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Background: Septoria tritici blotch (STB) is one of the most damaging wheat diseases worldwide, and the development of resistant cultivars is of paramount importance for sustainable crop management. However, the genetic basis of the resistance present in elite wheat cultivars remains largely unknown, which limits the implementation of this strategy. A collection of 285 wheat cultivars originating mostly from France was challenged with ten Zymoseptoria tritici isolates at the seedling stage. The collection was further evaluated in seven field trials across France using artificial inoculation., Results: Genome-wide association study resulted in the detection of 57 wheat QTL, among which 40 were detected at the seedling stage. Three quarters of these QTL were in genomic regions previously reported for to confer resistance to Z. tritici, but 10 QTL are novel and may be of special interest as new sources of resistance. Some QTL colocalise with major Stb resistance genes, suggesting their presence in the French elite winter wheat germplasm. Among them, the three QTL with the strongest effect colocalize with Stb6, Stb9 and Stb18. There was minimal overlap between the QTL detected at the seedling and adult plant stages, with only 1 out of 20 seedling QTL also being detected in field trials inoculated with the same isolate. This suggests that different resistance genes are involved at the seedling and adult plant stages., Conclusion: This work reveals the highly complex genetic architecture of French wheat resistance to STB and provides relatively small QTL intervals, which will be valuable for identifying the underlying causative genes and for marker-assisted selection., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Knowledge of genetic determinism and evolutionary dynamics mediating host-pathogen interactions is essential to manage fungal plant diseases. Studies on the genetic architecture of fungal pathogenicity often focus on large-effect effector genes triggering strong, qualitative resistance. It is not clear how this translates to predominately quantitative interactions. Here, we use the Zymoseptoria tritici-wheat model to elucidate the genetic architecture of quantitative pathogenicity and mechanisms mediating host adaptation. With a multi-host genome-wide association study, we identify 19 high-confidence candidate genes associated with quantitative pathogenicity. Analysis of genetic diversity reveals that sequence polymorphism is the main evolutionary process mediating differences in quantitative pathogenicity, a process that is likely facilitated by genetic recombination and transposable element dynamics. Finally, we use functional approaches to confirm the role of an effector-like gene and a methyltransferase in phenotypic variation. This study highlights the complex genetic architecture of quantitative pathogenicity, extensive diversifying selection and plausible mechanisms facilitating pathogen adaptation., (© 2024. The Author(s).)

Background: Investigations on plant-pathogen interactions require quantitative, accurate, and rapid phenotyping of crop diseases. However, visual assessment of disease symptoms is preferred over available numerical tools due to transferability challenges. These assessments are laborious, time-consuming, require expertise, and are rater dependent. More recently, deep learning has produced interesting results for evaluating plant diseases. Nevertheless, it has yet to be used to quantify the severity of Septoria tritici blotch (STB) caused by Zymoseptoria tritici-a frequently occurring and damaging disease on wheat crops., Results: We developed an image analysis script in Python, called SeptoSympto. This script uses deep learning models based on the U-Net and YOLO architectures to quantify necrosis and pycnidia on detached, flattened and scanned leaves of wheat seedlings. Datasets of different sizes (containing 50, 100, 200, and 300 leaves) were annotated to train Convolutional Neural Networks models. Five different datasets were tested to develop a robust tool for the accurate analysis of STB symptoms and facilitate its transferability. The results show that (i) the amount of annotated data does not influence the performances of models, (ii) the outputs of SeptoSympto are highly correlated with those of the experts, with a similar magnitude to the correlations between experts, and (iii) the accuracy of SeptoSympto allows precise and rapid quantification of necrosis and pycnidia on both durum and bread wheat leaves inoculated with different strains of the pathogen, scanned with different scanners and grown under different conditions., Conclusions: SeptoSympto takes the same amount of time as a visual assessment to evaluate STB symptoms. However, unlike visual assessments, it allows for data to be stored and evaluated by experts and non-experts in a more accurate and unbiased manner. The methods used in SeptoSympto make it a transferable, highly accurate, computationally inexpensive, easy-to-use, and adaptable tool. This study demonstrates the potential of using deep learning to assess complex plant disease symptoms such as STB., (© 2024. The Author(s).)

