- Open Access
Evolutionarily conserved partial gene duplication in the Triticeae tribe of grasses confers pathogen resistance
- Jeyaraman Rajaraman1Email authorView ORCID ID profile,
- Dimitar Douchkov1Email author,
- Stefanie Lück1,
- Götz Hensel1,
- Daniela Nowara1,
- Maria Pogoda1,
- Twan Rutten1,
- Tobias Meitzel1,
- Jonathan Brassac1,
- Caroline Höfle2,
- Ralph Hückelhoven2,
- Jörn Klinkenberg3,
- Marco Trujillo3, 6,
- Eva Bauer4,
- Thomas Schmutzer1,
- Axel Himmelbach1,
- Martin Mascher1,
- Barbara Lazzari5,
- Nils Stein1,
- Jochen Kumlehn1 and
- Patrick Schweizer^1
© The Author(s). 2018
- Received: 11 December 2017
- Accepted: 4 July 2018
- Published: 15 August 2018
The large and highly repetitive genomes of the cultivated species Hordeum vulgare (barley), Triticum aestivum (wheat), and Secale cereale (rye) belonging to the Triticeae tribe of grasses appear to be particularly rich in gene-like sequences including partial duplicates. Most of them have been classified as putative pseudogenes. In this study we employ transient and stable gene silencing- and over-expression systems in barley to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15).
The partial ARM1 gene is derived from a gene-duplication event in a common ancestor of the Triticeae and contributes to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis. In barley, allelic variants of HvARM1 but not of HvPUB15 are significantly associated with levels of powdery mildew infection. Both HvPUB15 and HvARM1 proteins interact in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. A genome-wide scan for partial gene duplicates reveals further events in barley resulting in stress-regulated, potentially neo-functionalized, genes.
The results suggest neo-functionalization of the partial gene copy HvARM1 increases resistance against powdery mildew infection. It further links plastid function with susceptibility to biotrophic pathogen attack. These findings shed new light on a novel mechanism to employ partial duplication of protein-protein interaction domains to facilitate the expansion of immune signaling networks.
- Partial gene duplication
- Disease resistance
- Triticeae grasses
Plants respond to pathogen attack by the activation of their innate immunity system, which is triggered by the perception of pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors . Successful plant pathogens manipulate their hosts by complex arsenals of secreted effector proteins, which suppress immunity and co-opt cellular host functions for accommodation and nutritional exploitation . Nonhost plant species, on the other hand, exhibit nonhost resistance (NHR), which protects them from the vast majority of attacks by pathogens that have adapted to different, more or less closely related, plant species . The outcome of pathogen-host interactions can vary from immune to highly susceptible, depending on the presence or absence of major resistance genes or on different levels of quantitative host resistance (QR). QR is usually determined by several quantitative trait loci (QTL) and may be partially explained by a manifestation of PAMP-triggered immunity (PTI). In contrast to effector-triggered immunity (ETI), it does not confer complete protection but may be more durable in the field [4–6].
All forms of host resistance are temporary results of the co-evolutionary arms race between host plants and their adapted pathogens. As such, pathogens evolve quickly and put enormous selection pressure on host genomes to keep pace with changing virulences [7–10], thereby resulting in strong selective pressure on new resistance and defense genes or gene variants. Besides alternative splicing, gene duplication is an efficient way to create novelty in genomes and is routinely observed for ETI-mediating nucleotide binding domain-leucine rich repeat domain (NB-LRR)-type major resistance genes as well as receptor-like kinases in pairs or clusters of tandem duplicates [11, 12]. Genes can be duplicated as complete or partial copies. In humans, partially duplicated genes have been recognized as a major cause of disease including different forms of cancer [13–15]. The possible contribution of partial gene duplicates to positive traits such as disease resistance is much less examined in animal genetics and apparently unexplored in plants [16–18]. The occurrence of partial gene copies is particularly relevant to the large and highly repetitive genomes of the Triticeae tribe of grasses including Hordeum vulgare ssp. vulgare (cultivated barley), Triticum aestivum (bread wheat), and Secale cereale (rye), which were described to be particularly rich in gene-like sequences including partial duplicates, most of which were classified as putative pseudogenes [19–21].
Cultivated barley is nonhost to the non-adapted wheat powdery mildew fungus Blumeria graminis f.sp. tritici (Bgt) but a host of the powdery mildew fungus B. graminis f.sp. hordei (Bgh), which causes up to 30% yield loss in the absence of genetic or chemical control of the disease [22, 23]. The epidemic spread of B. graminis is caused by the asexual propagation of the fungus, with a generation time of 5–7 days and massive production of conidiospores (Additional file 1: Figure S1). The interaction between different barley genotypes and Bgh isolates represents a well-studied model system for a fungal disease caused by an obligate biotrophic pathogen, and a growing number of host-response factors for defense or disease establishment have been identified [24–26]. The genome of Bgh was found to encode more than 500 candidate secreted effector proteins . In several pathogens, a growing number of effectors were found to target components of the plant ubiquitination machinery including plant U-box E3 ligases (PUBs) [28–34]. The covalent attachment of single ubiquitin moieties or polyubiquitin chains to lysine residues of eukaryotic protein substrates can have diverse effects on their fate. Ubiquitination most commonly results in the recognition and degradation of tagged proteins by the 26S proteasome, but it also mediates endosomal sorting into cellular compartments such as the lysosome or the plant vacuole, or contributes to DNA damage responses [35, 36]. The substrate specificity during ubiquitination is determined by the E3 ubiquitin ligases which can be subdivided into three categories, namely HECT, RING/U-box type, and cullin-RING ligases. These proteins mediate ubiquitin ligation in concert with the highly conserved ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2). Due to their central cellular function, components of the ubiquitination system represent central cellular hubs of protein regulation involved in all aspects of plant life. As such, beneficial or parasitic organisms may utilize the ubiquitination machinery  to establish susceptible interactions. On the other hand, higher plants exploit ubiquitin-mediated degradation of negative protein regulators of stress-hormone signaling for the initiation of defense responses [36–38].
In a phenotype-driven, transient RNA interference (RNAi) screen for the discovery of Rnr (for Required for nonhost resistance) genes to the non-adapted wheat powdery mildew fungus B. graminis f.sp. tritici (Bgt), we tested more than 631 barley genes, which were mostly associated with up-regulated transcripts in Bgt-attacked barley leaf epidermis . Reduced NHR was reflected by an increased percentage of transformed epidermal cells containing Bgt haustoria. This revealed 10 final Rnr gene candidates that significantly enhanced nonhost susceptibility upon silencing. Here we present an extensive evolutionary, genomics, and molecular functional study of the neo-functionalized partial gene copy Rnr5 encoding HvARM1 with homology to plant U-box protein 15 (HvPUB15). Besides its possible role in NHR that allowed its discovery in the NHR RNAi screening, functional analysis suggested Rnr5 as an important factor of QR against the adapted Bgh fungus.
Origin and evolution of HvARM1
Sequence overview of HvPUB15 and its partial duplicate HvARM1 in the barley genome
cDNA clone ID
HarvEST assembly #35 unigene Nr.
Full-coding sequence cDNA Acc. Nr.
