 profiling using P. hordei isolates with contrasting pathogenicity for the Rph3 host locus showed that the Rph3 43 gene was expressed only in interactions with Rph3 -avirulent isolates, a phenomenon also observed for 44 transcription activator-like effector-dependent genes known as executors conferring resistance to Xanthomonas 45 spp. Like the known transmembrane executors such as Bs3 and Xa7 heterologous expression of Rph3 in N. 46 benthamiana induced a cell death response. Given that Rph3 shares several features with executor genes, it 47 seems likely that P. hordei contains effectors similar to the transcription activator-like effectors that target host 48 executor genes. The isolation of Rph3 highlights convergent evolutionary processes in diverse plant-pathogen 49 interaction systems, where similar defence mechanisms evolved independently in monocots and dicots and 50 provide evidence for executor genes in the Triticeae tribe. 51 genes (“R genes”) encode NLRs, and the detailed mechanism of resistance associated with them contains unknown factors 38 . A significant knowledge gap concerns other molecular partners involved in the process of signalling by NLR proteins. Discovering these signalling components could improve the breeding and engineering of crops for disease resistance. Also, a more comprehensive understanding of the repertoire of plant resistance genes will enhance knowledge of plant-pathogen defence biology and facilitate diversification of strategies for disease control. In this study, we isolated the leaf rust resistance gene Rph3 in barley by positional cloning and mutagenesis. Rph3 encodes a putative transmembrane protein with no homology at mino acid level to any plant disease resistance gene isolated to date. We investigated the mechanism underlying this resistance gene and show that Rph3 is expressed only after a challenge by rust isolates containing the corresponding AvrRph3 gene. The Rph3 gene was sufficient to provide resistance to P. hordei and expression of the Rph3 gene causes cell death in barley and Nicotiana benthamiana . These results provide evidence for the existence of ‘executor’ genes in the Triticeae. a resistant response to the Rph3 -avirulent pathotype based on a specific marker detecting the Rph3 resistance allele. The transgenic experiments demonstrated that ORF2 complemented the lack of Rph3 in cv. Golden Promise. Taken together, high-resolution and physical delimitation, four independent mutants, and complementation results demonstrated that ORF2 was Rph3 . by P. avirulent Rph3 The Rph3 . Three additional haplotypes were identified as Hap2: WBDC094 and WBDC254; Hap3: WBDC238 and WBDC260; and Hap4: WBDC044. The identification of multiple sequence variations within wild barley suggests that additional allelic variants of Rph3 may exist. These findings also indicated that the Rph3 gene likely originated from wild barley in Israel, Syria, Jordan, or Greece, from which it was introgressed into cultivated barley germplasm. oryzae oryzae Bs3 and Bs4C conferring resistance X. campestris vesicatoria These Xanthomonas resistance genes are activated by corresponding transcriptional activator-like effectors (TALE) secreted by avirulent TALE-activated resistance genes designated “executor” genes they are involved in a plant immune response In this study, the Rph3 gene is only upon infection with an avirulent genotype of P. hordei . Like Rph3 , all currently cloned executor genes encode transmembrane proteins. The similarity in both expression profile and transmembrane domains suggests a similar resistance mechanism. We hypothesize that Rph3 -avirulent P. hordei pathotypes produce an effector, AvrRph3 , resistance protein 53,85 . Our study demonstrated that the RPH3 protein appears to cause cell death in barley and in the heterologous system N. benthamiana . The cell death can be directly prompted by Rph3 protein or indirectly via triggering a defence pathway. Previous work has shown that the executor genes ( Xa7 , Xa10 , Xa23 , Bs3 , and Bs4C ) trigger cell death in both their host (rice