Zymoseptoria tritici is the fungal pathogen responsible for Septoria tritici blotch on wheat. Disease outcome in this pathosystem is partly determined by isolate-specific resistance, where wheat resistance genes recognize specific fungal factors triggering an immune response. Despite the large number of known wheat resistance genes, fungal molecular determinants involved in such cultivar-specific resistance remain largely unknown. We identified the avirulence factor AvrStb9 using association mapping and functional validation approaches. Pathotyping AvrStb9 transgenic strains on Stb9 cultivars, near isogenic lines and wheat mapping populations, showed that AvrStb9 interacts with Stb9 resistance gene, triggering an immune response. AvrStb9 encodes an unusually large avirulence gene with a predicted secretion signal and a protease domain. It belongs to a S41 protease family conserved across different filamentous fungi in the Ascomycota class and may constitute a core effector. AvrStb9 is also conserved among a global Z. tritici population and carries multiple amino acid substitutions caused by strong positive diversifying selection. These results demonstrate the contribution of an 'atypical' conserved effector protein to fungal avirulence and the role of sequence diversification in the escape of host recognition, adding to our understanding of host-pathogen interactions and the evolutionary processes underlying pathogen adaptation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Amezrou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Septoria leaf blotch is a foliar wheat disease controlled by a combination of plant genetic resistances and fungicides use. R- gene-based qualitative resistance durability is limited due to gene-for-gene interactions with fungal avirulence ( Avr ) genes. Quantitative resistance is considered more durable but the mechanisms involved are not well documented. We hypothesize that genes involved in quantitative and qualitative plant-pathogen interactions are similar. A bi-parental population of Zymoseptoria tritici was inoculated on wheat cultivar 'Renan' and a linkage analysis performed to map QTL. Three pathogenicity QTL, Qzt-I05-1, Qzt-I05-6 and Qzt-I07-13 , were mapped on chromosomes 1, 6 and 13 in Z. tritici , and a candidate pathogenicity gene on chromosome 6 was selected based on its effector-like characteristics. The candidate gene was cloned by Agrobacterium tumefaciens -mediated transformation, and a pathology test assessed the effect of the mutant strains on 'Renan'. This gene was demonstrated to be involved in quantitative pathogenicity. By cloning a newly annotated quantitative-effect gene in Z. tritici that is effector-like, we demonstrated that genes underlying pathogenicity QTL can be similar to Avr genes. This opens up the previously probed possibility that 'gene-for-gene' underlies not only qualitative but also quantitative plant-pathogen interactions in this pathosystem., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Langlands-Perry, Pitarch, Lapalu, Cuenin, Bergez, Noly, Amezrou, Gélisse, Barrachina, Parrinello, Suffert, Valade and Marcel.)

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen., (© 2023. The Author(s).)

Crop pathogens pose severe risks to global food production due to the rapid rise of resistance to pesticides and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in the genetic composition of pathogen populations. Here we report the design of a microfluidics-based amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici. The fungus causes one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance. We optimized the primer design by integrating polymorphism data from 632 genomes of the same species. To test the performance of the assay, we genotyped 192 samples in two replicates. Analysis of the short-read sequence data generated by the assay showed a fairly stable success rate across samples to amplify a large number of loci. The performance was consistent between samples originating from pure genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed genotypes, we found that the assay recovers variations in allele frequencies. We also explored the potential of the amplicon assay to recover transposable element insertion polymorphism relevant for fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population structure across French wheat fields. Genomic monitoring of crop pathogens contributes to more sustainable crop protection and yields., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bellah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Quantitative resistance is considered more durable than qualitative resistance as it does not involve major resistance genes that can be easily overcome by pathogen populations, but rather a combination of genes with a lower individual effect. This durability means that quantitative resistance could be an interesting tool for breeding crops that would not systematically require phytosanitary products. Quantitative resistance has yet to reveal all of its intricacies. Here, we delve into the case of the wheat/Septoria tritici blotch (STB) pathosystem. Using a population resulting from a cross between French cultivar Renan, generally resistant to STB, and Chinese Spring, a cultivar susceptible to the disease, we built an ultra-dense genetic map that carries 148,820 single nucleotide polymorphism (SNP) markers. Phenotyping the interaction was done with two different Zymoseptoria tritici strains with contrasted pathogenicities on Renan. A linkage analysis led to the detection of three quantitative trait loci (QTL) related to resistance in Renan. These QTL, on chromosomes 7B, 1D, and 5D, present with an interesting diversity as that on 7B was detected with both fungal strains, while those on 1D and 5D were strain-specific. The resistance on 7B was located in the region of Stb8 and the resistance on 1D colocalized with Stb19 . However, the resistance on 5D was new, so further designated Stb20q . Several wall-associated kinases (WAK), nucleotide-binding and leucine-rich repeats (NB-LRR) type, and kinase domain carrying genes were present in the QTL regions, and some of them were expressed during the infection. These results advocate for a role of Stb genes in quantitative resistance and for resistance in the wheat/STB pathosystem being as a whole quantitative and polygenic.