Morex WGS contig Acc. Nr.a
Barke WGS contig Acc. Nr.a
Bowman WGS contig Acc. Nr.a
High-confidence barley gene IDa
Syntenic to Brachypodium distachyon, Oryza sativa, Sorghum bicolor
Syntenic to Ae. tauschii c
Allelic variants of HvARM1
The phylogenetic and functional (see below) data of ARM1 suggest that the gene is under selection for maintaining a quantitative level of resistance among Triticeae species to powdery mildew infection. We therefore analyzed gene variants (alleles) in a diverse collection of barley genotypes (Additional file 2: Tables S1–S3) for significant association with the severity of powdery mildew infection. Table 2 shows significantly associated single-nucleotide polymorphisms (SNPs) as well as gene-haplotype polymorphisms in two diverse, worldwide collections of barley landraces and cultivars. No association of HvPUB15 gene variants with the same trait was found in these populations. This result supports the view that HvARM1 — despite its partial nature — represents a functional gene protecting barley from powdery mildew attack, whereas the cellular functions of HvPUB15 may be more complex.
Function of HvARM1 during powdery mildew attack
Because HvARM1 was discovered in a TIGS screen for attenuated NHR , we first tested T3 progeny of three selected events for susceptibility to Bgt (Additional file 1: Figure S4c). Although there was a considerable variability between individuals per line, two lines exhibited higher susceptibility to the non-adapted fungus as compared to the control group of azygous segregant plants. In general, azygous plants are considered as better controls since they have undergone the same transformation procedures and lost the transgenic construct by segregation. Indeed, the transformation procedure had an impact on the Bgt interaction because the azygous control group was on average more susceptible than the Golden Promise wild type. Figure 2b additionally shows that the three selected events were also more susceptible to Bgh, compared to a population of control plants consisting of azygous segregants plus progeny from three azygous individuals identified in the T2 generation.
Bombardment with gene-specific RNAi and with over-expression (OEX) constructs for a direct comparison of altered HvARM1 versus HvPUB15 expression levels revealed that gene-specific HvARM1 silencing increased the relative susceptibility index (SI) to Bgh (Table 3), in line with the enhanced susceptibility observed in stable transgenic barley T3 plants. On the other hand, we found no significant effects of altering HvPUB15 mRNA levels on the interaction of transformed cells with Bgh, again indicating more complex, homoeostatic rather than defense-related functions of the encoded protein. Following powdery mildew inoculation, endogenous transcript levels of HvARM1 in peeled leaf epidermis were more strongly up-regulated above a basal level of expression compared to HvPUB15 (Additional file 1: Figure S5), which also suggests a defense-related role of HvARM1.
Localization and protein interactions of HvPUB15 and HvARM1
Genome-wide search for expressed partial gene duplicates
At least six species of the Triticeae tribe of grasses possess ARM1, a partial gene copy of a U-box/ARM-repeat E3 ligase closely related to OsPUB15 of rice . The rice genome also contains a number of “ARM-repeat only” genes, but none of them appears to represent a partial copy of OsPUB15, because BlastN analysis of the ARM-repeat region of OsPUB15 (positions 2000–2952 in cDNA Acc. AK106557.1) at the National Center for Biotechnology Information (NCBI) did not produce significant hits for any other rice gene. By contrast, the same query sequence revealed HvARM1 as the most significant hit (86% identity) in barley. Sequence analysis in cultivated barley, wheat, and rye, and in three diploid wild wheat species suggest a monophyletic origin of ARM1. The large and highly repetitive genomes of the cultivated Triticeae species barley, wheat, and rye are known to be rich in gene-like sequences including partial duplicates, and most of them were classified as putative pseudogenes [19, 20]. The classification criteria for these putative pseudogenes were (1) non-syntenic map positions among grasses and (2) unique occurrence in one species or in one of the three subgenomes of hexaploid wheat. Illegitimate meiotic crossing over and subsequent sequence capture by transposable elements, as well as random sequence insertion during non-homologous end joining for double-strand break DNA repair, are the two proposed major events leading to non-tandem (partial) gene duplicates . By contrast, sub- or neo-functionalized, expressed and full-length gene duplicates often exist as tandemly repeated gene pairs or clusters of genes, as a result of unequal crossover during meiosis that is often followed by gene conversions [51, 52]. As shown in Table 1, the full-length genes HvPUB15 and AetPUB15 share syntenic map positions on the long arm of homologous chromosome group 3 [53, 54]. The partially duplicated HvARM1 gene was mapped at a distance of approximately 95 Mbp from HvPUB15 on chromosome 3H, and all six analyzed Triticeae ARM1 genes contain a non-repetitive, unknown sequence in exon 1 that is not present in the corresponding PUB15-like genes. Taken together this suggests that an event of DNA double-strand break repair in a common ancestor of Triticeae species gave rise to ARM1.
Marker-trait associations of HvARM1 and HvPUB15 in diverse collections of cultivated H. vulgare ssp. vulgare
Holm corr. pe
PM_ max_2_isol rel_Rol
PM_ max_2_isol rel_MRX
PM_ max_2_isol rel_Rol
PM_ max_2_isol rel_MRX
PM_ max_2_isol rel_MRX
PM_ max_2_isol rel_MRX
PM_ max_2_isol rel_MRX
PM_ max_2_isol rel_Rol
PM_ max_2_isol rel_MRX
PM_ max_2_isol rel_Rol
Effect of TIGS and transient over-expression (OEX) of HvPUB15, HvARM1, and genes encoding their interacting proteins on QR against B. graminis f.sp. hordei
Rel. SI (log2)a
p (t test)b
Rel. SI (log2)d
p (t test)b
U-box/ARM E3 protein ligase (HvPUB15)
0.17 ± 0.41
0.26 ± 0.19
ARM-repeat protein (HvARM1)
1.16 ± 0.22
−0.01 ± 0.21
Thylakoid formation 1 (Thf1)
−1.35 ± 0.59
0.47 ± 12.1
ATP-dependent Clp-protease adaptor (ClpS1)
−0.60 ± 0.41
0.70 ± 0.06
1.14 ± 0.25
Class III peroxidase TaPrx103
−1.06 ± 0.15
Degradation of putative HvPUB15 substrate proteins by transient OEX of HvPUB15
Rel. cell no. (log2)a
p (t test)b
Rel. cell no. (log2)a
p (t test)b
−0.59 ± 0.11
0.03 ± 0.28
HvPUB15 + HvARM1
−0.42 ± 0.07
−0.64 ± 0.56
−0.30 ± 0.31
0.33 ± 0.19
Transient over-expression of HvARM1 enhances resistance in T. aestivum against B. graminis f.sp. tritici
Relative SI (log2)a
p (t test)b
−0.37 ± 0.12
d HvARM1 ∆ATG
0.19 ± 0.13
The HvARM1 and HvPUB15 proteins interacted in yeast and in plants with the plastid-localized proteins HvClpS1 and HvThf1, and both appear to be susceptibility-related factors based on TIGS and transient OEX results (Table 3). The observation that transcripts of both HvClpS1 and HvThf1 were down-regulated in the epidermis of powdery mildew-attacked leaves might reflect an attempt of the plant to reduce the levels of susceptibility-related factors (Additional file 1: Figure S10 and Additional file 2: Table S7). In contrast to HvClpS1, the degradation of HvThf1 appeared to be mediated by HvPUB15, which suggests this protein as the strongest candidate for a susceptibility-related process involving HvPUB15 and being antagonized by HvARM1. However, because HvPUB15/HvARM1 co-expression did not suppress PUB15-induced HvThf1:YFP degradation, we cannot propose a simple model for a direct antagonistic mode of action of HvARM1. The THF1 protein of A. thaliana was found to be localized in the plastid stroma and at its outer membrane facing the cytoplasm, where it was proposed to play a role in sugar sensing . This is relevant with respect to the high demand of powdery mildew-infected cells for energy equivalents to transport large amounts of glucose into haustoria, a process that depends on SWEET sugar transporters and other factors [64–66]. Further support for the involvement of Thf1 in disease responses comes from the finding that the closest wheat homolog to HvThf1, designated as TaToxABP1, is a binding protein and a target of Toxin A produced by the necrotrophic, tan-spot fungal pathogen Pyrenophora tritici-repentis . Toxin A treatment also triggered an oxidative burst in leaves of wheat and barley [68, 69], thereby providing a link of Thf1 function with reactive oxygen species (ROS) control, at least in chloroplasts, and proposes a mode of action of Toxin A. Also, the interaction of the Thf1 protein with I2-like coiled-coil (CC) domains of several NB-LRR-type resistance proteins leading to their destabilization has been reported . Finally, the link of protein turnover by proteasomal degradation with chloroplast biology was recently established by reports on the role of the closest HvPUB15 homolog in A. thaliana designated as AtPUB4, and of AtCHIP, in plastid quality control and degradation of the caseinolytic plastid peptidase AtClpP4, respectively [42, 59, 71]. Mutants of AtPUB4 showed reduced resilience against abiotic stress, indicative of compromised plastid-based control of ROS generation. Plants silenced in or over-expressing AtCHIP exhibited a chlorotic phenotype indicating a strict requirement of accurate control of AtClpP4 levels for cellular homoestasis. In contrast to Thf1, no published information supporting a role of ClpS1 in plant-pathogen interactions is currently available.