or pepper) and N. benthamiana . Although Xa27 was reported to triggers cell death only in rice 54 , we found that it does trigger cell death in N. benthamiana when driven by the MasΩ promoter, suggesting that expression level is essential for function. XA27 was found in apoplast, in endoplasmic reticulum RPH3 sequence of Rph3 and its most similar gene ORF10 ) imply ancient, independent origins. The Rph3 resistance allele was detected based on sequence analysis in wild barley accessions collected from the Eastern Mediterranean and Greece. The gene in modern cultivars originated from two donors, cv. Aim and landrace Estate, both of which are spring type, six-rowed, and came from Egypt 22,104 . The two lines are accessioned as HOR 2470 and HOR 2476 in the barley collection at IPK, and their pedigrees are unknown. The best explanation would be that the gene was introgressed into cultivated barley from wild barley in or around Egypt via hybridization. This hybridization could have been a result of deliberate crossing by a farmer/breeder to introduce a new beneficial allele or random outcrossing between a cultivar and wild relatives growing as a weed in the vicinity followed by deliberate selection by a farmer. It is impossible to separate these hypotheses due to the lack of information about the origin of both accessions. The sequence identity of Rph3 among all 41 resistant lines of cultivated barleys from diverse sources indicates a single introgression event. Of interest, the alleles Rph3.c , Rph3.aa , and Rph3.w were designated based on differing origins 57 all show identical specificity with Australian isolates of P. hordei and all were found to share 100% sequence identity. in Quantification of fungal biomass was performed by chitin Ayliffe et al. 106 Infected tissues from four biological replicates cv. Bowman to produce measurements fluorometer 485-nm adsorption, 535-nm emission wavelength and 1.0-sec measurement time. µM MyFi reaction Australia), and 20 ng of genomic DNA. Thermocycling conditions consisted C for 10 followed by 30 cycles of 94 o C for 30 seconds, 55-60 o C for 30 72 o C for 30 seconds, followed by a final extension at 72 o C for 10 minutes. PCR products were digested for three hours under The digested products were monitored by electrophoresis on an agarose gel and visualized by staining 6x GelRed® USA) (1.5 µl/100 ml agarose gel). first of the inoculated plants from the lines used were harvested at different time points with three biological replicates, flash-frozen in liquid nitrogen, stored at o C RNA extraction. RNA extracted samples using TRIzol TM Reagent (Thermo 719 Fisher Scientific Ltd) following the manufacturer’s instructions. The genomic DNA was digested using DNase I RNA checked on agarose 1.5% gels, reviewed on NanoDrop TM Spectrophotometer Fisher Scientific Ltd). RT-qPCR Luna® Universal One- Step RT-qPCR Kit (New England Biolabs®) instructions CFX TM Real-Time PCR Detection ADP-Ribosylation Factor ADPRF reference gene, RT-qPCR data analyzed Cq SI Table S17. Bioscience’s clonal gene synthesis service, using codon optimization for expression in N. benthamiana , of the Bsa I and Bpi I internal restriction sites. The coding sequences were cloned into the pTwist-Kan- High-copy vector, including two flanking Bsa I restriction sites for subsequent Golden Gate cloning. The resulting plasmids were used in the Golden Gate assembly with pICH85281 ( mannopine synthase + Ω promoter ( Mas Ω ), Addgene no. 50272), pICSL50009 (6xHA, TSL Synbio), pICSL60008 (Arabidopsis heat shock protein terminator, HSPter, TSL Synbio), and the binary vector pICH47732 (Addgene no. 48000). The Rph3 L93F and Rph3 P126L mutagenesis High-Fidelity DNA Polymerase Fisher), pTwist-Kan-High-copy::Rph3 WT primers flanking mutation Rph3_rv. full-length cloned pICSL01005 (TSL Synbio) PCR amplification of Rph3 E72* truncated primers Rph3_fw Rph3_E72*_rv (5’- resulting fragment was purified and used in a Golden Gate the pICSL01005 vector (TSL synbio). used for subsequent binary in a similar assembly reaction described for Rph3 WT