Background: Tunisia is considered a secondary center of diversification of durum wheat and has a large number of abandoned old local landraces. An accurate investigation and characterization of the morphological and genetic features of these landraces would allow their rehabilitation and utilization in wheat breeding programs. Here, we investigated a diverse collection of 304 local accessions of durum wheat collected from five regions and three climate stages of central and southern Tunisia., Results: Durum wheat accessions were morphologically characterized using 12 spike- and grain-related traits. A mean Shannon-Weaver index (H') of 0.80 was obtained, indicating high level of polymorphism among accessions. Based on these traits, 11 local landraces including Mahmoudi, Azizi, Jneh Khotifa, Mekki, Biskri, Taganrog, Biada, Badri, Richi, Roussia and Souri were identified. Spike length (H' = 0.98), spike shape (H' = 0.86), grain size (H' = 0.94), grain shape (H' = 0.87) and grain color (H' = 0.86) were the most polymorphic morphological traits. The genetic diversity of these accessions was assessed using 10 simple sequence repeat (SSR) markers, with a polymorphic information content (PIC) of 0.69. Levels of genetic diversity were generally high (I = 0.62; He = 0.35). In addition, population structure analysis revealed 11 genetic groups, which were significantly correlated with the morphological characterization. Analysis of molecular variance (AMOVA) showed high genetic variation within regions (81%) and within genetic groups (41%), reflecting a considerable amount of admixture between landraces. The moderate (19%) and high (59%) levels of genetic variation detected among regions and among genetic groups, respectively, highlighted the selection practices of farmers. Furthermore, Mahmoudi accessions showed significant variation in spike density between central Tunisia (compact spikes) and southern Tunisia (loose spikes with open glume), may indicate an adaptation to high temperature in the south., Conclusion: Overall, this study demonstrates the genetic richness of local durum wheat germplasm for better in situ and ex situ conservation and for the subsequent use of these accessions in wheat breeding programs.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch, a major pathogen of wheat globally and the most damaging pathogen of wheat in Europe. A gene-for-gene (GFG) interaction between Z. tritici and wheat cultivars carrying the Stb6 resistance gene has been postulated for many years, but the genes have not been identified. We identified AvrStb6 by combining quantitative trait locus mapping in a cross between two Swiss strains with a genome-wide association study using a natural population of c. 100 strains from France. We functionally validated AvrStb6 using ectopic transformations. AvrStb6 encodes a small, cysteine-rich, secreted protein that produces an avirulence phenotype on wheat cultivars carrying the Stb6 resistance gene. We found 16 nonsynonymous single nucleotide polymorphisms among the tested strains, indicating that AvrStb6 is evolving very rapidly. AvrStb6 is located in a highly polymorphic subtelomeric region and is surrounded by transposable elements, which may facilitate its rapid evolution to overcome Stb6 resistance. AvrStb6 is the first avirulence gene to be functionally validated in Z. tritici, contributing to our understanding of avirulence in apoplastic pathogens and the mechanisms underlying GFG interactions between Z. tritici and wheat., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Partial resistance quantitative trait loci (QTLs) Rphq11 and rphq16 against Puccinia hordei isolate 1.2.1 were previously mapped in seedlings of the mapping populations Steptoe/Morex and Oregon Wolfe Barleys, respectively. In this study, QTL mapping was performed at adult plant stage for the two mapping populations challenged with the same rust isolate. The results suggest that Rphq11 and rphq16 are effective only at seedling stage, and not at adult plant stage. The cloning of several genes responsible for partial resistance of barley to P. hordei will allow elucidation of the molecular basis of this type of plant defence. A map-based cloning approach requires to fine-map the QTL in a narrow genetic window. In this study, Rphq11 and rphq16 were fine-mapped using an approach aiming at speeding up the development of plant material and simplifying its evaluation. The plant materials for fine-mapping were identified from early plant materials developed to produce QTL-NILs. The material was first selected to carry the targeted QTL in heterozygous condition and susceptibility alleles at other resistance QTLs in homozygous condition. This strategy took four to five generations to obtain fixed QTL recombinants (i.e., homozygous resistant at the Rphq11 or rphq16 QTL alleles, homozygous susceptible at the non-targeted QTL alleles). In less than 2 years, Rphq11 was fine-mapped into a 0.2-cM genetic interval and a 1.4-cM genetic interval for rphq16 . The strongest candidate gene for Rphq11 is a phospholipid hydroperoxide glutathione peroxidase. Thus far, no candidate gene was identified for rphq16 .