Besides the PUB15/ARM1 gene pair, we found evidence for seven additional gene-duplication events across the barley genome that gave rise to novel, expressed genes encoding truncated proteins (Additional file 1: Figure S13). This number may be underestimated because the search was based on a library of 23,614 full-length cDNA clones, which covers approximately 50–66% of the entire predicted gene space. A more comprehensive study of partial gene duplications will have to await improved gene models of the barley reference sequence, as compared to the current annotation . The PUB15/ARM1 gene pair may also not be the only case of evolutionarily conserved duplication/gene rearrangements in Triticeae, because we also found similarly conserved events in a receptor-like kinase and a CASP-like protein (Fig. 6). Future expression studies and functional tests of these partially duplicated or rearranged genes across Triticeae may reveal a more comprehensive picture of their potential to support host survival.
The results presented here suggest that ARM1 is a case of gene neo-functionalization after a non-tandem, partial gene-duplication event that gained a role in quantitative resistance against B. graminis and maybe other pathogenic fungi. The ARM1 most likely originates from a partial duplication of the E3 ligase PUB15, which occurred in a common ancestor of the Triticeae tribe of grasses. At least in barley, the HvARM1-interacting protein and proposed substrate of HvPUB15, the plastid-localized HvThf1, links susceptibility to biotrophic pathogens with homeostatic protein function in plastids. Our findings shed new light on a novel mechanism to employ partial duplication of the protein-protein interaction domain to facilitate the expansion of immune signaling networks. The genome-wide search for further neo-functionalized gene duplicates encoding truncated proteins may uncover a yet poorly explored aspect of plant genome dynamics, which might be relevant for plant-stress responses in general and for plant-pathogen co-evolution in particular.
A more detailed description of materials and methods used in this study is provided in Additional file 3: Methods S1.
Plant and fungal material
TIGS and transient OEX experiments were done in 7-day-old seedlings of spring barley Golden Promise, except for OEX of site-directed mutagenesis (SDM) and TIGS of HvThf1 and HvClpS1 where the closely related genotype Maythorpe was used. Stable transgenic barley plants of cv. Golden Promise were generated as described . Bombarded leaf segments or transgenic plants were inoculated with Swiss Bgt field isolate FAL 92315, or Swiss Bgh field isolate CH4.8 throughout the study.
Sequence alignment, phylogenetic analysis, and estimation of pressures of selection
PUB15 and ARM1 orthologs were obtained from five Triticeae species, besides barley, including Secale cereale [21, 74], Triticum aestivum , and its three potential wild progenitors T. urartu (A genome), Aegilops speltoides (B genome), and Ae. tauschii (D genome). The sequences were retrieved by BlastN search of the H. vulgare sequences (AK361754 and AK371875, respectively) against the whole genome assembly databases (http://webblast.ipk-gatersleben.de/ryeselect/ for S. cereale and https://urgi.versailles.inra.fr/blast/blast.php for T. aestivum and its relatives). The rice ortholog, OsPUB15 (XM_015795011), was downloaded as outgroup sequence. All 17 coding sequences could be fully acquired but for the Ae. speltoides ARM1 sequence lacking the last exon.
The sequences were aligned with MAFFT v7.308  using the default settings within Geneious 10.0.9 (https://www.geneious.com)  followed by manual adjustment. The model of sequence evolution was determined with jModeltest 2.1.10 . The best-fit model, identified with the Akaike information criterion AIC [79, 80], was the general time-reversible (GTR)  with rates variation according to a gamma distribution . The maximum likelihood (ML) phylogenetic tree was calculated with RAxML v8.2.7  using the GTRGAMMA model and 100 bootstrap replicates (options –f a and –x).
Pressures of selection were investigated at the codon level throughout the phylogeny for both proteins separately. The pressure of selection can be estimated by ω (non-synonymous substitution rate divided by synonymous substitution rate, dN/dS). An ω < 1 suggests that the site is under negative, or purifying, selection, while an ω > 1 indicates that positive selection is occurring, and ω = 1 for neutral changes. Values of ω were estimated for each non-ambiguous codon using the codeml program within the package PAML4.9 h . Following Jeffares et al. , codon site models M0 [86, 87], allowing only one class of ω, M1a [88, 89], allowing two categories of sites (0 < ω0 < 1 and ω1 = 1 with proportions p0 and p1 = 1- p0), and M2a [88, 89], which includes a proportion of sites under positive selection, were tested. Likelihood ratio tests (LRTs) were performed to compare models M0 and M1a, as a test for variation of ω among sites, and models M1a and M2a, as a test for positive selection, against a χ2 distribution (with one and two degrees of freedom, respectively). Both proteins showed variations of ω among sites with M1a versus M0 LRTs significant. But with nearly identical likelihoods for models M1a and M2a, PUB15 and ARM1, though to a lesser extent, proved to be under purifying selection with a small portion of sites under neutral selection. Sequence information is available at https://doi.org/10.6084/m9.figshare.c.4092686.v1.