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Global food production is reduced by at least 10% by a wide range of microbial pathogens of plants 1,2 .

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Deployment of resistance genes has long been considered the most cost-effective and environmentally friendly 54 method to protect crops against pathogens 1,3,4 . However, the effectiveness of resistance genes is often limited 55 to a few years as pathogens evolve rapidly to acquire virulence that erodes or defeats genetic protection 5 . The 56 constant conflict between host plants and their pathogens shapes genetic diversity in both organisms. Rust 57 pathogens are obligate biotrophic fungi that can grow and reproduce only on living host tissues 6 . They cause 58 devastating losses in agricultural production worldwide 5,7 , and remain a major threat to cereal production 59 because of the ongoing evolution of virulence that overcomes genetic resistance and can lead to complete crop 60 loss in extreme epidemic situations 8 .

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To date, 106 loci conferring resistance to the leaf rust pathogens of wheat (Puccinia triticina) and barley (P. 63 hordei) have been formally catalogued 5 . Resistance alleles for only ten of these genes have been cloned with 64 six encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors 9-15 . The three remaining genes 4 genes ("R genes") encode NLRs, and the detailed mechanism of resistance associated with them contains 90 unknown factors 38 . A significant knowledge gap concerns other molecular partners involved in the process of 91 signalling by NLR proteins. Discovering these signalling components could improve the breeding and engineering 92 of crops for disease resistance. Also, a more comprehensive understanding of the repertoire of plant resistance 93 genes will enhance knowledge of plant-pathogen defence biology and facilitate diversification of strategies for 94 disease control.

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In this study, we isolated the leaf rust resistance gene Rph3 in barley by positional cloning and mutagenesis.

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Rph3 encodes a putative transmembrane protein with no homology at mino acid level to any plant disease 98 resistance gene isolated to date. We investigated the mechanism underlying this resistance gene and show that

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Rph3 is an incompletely dominant gene that confers resistance to P. hordei. Barley line BW746 105 (Bowman*11/Estate) is near-isogenic to cultivar (cv.) Bowman and carries the Rph3.c allele from the landrace 106 Estate. Having ten backcrosses to cv. Bowman, this line comprises more than 99% of the recipient cultivar 107 genome. Inoculation with P. hordei pathotypes 5453 P+ (AvrRph3), and 200 P-(AvrRph3) on seedlings showed 108 that cv. Bowman is susceptible, and BW746 is resistant to P. hordei pathotypes 5453 P+ (AvrRph3) (Fig. 1A) and 126 Scarlett (Rph3) and cv. Tallon (rph3) was used to investigate the chromosome region encompassing Rph3 (SI 127 Appendix, Table S2). The entire population was genotyped with markers previously reported near the Rph3 locus 128 in chromosome arm 7HL 40 . Tunable Genotyping-by-Sequencing (tGBS) was used on 42 representative RILs from 129 both resistant and susceptible phenotypic classes (21 lines for each) carrying recombinant chromosomes in the 6 delimitation of the Rph3 gene was carried out in cv. Barke that has been shown to carry the resistance allele 142 based on the multi-pathotype test in this study (SI Appendix, Table S3)

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Golden Promise. Taken together, high-resolution and physical delimitation, four independent mutants, and 182 complementation results demonstrated that ORF2 was Rph3.

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Rph3 is induced by P. hordei isolates avirulent for Rph3. The Rph3 transcript was not found in any published 185 barley RNAseq, full-length cDNA, or expressed sequence tag (EST) database. Transcript of Rph3 was detected in 186 leaves of resistant line BW746 inoculated with P. hordei pathotypes avirulent for Rph3 by RT-qPCR (Fig. 3). In

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A BLASTX search against the non-redundant database using the cDNA of Rph3 as a query returned seven hits 210 with different levels of identity. The 9-cis-epoxycarotenoid dioxygenase (HORVU_NCED) protein from barley 211 shares 46% identity with RPH3. Two sequences with similarity to RPH3 were retrieved from Aegilops tauschii,

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The phylogenetic relationship between the RPH3 protein and the four cereal homologs suggests that the RPH3 222 protein evolved the ability to confer resistance against P. hordei within barley after the divergence of wheat and     transport, such as vesicle-mediated (padj=8.4e-25) and protein transport (padj=5.2e-15). In contrast, enrichment 278 in down-regulated genes was localized to the plastid (padj=7.5e-36) and associated with photosynthesis 279 (padj=2.1e-4) (SI Appendix, Table S10). This indicates that Rph3-mediated resistance is correlated with up-280 regulation of endomembrane trafficking components, which might contribute to the immune response. to pathotype 5453 P+ (avirulent for Rph3) of P. hordei were the same (SI Appendix, Fig. SI15). Therefore, we 296 conclude that all of these stocks originated from the same ancestor and transcribed one unique isoform of Rph3.