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover, our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver., Competing Interests: The authors declare that no competing interests exist.

Key Message: Rphq2, a minor gene for partial resistance to Puccinia hordei , was physically mapped in a 188 kbp introgression with suppressed recombination between haplotypes of rphq2 and Rphq2 barley cultivars., Abstract: Partial and non-host resistances to rust fungi in barley (Hordeum vulgare) may be based on pathogen-associated molecular pattern (PAMP)-triggered immunity. Understanding partial resistance may help to understand non-host resistance, and vice versa. We constructed two non-gridded BAC libraries from cultivar Vada and line SusPtrit. Vada is immune to non-adapted Puccinia rust fungi, and partially resistant to P. hordei. SusPtrit is susceptible to several non-adapted rust fungi, and has been used for mapping QTLs for non-host and partial resistance. The BAC libraries help to identify genes determining the natural variation for partial and non-host resistances of barley to rust fungi. A major-effect QTL, Rphq2, for partial resistance to P. hordei was mapped in a complete Vada and an incomplete SusPtrit contig. The physical distance between the markers flanking Rphq2 was 195 Kbp in Vada and at least 226 Kbp in SusPtrit. This marker interval was predicted to contain 12 genes in either accession, of which only five genes were in common. The haplotypes represented by Vada and SusPtrit were found in 57 and 43%, respectively, of a 194 barley accessions panel. The lack of homology between the two haplotypes probably explains the suppression of recombination in the Rphq2 area and limit further genetic resolution in fine mapping. The possible candidate genes for Rphq2 encode peroxidases, kinases and a member of seven-in-absentia protein family. This result suggests that Rphq2 does not belong to the NB-LRR gene family and does not resemble any of the partial resistance genes cloned previously.

Quantitative resistance (QR) refers to a resistance that is phenotypically incomplete and is based on the joined effect of several genes, each contributing quantitatively to the level of plant defense. Often, QR remains durably effective, which is the primary driver behind the interest in it. The various terms that are used to refer to QR, such as field resistance, adult plant resistance, and basal resistance, reflect the many properties attributed to it. In this article, we discuss aspects connected to those attributions, in particular the hypothesis that much of the QR to biotrophic filamentous pathogens is basal resistance, i.e., poor suppression of PAMP-triggered defense by effectors. We discuss what role effectors play in suppressing defense or improving access to nutrients. Based on the functions of the few plant proteins identified as involved in QR, vesicle trafficking and protein/metabolite transportation are likely to be common physiological processes relevant to QR.