Exome capture sequencing
Genomic DNA was extracted from barley leaf material from a single plant for each accession and used for the hybridization with the barley SeqCap Ez oligo pool (Design Name: 120426_Barley_BEC_D04, . Quality-trimmed reads were mapped to the reference genome (http://webblast.ipk-gatersleben.de/barley_ibsc/downloads/) with Burrows-Wheeler Aligner (BWA) v0.7.5a using the mem algorithm with default parameters  and retaining only properly paired reads. Variant calling and realignment around indels were performed with Genome Analysis Toolkit (GATK), version 2.7.4 (https://software.broadinstitute.org/gatk/). Variant calls were filtered for high quality and ≥ 80% of samples being represented at each locus, and a dataset of 449,585 SNPs was produced, suitable for genetic association analysis of the two genes under investigation (full information about genome-wide variants from this dataset will be published elsewhere).
Association genetic analysis
Association of SNP and gene haplotypes (marker) of HvARM1 and HvPUB15 with the severity of Bgh infection (trait) was calculated based on genetic and phenotypic data of two diverse collections of cultivated barley (H. vulgare ssp. vulgare). Bgh infection values were determined in a detached leaf assay using second leaves of approximately 12-day-old seedlings, as described . First, a worldwide collection of 76 landraces (WHEALBI_LRC) was inoculated either with isolate JKI-75 or JKI-242, which exhibit a complex and complementing virulence spectra . Second, a worldwide collection of 127 cultivars (WHEALBI_CULT) was inoculated with the same two Bgh isolates. Both populations consisted of single seed-derived lines, and an average of 5 parallel plants per line was used in each inoculation assay. For passport data of all lines see Additional file 2: Table S1. Seven days after inoculation, disease was scored by estimating the percentage of leaf area covered by fungal mycelium. Because disease scores were variable between different inoculation experiments, they were normalized to internal standards cv. Roland or Morex, as indicated. Phenotypic data of all isolate-genotype combinations are based on two independent inoculation series. SNP calls were derived from exome capture resequencing, and haplotypes were calculated based on the combination of SNP calls per gene. SNP-trait and haplotype-trait associations were calculated in TASSEL v4.1 using a mixed linear model with kinship as random effect. Marker data for kinship calculations were derived from 4032 polymorphic genotyping by sequencing (GBS) markers. Marker-trait associations were assumed significant if the Holm’s-corrected p value was < 0.05 (number or SNP or haplotypes/gene = number of tests).
TIGS and transient over-expression
TIGS constructs were generated and transferred by particle bombardment into leaf epidermal cells of 7-day-old barley seedlings as described . Leaf segments were inoculated 3 days after the bombardment with Bgh at a density of 140–180 conidia mm–2. Transformed GUS-stained epidermal cells as well as haustoria-containing transformed (susceptible) cells were counted 48 h after inoculation, and TIGS effects on the susceptibility index (SI) were statistically analyzed .
For verification of transgene effects, HvARM1 was excised from a subclone of the bacterial artificial chromosome (BAC) HVVMRXALLhA0581d24 (Acc. Nr. KM979563) as a StuI/SphI fragment and inserted into SmaI/SphI sites of pIPKTA09 . For transient OEX of candidate genes, full-coding sequences were polymerase chain reaction (PCR) amplified from cDNA and inserted as an XbaI fragment into the multiple cloning site of pIPKTA09. Mutations (HvARM1-L286H and HvARM1-L308K) were introduced by SDM using the QuikChange Kit (Stratagene, San Diego, CA, USA). The resulting sequence-verified constructs were bombarded into barley as described for BAC clones. For PCR primers used in this study see Additional file 1: Table S9.
For the HvThf1 and HvClpS1 protein degradation assay, 4 μg each of plasmid DNA encoding HvPUB15 or HvARM1 plus Thf1:YFP or ClpS1:YFP plus pUbiGUS  were co-bombarded into 7-day-old barley cv. Golden Promise. The numbers of YFP-fluorescing cells with plastid-localized signals were counted 24 h after particle bombardment, followed by GUS-staining . The numbers of GUS-expressing cells were used for normalization of the YFP signal.
Inoculation and evaluation of transgenic plants
Phenotypic evaluation of Bgh and Bgt interactions was done microscopically on second, detached leaves of 12–14 day-old plants placed on phytoagar plates (23,2 cm × 23,2 cm) inoculated at a spore density of 30–40 conidia mm− 2. Inoculated leaf segments were incubated for 48 h (Bgh) or 72 h (Bgt) followed by staining with Coomassie brilliant blue R 250 . The number of growing colonies/leaf area was counted under a standard bright field microscope at 100× magnification.
Yeast two-hybrid experiments
Yeast two-hybrid screening was performed according to the Yeast Handbook and manual of Matchmaker™ Library Construction and Screening Kits (Takara/Clontech Laboratories, Saint-Germain-en-Laye, France). The full-length coding sequence of HvARM1 (1–442 AA) was used to screen a library of 7 × 106 mating events according to . For targeted Y2H assays, the coding region (1–831 AA) of HvPUB15 was used to test positive prey clones of the HvARM1 screening.
Bimolecular fluorescence complementation and co-immunoprecipitation
For bimolecular fluorescence complementation (BiFC) of HvARM1 and HvPUB15 proteins with potential plastid interactors, Nicotiana benthamiana plants were grown and agro-infiltrated as described in detail in Additional file 3: Methods S1. For BiFC with HvThf1 and HvClpS1, the wild-type full-length sequences of HvPUB15 or HvARM1, U-box mutants of HvPUB15, the ARM domain (351 to 831 AA) only of HvPUB15, or HvThf1 without N-terminal plastid import signal (-SP) were cloned into 35S::GWVYNE-pBar and 35S::GWVYCE-pBar GATEWAY destination vectors containing the N- and C-terminal split parts of the enhanced YFP protein Venus, respectively . BiFC constructs were transiently co-expressed by infiltration of Agrobacterium tumefaciens transformed with the corresponding binary vectors, and examined by confocal laser scanning microscopy (CLSM) 48 h after infiltration. For the development of U-box mutants, a DNA fragment between 709 and 739 bp (from ATG) on the U-box domain of HvPUB15 was excised using BsaXI and replaced by ligating synthetic oligos carrying the respective U-box mutation.
For co-immunoprecipitation (Co-IP), YFP-tagged HvARM1 and HvPUB15ARM under the control of the 35S promoter were generated by cloning the full-coding sequence of HvARM1 (1–442 AA) or the ARM-repeat region of HvPUB15 (351–831 AA) into pEARLEYGATE104 (Earley et al., 2006). cMyc-Tagged HvThf1 (1–294 AA) and HvClpS1 (1–161 AA) under the control of the CaMV 35S promoter were generated by cloning into pGWB418 . Mesophyll-protoplast transformation and co-immunoprecipitation was done as described .
Subcellular localization of fluorescent proteins
For subcellular localization, full-length sequences of HvPUB15, HvARM1, HvThf1, and HvClpS1 were N- and C-terminally fused in-frame to YFP in pIPKTA48 and pIPKTA49 vectors (Additional file 1: Figures S11 and S12). Resulting YFP-fusion constructs were transiently expressed in 7-day-old barley leaf segments by particle bombardment and examined after 12–24 h of incubation with or without B. graminis inoculation using CLSM.