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Analysis of GBS markers using a worldwide barley collection of 20,607 accessions identified a single paired GBS 299 marker landing on the Rph3 gene. This paired GBS marker (gRph3_I1E2 and gRph3_E2I2; SI Appendix, Table S13) 300 was detected in 134 accessions comprising 32 landraces, 70 cultivars, 14 breeding lines, 15 wild accessions, one 301 semi-wild accession, and two other genotypes (SI Appendix, Table S14). The landraces and breeding lines with 302 Rph3 were from many parts of the world, but the cultivars were mostly from Europe (especially Germany with

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The second subgroup consists of genes whose expression is induced exclusively in the presence of avirulent strains 334 or pathotypes. This phenomenon has been reported for genes conferring resistance to plant viruses, bacteria, and 335 fungi, and the barley gene Rph3 belongs in this sub-group.

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A particular induction of a resistance gene to an avirulent pathogen in the last sub-group has been observed in only

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Cloning studies have shown that non-durable resistance genes tend to be NLRs. The current study demonstrates 453 that other types of resistance genes are also vulnerable to evolving pathogens and that much remains to be 454 learnt about the durability of resistance genes. This study showed that Rph3 transcription is induced only by 455 avirulent P. hordei pathotypes. Rph3 encodes a transmembrane executor that induces host cell death in similar Quantification of fungal biomass in infected tissues. Quantification of fungal biomass was performed by chitin 504 measurement as described by Ayliffe et al. 106 . Infected leaf tissues from four biological replicates of cv. Bowman 505 and BW746 were harvested at 2, 4, and 8 dpi, weighed and placed in 15-ml Falcon tubes. One M KOH containing 506 0.1% Silwet L-77 (Lehle Seeds, U.S.A.) was added to cover the tissue entirely. After autoclaving, the tissues were 507 washed and neutralized as described in the "histological analysis" section. Subsequently, the liquid was poured     Table S4). The segregants were genotyped using DNA 549 markers flanking the Rph3 locus (MLOC_005 and MLOC_040). Progeny in which a recombination event had 550 occurred between these markers were further genotyped using internal DNA markers to define the recombination 551 site. All recombinants were self-pollinated to select homozygous recombinants using appropriate DNA markers, 552 and the homozygotes were challenged with Rph3-avirulent P. hordei pathotype 5453 P+ and were scored for rust 553 response based on our phenotyping platform to have unequivocal phenotypic data. Additionally, homozygous 554 recombinants scored for all internal DNA markers.

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An international barley collection of 78 accessions representing different sources and alleles of Rph3 based on 556 previous research was subjected to multi-pathotype tests to study the allelic variation (SI Appendix, Table S12).

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Each accession was initially been multiplied from a single seed to ensure genetic purity. Genotypic and 558 phenotypic data were collected from each pure line.      Table S5).

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Genetic diversity at the Rph3 locus amplify 4,882 bp were the same as described in the "DNA marker analysis" section but with the elongation step 644 lasting for 5 minutes instead of 30 seconds. The amplicons were purified using AMPure XP magnetic beads 645 (Beckman Coulter Life Sciences, USA). The sequencing template was subjected to Sanger sequencing using 28 646 internal primers (14 forward and 14 reverse primers) (SI Appendix, Table S11) that were designed from the 647 reference DNA sequence of cv. Barke.

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Fisher Scientific Ltd) following the manufacturer's instructions. The genomic DNA was digested using DNase I 720 (Sigma-Aldrich). RNA quality was checked on agarose 1.5% gels, and quantity was reviewed on a NanoDrop TM 721 1000 Spectrophotometer (Thermo Fisher Scientific Ltd). RT-qPCR was performed using Luna® Universal One-

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Step RT-qPCR Kit (New England Biolabs®) following instructions from the manufacturer and the CFX TM Real-Time

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PCR Detection System (Bio-RAD). ADP-Ribosylation Factor (ADPRF) was used as a reference gene, and RT-qPCR 724 data were analyzed using the ∆∆Cq method. Primer sequences for RT-qPCR are listed in SI Appendix, Table S17.

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The RNA samples were initially quantified using a NanoDrop TM 1000 spectrophotometer (Thermo Fisher

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Scientific Ltd), degradation and potential contamination were checked on 1.5% agarose gel, and RNA integrity 733 and quantitation were measured using an Agilent 2100 analyser. A library was prepared using the NEBNext®