Background: Zymoseptoria tritici is a hemibiotrophic ascomycete fungus causing leaf blotch of wheat that often decreases yield severely. Populations of the fungus are known to be highly diverse and poorly differentiated from each other. However, a genotyping tool is needed to address further questions in large collections of isolates, regarding regional population structure, adaptation to anthropogenic selective pressures, and dynamics of the recently discovered accessory chromosomes. This procedure is limited by costly and time-consuming simplex PCR genotyping. Recent development of genomic approaches and of larger sets of SSRs enabled the optimization of microsatellite multiplexing., Findings: We report here a reliable protocol to amplify 24 SSRs organized in three multiplex panels, and covering all Z. tritici chromosomes. We also propose an automatic allele assignment procedure, which allows scoring alleles in a repeatable manner across studies and laboratories. All together, these tools enabled us to characterize local and worldwide populations and to calculate diversity indexes consistent with results reported in the literature., Conclusion: This easy-to-use, accurate, repeatable, economical, and faster technical strategy can provide useful genetic information for evolutionary inferences concerning Z. tritici populations. Moreover, it will facilitate the comparison of studies from different scientific groups.

Key Message: We developed 'Golden SusPtrit', i.e., a barley line combining SusPtrit's high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation. Nonhost and partial resistance to Puccinia rust fungi in barley are polygenically inherited. These types of resistance are principally prehaustorial, show high diversity between accessions of the plant species and are genetically associated. To study nonhost and partial resistance, as well as their association, candidate gene(s) for resistance must be cloned and tested in susceptible material where SusPtrit would be the line of choice. Unfortunately, SusPtrit is not amenable to Agrobacterium-mediated transformation. Therefore, a doubled haploid (DH) mapping population (n = 122) was created by crossing SusPtrit with Golden Promise to develop a 'Golden SusPtrit', i.e., a barley line combining SusPtrit's high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation. We identified nine genomic regions occupied by resistance quantitative trait loci (QTLs) against four non-adapted rust fungi and P. hordei isolate 1.2.1 (Ph.1.2.1). Four DHs were selected for an Agrobacterium-mediated transformation efficiency test. They were among the 12 DH lines most susceptible to the tested non-adapted rust fungi. The most efficiently transformed DH line was SG062N (11-17 transformants per 100 immature embryos). The level of non-adapted rust infection on SG062N is either similar to or higher than the level of infection on SusPtrit. Against Ph.1.2.1, the latency period conferred by SG062N is as short as that conferred by SusPtrit. SG062N, designated 'Golden SusPtrit', will be a valuable experimental line that could replace SusPtrit in nonhost and partial resistance studies, especially for stable transformation using candidate genes that may be involved in rust-resistance mechanisms.

ABSTRACT Partial resistance is a quantitative type of resistance that, by definition of Parlevliet, is not based on hypersensitivity. It is largely pathotype nonspecific, although some minor isolate-specific responses have been reported. In order to elucidate the isolate specificity of individual genes for partial resistance, three barley recombinant inbred line mapping populations were analyzed for resistance to the leaf rust fungus Puccinia hordei. The mapping populations were inoculated with one isolate avirulent and two isolates virulent to resistance gene Rph7g. Six significant quantitative trait loci (QTLs) were detected. Of these, two (Rphq3 and Rphq11) were detected with only the avirulent isolate (1.2.1.) and one (Rphq18) only with both virulent isolates (CO-04 and 28.1). The effectiveness of these QTLs was tested with 14 isolates, using a tester set of genotypes containing alleles for resistance or susceptibility for these QTLs. QTL Rphq18 was effective to only two isolates, CO-04 and 28.1, whereas Rphq3 and Rphq11 were ineffective to CO-04 and 28.1 but effective to all other isolates, except one. This resulted in a significant Person's differential interaction, which is a hallmark of a gene-for-gene interaction. The minor gene-for-minor gene interaction is not based on hypersensitivity and there is no evidence that the resistance is based on genes belonging to the nucleotide-binding leucine-rich repeat class.