Genome-wide search for expressed partial gene duplicates
To identify expressed partial gene duplicates, a local database was generated using 23,614 full-length cDNA sequences of barley , and a Blast search was carried out against itself using the megablast tool in the Galaxy platform  with the recommended settings, except the percent identity cutoff was set to 80–99%. To exclude pairs of genes sharing only functional domains, an alignment-to-shorter-gene length ratio of at least 0.8 was set. To identify the open reading frames, a tBlastx analysis of the pairs was carried out using the Galaxy platform. Manual curation was done to exclude non-spliced transcripts and chimeric as well as partial clones. Identified full-length and partial genes were aligned using MAUVE and the MUSCLE algorithm (MegAlign Pro, Version 15.1.0 (155) DNASTAR).
We would like to thank Gabi Brantin, Manuela Knauft, Sonja Gentz, and Cornelia Marthe for their excellent technical assistance.
This work was supported by the German Ministry for Education and Research, grant acronyms GABI-nonhost (to P.S.) and GABI-phenome (to P.S., J.KU. and R.H.), by German DFG (ERA-PG project TritNONHOST grant number DFG Schw 848/2–1 to P.S.), by EU FP6 project BIOEXPLOIT (to P.S.), and by EU FP7 project WHEALBI (to P.S. and N.S).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional files. Sequence information for cDNA clones HO23D08 and HO14H18 described in Table 1 is provided at https://www.ncbi.nlm.nih.gov/nuccore under accession numbers DN188354.1 and CK569805.1, respectively . Microarray data are available at ArrayExpress, https://www.ebi.ac.uk/arrayexpress under accession number E-MTAB-2916 . All sequences used for phylogenetic analysis are available at https://doi.org/10.6084/m9.figshare.c.4092686.v1 . Off-target prediction software si-Fi21 described in Additional file 1: Figure S4b and Additional file 3: Methods S1 is available at https://doi.org/10.5447/ipk/2017/9 . Nucleotide sequences with accession number AK361754 and AK371875 described in Table 1 are publicly available at https://www.ncbi.nlm.nih.gov/nuccore. Whole genome shotgun (WGS) sequence contigs with accession number CAJW010005672, CAJX010121345, CAJV010187631, CAJV010187631, CAJV010187631 and CAJX010121345 described in Table 1 are publicly available at https://www.ebi.ac.uk/. High-confidence barley genes HORVU3Hr1G113910, HORVU3Hr1G081380, HORVU3Hr1G117760, HORVU2Hr1G041260, HORVU7Hr1G020580, HORVU2Hr1G080670, HORVU3Hr1G059130, HORVU2Hr1G003460 described in Table 1 and Additional file 1: Table S8 are publicly available at http://barlex.barleysequence.org.
PS designed the research, analyzed data, and wrote the article. JKU, RH, MM, MT, and NS designed the research. JR analyzed data and wrote the article. RH, DD, JB, JKU, and GH discussed results and edited the manuscript. JR, DD, SL, and GH performed research (TIGS and transgenic plants). DD and EB performed research (BAC clone sequencing and rye whole genome sequencing). JR and CH performed research (yeast two-hybrid screen). JR, JKL, and TM performed research (in vivo protein interactions). JR and TR performed research (protein localization). BL, AH, TS, and MM performed research (exome capture sequencing, and genotyping by sequencing). DN and MP performed research (association mapping). PS and JB performed the phylogenetic analysis. All authors read and approved the final manuscript.
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- Macho AP, Zipfel C. Plant PRRs and the activation of innate immune signaling. Mol Cell. 2014;54:263–72.PubMedView ArticleGoogle Scholar
- Toruno TY, Stergiopoulos I, Coaker G. Plant-pathogen effectors: cellular probes interfering with plant defenses in spatial and temporal manners. Annu Rev Phytopathol. 2016;54:419–41.PubMedPubMed CentralView ArticleGoogle Scholar
- Schulze-Lefert P, Panstruga R. A molecular evolutionary concept connecting nonhost resistance, pathogen host range, and pathogen speciation. Trends Plant Sci. 2011;16:117–25.PubMedView ArticleGoogle Scholar
- St Clair DA. Quantitative disease resistance and quantitative resistance loci in breeding. Annu Rev Phytopathol. 2010;48:247–68.PubMedView ArticleGoogle Scholar
- Kou YJ, Wang SP. Broad-spectrum and durability: understanding of quantitative disease resistance. Curr Opin Plant Biol. 2010;13:181–5.PubMedView ArticleGoogle Scholar
- Niks RE, Qi XQ, Marcel TC. Quantitative resistance to biotrophic filamentous plant pathogens: concepts, misconceptions, and mechanisms. Annu Rev Phytopathol. 2015;53:445–70.PubMedView ArticleGoogle Scholar
- Yahiaoui N, Brunner S, Keller B. Rapid generation of new powdery mildew resistance genes after wheat domestication. Plant J. 2006;47:85–98.PubMedView ArticleGoogle Scholar
- Sperschneider J, Ying H, Dodds PN, Gardiner DM, Upadhyaya NM, Singh KB, Manners JM, Taylor JM. Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors. Front Plant Sci. 2014;5:372.PubMedPubMed CentralView ArticleGoogle Scholar
- Badouin H, Gladieux P, Gouzy J, Siguenza S, Aguileta G, Snirc A, Le Prieur S, Jeziorski C, Branca A, Giraud T. Widespread selective sweeps throughout the genome of model plant pathogenic fungi and identification of effector candidates. Mol Ecol. 2017;26:2041–62.PubMedView ArticleGoogle Scholar
- Plissonneau C, Benevenuto J, Mohd-Assaad N, Fouché S, Hartmann FE, Croll D. Using population and comparative genomics to understand the genetic basis of effector-driven fungal pathogen evolution. Front Plant Sci. 2017;8:119. https://doi.org/10.3389/fpls.2017.00119.PubMedPubMed CentralView ArticleGoogle Scholar
- Wei FS, Wong RA, Wise RP. Genome dynamics and evolution of the Mla (powdery mildew) resistance locus in barley. Plant Cell. 2002;14:1903–17.PubMedPubMed CentralView ArticleGoogle Scholar
- Hu PS, Wise RP. Diversification of Lrk/Tak kinase gene clusters is associated with subfunctionalization and cultivar-specific transcript accumulation in barley. Funct Integr Genomics. 2008;8:199–209.PubMedView ArticleGoogle Scholar
- Hu XY, Burghes AH, Ray PN, Thompson MW, Murphy EG, Worton RG. Partial gene duplication in Duchenne and Becker muscular dystrophies. J Med Genet. 1988;25:369–76.PubMedPubMed CentralView ArticleGoogle Scholar
- Strout MP, Marcucci G, Bloomfield CD, Caligiuri MA. The partial tandem duplication of ALL1 (MLL) is consistently generated by Alu-mediated homologous recombination in acute myeloid leukemia. Proc Natl Acad Sci U S A. 1998;95:2390–5.PubMedPubMed CentralView ArticleGoogle Scholar
- Devlin RH, Deeb S, Brunzell J, Hayden MR. Partial gene duplication involving exon-alu interchange results in lipoprotein-lipase deficiency. Am J Hum Genet. 1990;46:112–9.PubMedPubMed CentralGoogle Scholar
- Kitano T, Tian W, Umetsu K, Yuasa I, Yamazaki K, Saitou N, Osawa M. Origin and evolution of gene for prolactin-induced protein. Gene. 2006;383:64–70.PubMedView ArticleGoogle Scholar
- Grishkevich V, Yanai I. Gene length and expression level shape genomic novelties. Genome Res. 2014;24:1497–503.PubMedPubMed CentralView ArticleGoogle Scholar
- Korithoski B, Kolaczkowski O, Mukherjee K, Kola R, Earl C, Kolaczkowski B. Evolution of a novel antiviral immune-signaling interaction by partial-gene duplication. PLoS One. 2015;10:e0137276.PubMedPubMed CentralView ArticleGoogle Scholar
- Wicker T, Mayer KFX, Gundlach H, Martis M, Steuernagel B, Scholz U, Simkova H, Kubalakova M, Choulet F, Taudien S, et al. Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley, and their relatives. Plant Cell. 2011;23:1706–18.PubMedPubMed CentralView ArticleGoogle Scholar
- Akhunov ED, Sehgal S, Liang HQ, Wang SC, Akhunova AR, Kaur G, Li WL, Forrest KL, See D, Simkova H, et al. Comparative analysis of syntenic genes in grass genomes reveals accelerated rates of gene structure and coding sequence evolution in polyploid wheat. Plant Physiol. 2013;161:252–65.PubMedView ArticleGoogle Scholar
- Bauer E, Schmutzer T, Barilar I, Mascher M, Gundlach H, Martis MM, Twardziok SO, Hackauf B, Gordillo A, Wilde P, et al. Towards a whole-genome sequence for rye (Secale cereale L.). Plant J. 2017;89:853–69.PubMedView ArticleGoogle Scholar
- Panstruga R, Schulze-Lefert P. Live and let live: insights into powdery mildew disease and resistance. Mol Plant Pathol. 2002;3:495–502.PubMedView ArticleGoogle Scholar
- Oerke EC. Crop losses to pests. J Agric Sci. 2006;144:31–43.View ArticleGoogle Scholar
- Huckelhoven R. Cell wall-associated mechanisms of disease resistance and susceptibility. Annu Rev Phytopathol. 2007;45:101–27.PubMedView ArticleGoogle Scholar
- Collinge DB. Cell wall appositions: the first line of defence. J Exp Bot. 2009;60:351–2.PubMedView ArticleGoogle Scholar
- Huckelhoven R, Panstruga R. Cell biology of the plant-powdery mildew interaction. Curr Opin Plant Biol. 2011;14:738–46.PubMedView ArticleGoogle Scholar
- Pedersen C, van Themaat EVL, McGuffin LJ, Abbott JC, Burgis TA, Barton G, Bindschedler LV, Lu XL, Maekawa T, Wessling R, et al. Structure and evolution of barley powdery mildew effector candidates. BMC Genomics. 2012;13:694.PubMedPubMed CentralView ArticleGoogle Scholar
- Abramovitch RB, Janjusevic R, Stebbins CE, Martin GB. Type III effector AvrPtoB requires intrinsic E3 ubiquitin ligase activity to suppress plant cell death and immunity. Proc Natl Acad Sci U S A. 2006;103:2851–6.PubMedPubMed CentralView ArticleGoogle Scholar
- Angot A, Peeters N, Lechner E, Vailleau F, Baud C, Gentzbittel L, Sartorel E, Genschik P, Boucher C, Genin SP. Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants. Proc Natl Acad Sci U S A. 2006;103:14620–5.PubMedPubMed CentralView ArticleGoogle Scholar
- Rosebrock TR, Zeng LR, Brady JJ, Abramovitch RB, Xiao FM, Martin GB. A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity. Nature. 2007;448:370–U313.PubMedPubMed CentralView ArticleGoogle Scholar
- Groll M, Schellenberg B, Bachmann AS, Archer CR, Huber R, Powell TK, Lindow S, Kaiser M, Dudler R. A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism. Nature. 2008;452:755–8.PubMedView ArticleGoogle Scholar
- Spallek T, Robatzek S, Gohre V. How microbes utilize host ubiquitination. Cell Microbiol. 2009;11:1425–34.PubMedView ArticleGoogle Scholar
- Bos JIB, Armstrong MR, Gilroy EM, Boevink PC, Hein I, Taylor RM, Tian ZD, Engelhardt S, Vetukuri RR, Harrower B, et al. Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1. Proc Natl Acad Sci U S A. 2010;107:9909–14.PubMedPubMed CentralView ArticleGoogle Scholar
- Nomura K, Mecey C, Lee YN, Imboden LA, Chang JH, He SY. Effector-triggered immunity blocks pathogen degradation of an immunity-associated vesicle traffic regulator in Arabidopsis. Proc Natl Acad Sci U S A. 2011;108:10774–9.PubMedPubMed CentralView ArticleGoogle Scholar
- Vierstra RD. The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol. 2009;10:385–97.PubMedView ArticleGoogle Scholar
- Trujillo M, Shirasu K. Ubiquitination in plant immunity. Curr Opin Plant Biol. 2010;13:402–8.PubMedView ArticleGoogle Scholar
- Schweizer P. Nonhost resistance of plants to powdery mildew — new opportunities to unravel the mystery. Physiol Mol Plant Pathol. 2007;70:3–7.View ArticleGoogle Scholar
- Sadanandom A, Bailey M, Ewan R, Lee J, Nelis S. The ubiquitin-proteasome system: central modifier of plant signalling. New Phytol. 2012;196:13–28.PubMedView ArticleGoogle Scholar
- Douchkov D, Lück S, Johrde A, Nowara D, Himmelbach A, Rajaraman J, Stein N, Sharma R, Kilian B, Schweizer P. Discovery of genes affecting resistance of barley to adapted and non-adapted powdery mildew fungi. Genome Biol. 2014;15:518.PubMedPubMed CentralView ArticleGoogle Scholar
- Azevedo C, Santos-Rosa MJ, Shirasu K. The U-box protein family in plants. Trends Plant Sci. 2001;6:354–8.PubMedView ArticleGoogle Scholar
- Mudgil Y, Shiu SH, Stone SL, Salt JN, Goring DR. A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family. Plant Physiol. 2004;134:59–66.PubMedPubMed CentralView ArticleGoogle Scholar
- Zeng LR, Park CH, Venu RC, Gough J, Wang GL. Classification, expression pattern, and E3 ligase activity assay of rice U-box-containing proteins. Mol Plant. 2008;1:800–15.PubMedView ArticleGoogle Scholar
- Bernhardt N, Brassac J, Kilian B, Blattner FR. Dated tribe-wide whole chloroplast genome phylogeny indicates recurrent hybridizations within Triticeae. BMC Evol Biol. 2017;17:141.PubMedPubMed CentralView ArticleGoogle Scholar
- Park JJ, Yi J, Yoon J, Cho LH, Ping J, Jeong HJ, Cho SK, Kim WT, An G. OsPUB15, an E3 ubiquitin ligase, functions to reduce cellular oxidative stress during seedling establishment. Plant J. 2011;65:194–205.PubMedView ArticleGoogle Scholar
- Close TJ, Bhat PR, Lonardi S, Wu Y, Rostoks N, Ramsay L, Druka A, Stein N, Svensson JT, Wanamaker S, et al. Development and implementation of high-throughput SNP genotyping in barley. BMC Genomics. 2009;10:582.PubMedPubMed CentralView ArticleGoogle Scholar
- Huang J, Taylor JP, Chen JG, Uhrig JF, Schnell DJ, Nakagawa T, Korth KL, Jones AM. The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis. Plant Cell. 2006;18:1226–38.PubMedPubMed CentralView ArticleGoogle Scholar
- Nishimura K, Asakura Y, Friso G, Kim J, Oh SH, Rutschow H, Ponnala L, van Wijk KJ. ClpS1 Is a conserved substrate selector for the chloroplast Clp protease system in Arabidopsis. Plant Cell. 2013;25:2276–301.PubMedPubMed CentralView ArticleGoogle Scholar
- Koegl M, Hoppe T, Schlenker S, Ulrich HD, Mayer TU, Jentsch S. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell. 1999;96:635–44.PubMedView ArticleGoogle Scholar
- Nelson BK, Cai X, Nebenfuhr A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007;51:1126–36.PubMedView ArticleGoogle Scholar
- Katju V, Lynch M. The structure and early evolution of recently arisen gene duplicates in the Caenorhabditis elegans genome. Genetics. 2003;165:1793–803.PubMedPubMed CentralGoogle Scholar
- Leister D. Tandem and segmental gene duplication and recombination in the evolution of plant disease resistance genes. Trends Genet. 2004;20:116–22.PubMedView ArticleGoogle Scholar
- Himmelbach A, Liu L, Zierold U, Altschmied L, Maucher H, Beier F, Muller D, Hensel G, Heise A, Schutzendubel A, et al. Promoters of the barley germin-like GER4 gene cluster enable strong transgene expression in response to pathogen attack. Plant Cell. 2010;22:937–52.PubMedPubMed CentralView ArticleGoogle Scholar
- Luo MC, Gu YQ, You FM, Deal KR, Ma YQ, Hu YQ, Huo NX, Wang Y, Wang JR, Chen SY, et al. A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor. Proc Natl Acad Sci U S A. 2013;110:7940–5.PubMedPubMed CentralView ArticleGoogle Scholar
- Mascher M, Richmond TA, Gerhardt DJ, Himmelbach A, Clissold L, Sampath D, Ayling S, Steuernagel B, Pfeifer M, D'Ascenzo M, et al. Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond. Plant J. 2013;76:494–505.PubMedPubMed CentralView ArticleGoogle Scholar
- Blanc G, Wolfe KH. Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution. Plant Cell. 2004;16:1679–91.PubMedPubMed CentralView ArticleGoogle Scholar
- Roulin A, Auer PL, Libault M, Schlueter J, Farmer A, May G, Stacey G, Doerge RW, Jackson SA. The fate of duplicated genes in a polyploid plant genome. Plant J. 2013;73:143–53.PubMedView ArticleGoogle Scholar
- Hughes TE, Langdale JA, Kelly S. The impact of widespread regulatory neofunctionalization on homeolog gene evolution following whole-genome duplication in maize. Genome Res. 2014;24:1348–55.PubMedPubMed CentralView ArticleGoogle Scholar
- Panchy N, Lehti-Shiu M, Shiu SH. Evolution of gene duplication in plants. Plant Physiol. 2016;171:2294–316.PubMedPubMed CentralGoogle Scholar
- Woodson JD, Joens MS, Sinson AB, Gilkerson J, Salome PA, Weigel D, Fitzpatrick JA, Chory J. Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts. Science. 2015;350:450–4.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhou J, Lu D, Xu G, Finlayson SA, He P, Shan L. The dominant negative ARM domain uncovers multiple functions of PUB13 in Arabidopsis immunity, flowering, and senescence. J Exp Bot. 2015;66:3353–66.PubMedPubMed CentralView ArticleGoogle Scholar
- van der Hoorn RAL, Kamoun S. From Guard to Decoy: A new model for perception of plant pathogen effectors. Plant Cell. 2008;20:2009–17.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim SH, Qi D, Ashfield T, Helm M, Innes RW. Using decoys to expand the recognition specificity of a plant disease resistance protein. Science. 2016;351:684–7.PubMedView ArticleGoogle Scholar
- Wang J, Qu B, Dou S, Li L, Yin DD, Pang ZQ, Zhou ZZ, Tian MM, Liu GZ, Xie Q, et al. The E3 ligase OsPUB15 interacts with the receptor-like kinase PID2 and regulates plant cell death and innate immunity. BMC Plant Biol. 2015;15:49.PubMedPubMed CentralView ArticleGoogle Scholar
- Scholes JD, Lee PJ, Horton P, Lewis DH. Invertase: understanding changes in the photosynthetic and carbohydrate metabolism of barley leaves infected with powdery mildew. New Phytol. 1994;126:213–22.View ArticleGoogle Scholar
- Chen LQ, Hou BH, Lalonde S, Takanaga H, Hartung ML, Qu XQ, Guo WJ, Kim JG, Underwood W, Chaudhuri B, et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature. 2010;468:527–32.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen LQ, Qu XQ, Hou BH, Sosso D, Osorio S, Fernie AR, Frommer WB. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science. 2012;335:207–11.PubMedView ArticleGoogle Scholar
- Manning VA, Hardison LK, Ciuffetti LM. Ptr ToxA interacts with a chloroplast-localized protein. Mol Plant-Microbe Interact. 2007;20:168–77.PubMedView ArticleGoogle Scholar
- Manning VA, Chu AL, Scofield SR, Ciuffetti LM. Intracellular expression of a host-selective toxin, ToxA, in diverse plants phenocopies silencing of a ToxA-interacting protein, ToxABP1. New Phytol. 2010;187:1034–47.PubMedView ArticleGoogle Scholar
- Pandelova I, Figueroa M, Wilhelm LJ, Manning VA, Mankaney AN, Mockler TC, Ciuffetti LM. Host-selective toxins of Pyrenophora tritici-repentis induce common responses associated with host susceptibility. PLoS One. 2012;7:e40240.PubMedPubMed CentralView ArticleGoogle Scholar
- Hamel L-P, Sekine K-T, Wallon T, Sugiwaka Y, Kobayashi K, Moffett P. The chloroplastic protein THF1 interacts with the coiled-coil domain of the disease resistance protein N ' and regulates light-dependent cell death. Plant Physiol. 2016;171:658–74.PubMedPubMed CentralView ArticleGoogle Scholar
- Wei J, Qiu X, Chen L, Hu W, Hu R, Chen J, Sun L, Li L, Zhang H, Lv Z, Shen G. The E3 ligase AtCHIP positively regulates Clp proteolytic subunit homeostasis. J Exp Bot. 2015;66:5809–20.PubMedView ArticleGoogle Scholar
- Mascher M, Gundlach H, Himmelbach A, Beier S, Twardziok SO, Wicker T, Radchuk V, Dockter C, Hedley PE, Russell J, et al. A chromosome conformation capture ordered sequence of the barley genome. Nature. 2017;544:427–33.PubMedView ArticleGoogle Scholar
- Hensel G, Valkov V, Middlefell-Williams J, Kumlehn J. Efficient generation of transgenic barley: the way forward to modulate plant-microbe interactions. J Plant Physiol. 2008;165:71–82.PubMedView ArticleGoogle Scholar
- Schmutzer T. Scaffolds of rye (Secale cereale L.) inbred line Lo7– version 2. In: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland OT Gatersleben, Corrensstraße 3, 06466, Germany; 2016. https://doi.org/10.5447/ipk/2016/56.Google Scholar
- International Wheat Genome Sequencing Consortium. A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science. 2014;345:1251788.View ArticleGoogle Scholar
- Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80.PubMedPubMed CentralView ArticleGoogle Scholar
- Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647–9.PubMedPubMed CentralView ArticleGoogle Scholar
- Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012;9:772.PubMedPubMed CentralView ArticleGoogle Scholar
- Akaike H. Information theory and an extension of the maximum likelihood principle. In: Parzen E, Tanabe K, Kitagawa G, editors. Selected papers of Hirotugu Akaike. New York: Springer; 1998. p. 199–213.View ArticleGoogle Scholar
- Akaike H. A new look at the statistical model identification. In: Parzen E, Tanabe K, Kitagawa G, editors. Selected papers of Hirotugu Akaike. New York: Springer; 1998. p. 215–22.Google Scholar
- Lanave C, Preparata G, Saccone C, Serio G. A new method for calculating evolutionary substitution rates. J Mol Evol. 1984;20:86–93.PubMedView ArticleGoogle Scholar
- Yang ZH. Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J Mol Evol. 1994;39:306–14.PubMedView ArticleGoogle Scholar
- Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–3.PubMedPubMed CentralView ArticleGoogle Scholar
- Yang Z. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci. 1997;13:555–6.PubMedGoogle Scholar
- Jeffares DC, Tomiczek B, Sojo V, dos Reis M. A beginners guide to estimating the non-synonymous to synonymous rate ratio of all protein-coding genes in a genome. Methods Mol Biol. 2015;1201:65–90.PubMedView ArticleGoogle Scholar
- Goldman N, Yang Z. A codon-based model of nucleotide substitution for protein-coding DNA sequences. Mol Biol Evol. 1994;11:725–36.PubMedGoogle Scholar
- Yang Z, Nielsen R. Synonymous and nonsynonymous rate variation in nuclear genes of mammals. J Mol Evol. 1998;46:409–18.PubMedView ArticleGoogle Scholar
- Nielsen R, Yang Z. Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics. 1998;148:929–36.PubMedPubMed CentralGoogle Scholar
- Yang Z, Wong WS, Nielsen R. Bayes empirical bayes inference of amino acid sites under positive selection. Mol Biol Evol. 2005;22:1107–18.PubMedView ArticleGoogle Scholar
- Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25:1754–60.Google Scholar
- Spies A, Korzun V, Bayles R, Rajaraman J, Himmelbach A, Hedley PE, Schweizer P. Allele mining in barley genetic resources reveals genes of race-non-specific powdery mildew resistance. Front Plant Sci. 2011;2:113.PubMedGoogle Scholar
- Šurlan-Momirović G, Flath K, Silvar C, Branković G, Kopahnke D, Knežević D, Schliephake E, Ordon F, Perović D. Exploring the Serbian GenBank barley (Hordeum vulgare L. subsp. vulgare) collection for powdery mildew resistance. Genet Resour Crop Evol. 2016;63:275–87.View ArticleGoogle Scholar
- Douchkov D, Nowara D, Zierold U, Schweizer P. A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. Mol Plant-Microbe Interact. 2005;18:755–61.PubMedView ArticleGoogle Scholar
- Zimmermann G, Baumlein H, Mock HP, Himmelbach A, Schweizer P. The multigene family encoding germin-like proteins of barley. Regulation and function in basal host resistance. Plant Physiol. 2006;142:181–92.PubMedPubMed CentralView ArticleGoogle Scholar
- Schweizer P, Gees R, Mosinger E. Effect of jasmonic acid on the interaction of barley (Hordeum vulgare L.) with the powdery mildew Erysiphe graminis f.sp. hordei. Plant Physiol. 1993;102:503–11.PubMedPubMed CentralView ArticleGoogle Scholar
- Hoefle C, Huesmann C, Schultheiss H, Bornke F, Hensel G, Kumlehn J, Hückelhoven R. A barley ROP GTPase ACTIVATING PROTEIN associates with microtubules and regulates entry of the barley powdery mildew fungus into leaf epidermal cells. Plant Cell. 2011;23:2422–39.PubMedPubMed CentralView ArticleGoogle Scholar
- Thormahlen I, Meitzel T, Groysman J, Ochsner AB, von Roepenack-Lahaye E, Naranjo B, Cejudo FJ, Geigenberger P. Thioredoxin f1 and NADPH-dependent thioredoxin reductase C have overlapping functions in regulating photosynthetic metabolism and plant growth in response to varying light conditions. Plant Physiol. 2015;169:1766–86.PubMedPubMed CentralGoogle Scholar
- Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 2007;104:34–41.Google Scholar
- Stegmann M, Anderson RG, Ichimura K, Pecenkova T, Reuter P, Zarsky V, McDowell JM, Shirasu K, Trujillo M. The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis. Plant Cell. 2012;24:4703–16.PubMedPubMed CentralView ArticleGoogle Scholar
- Matsumoto T, Tanaka T, Sakai H, Amano N, Kanamori H, Kurita K, Kikuta A, Kamiya K, Yamamoto M, Ikawa H, et al. Comprehensive sequence analysis of 24,783 barley full-length cDNAs derived from 12 clone libraries. Plant Physiol. 2011;156:20–8.PubMedPubMed CentralView ArticleGoogle Scholar
- Afgan E, Baker D, van den Beek M, Blankenberg D, Bouvier D, Cech M, Chilton J, Clements D, Coraor N, Eberhard C, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 2016;44:W3–W10.PubMedPubMed CentralView ArticleGoogle Scholar
- Zierold U, Scholz U, Schweizer P. Transcriptome analysis of mlo-mediated resistance in the epidermis of barley. Mol Plant Pathol. 2005;6:139–51.PubMedView ArticleGoogle Scholar
- Delventhal R, Rajaraman J, Stefanato FL, Rehman S, Aghnoum R, McGrann GRD, Bolger M, Usadel B, Hedley PE, Boyd L, et al. A comparative analysis of nonhost resistance across the two Triticeae crop species wheat and barley. BMC Plant Biol. 2017;17:232.PubMedPubMed CentralView ArticleGoogle Scholar
- Brassac J: ARM1 and PUB15 coding sequences for phylogenetic tree. In Data Sets figshare; 2018. https://doi.org/10.6084/m9.figshare.c.4092686.v1.
- Lueck S: siFi_ Software for long double-stranded RNAi-target design and off-target prediction. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland OT Gatersleben, Corrensstraße 3, 06466, Germany; 2017. https://doi.org/10.5447/ipk/2017/9.
- Schweizer P, Pokorny J, Abderhalden O, Dudler R. A transient assay system for the functional assessment of defense-related genes in wheat. Mol Plant-Microbe Interact. 1999;12:647–54.View ArticleGoogle Scholar