The potential of the model grass Brachypodium distachyon L. (Brachypodium) for studying grass-pathogen interactions is still underexploited. We aimed to identify genomic regions in Brachypodium associated with quantitative resistance to the false brome rust fungus Puccinia brachypodii . The inbred lines Bd3-1 and Bd1-1, differing in their level of resistance to P. brachypodii, were crossed to develop an F(2) population. This was evaluated for reaction to a virulent isolate of P. brachypodii at both the seedling and advanced growth stages. To validate the results obtained on the F(2), resistance was quantified in F(2)-derived F(3) families in two experiments. Disease evaluations showed quantitative and transgressive segregation for resistance. A new AFLP-based Brachypodium linkage map consisting of 203 loci and spanning 812 cM was developed and anchored to the genome sequence with SSR and SNP markers. Three false brome rust resistance QTLs were identified on chromosomes 2, 3, and 4, and they were detected across experiments. This study is the first quantitative trait analysis in Brachypodium. Resistance to P. brachypodii was governed by a few QTLs: two acting at the seedling stage and one acting at both seedling and advanced growth stages. The results obtained offer perspectives to elucidate the molecular basis of quantitative resistance to rust fungi.

Purple false brome grass (Brachypodium distachyon) has recently emerged as a model system for temperate grasses and is also a potential model plant to investigate plant interactions with economically important pathogens such as rust fungi. We determined the host status of five Brachypodium species to three isolates of Puccinia brachypodii, the prevalent rust species on Brachypodium sylvaticum in nature, and to one isolate each of three formae speciales of the stripe rust fungus P. striiformis. Two P. striiformis isolates produced sporulating lesions, both in only one of the tested interactions, suggesting a marginal host status of B. distachyon. P. brachypodii formed sporulating uredinia on the five Brachypodium species tested, and a range of reactions was observed. Surprisingly, the B. sylvaticum-derived rust isolates were more frequently pathogenic to B. distachyon than to their original host species. The B. distachyon diploid inbred lines, developed and distributed as reference material to the Brachypodium research community, include susceptible and resistant genotypes to at least three of the four P. brachypodii isolates tested. This creates the opportunity to use B. distachyon/P. brachypodii as a model pathosystem. In one B. distachyon accession, heavy infection by the loose smut fungus Ustilago bromivora occurred. That pathogen could also serve as a model pathogen of Brachypodium.

Background: The barley-Puccinia hordei (barley leaf rust) pathosystem is a model for investigating partial disease resistance in crop plants and genetic mapping of phenotypic resistance has identified several quantitative trait loci (QTL) for partial resistance. Reciprocal QTL-specific near-isogenic lines (QTL-NILs) have been developed that combine two QTL, Rphq2 and Rphq3, the largest effects detected in a recombinant-inbred-line (RIL) population derived from a cross between the super-susceptible line L94 and partially-resistant line Vada. The molecular mechanism underpinning partial resistance in these QTL-NILs is unknown., Results: An Agilent custom microarray consisting of 15,000 probes derived from barley consensus EST sequences was used to investigate genome-wide and QTL-specific differential expression of genes 18 hours post-inoculation (hpi) with Puccinia hordei. A total of 1,410 genes were identified as being significantly differentially expressed across the genome, of which 55 were accounted for by the genetic differences defined by QTL-NILs at Rphq2 and Rphq3. These genes were predominantly located at the QTL regions and are, therefore, positional candidates. One gene, encoding the transcriptional repressor Ethylene-Responsive Element Binding Factor 4 (HvERF4) was located outside the QTL at 71 cM on chromosome 1H, within a previously detected eQTL hotspot for defence response. The results indicate that Rphq2 or Rphq3 contains a trans-eQTL that modulates expression of HvERF4. We speculate that HvERF4 functions as an intermediate that conveys the response signal from a gene(s) contained within Rphq2 or Rphq3 to a host of down-stream defense responsive genes. Our results also reveal that barley lines with extreme or intermediate partial resistance phenotypes exhibit a profound similarity in their spectrum of Ph-responsive genes and that hormone-related signalling pathways are actively involved in response to Puccinia hordei., Conclusions: Differential gene expression between QTL-NILs identifies genes predominantly located within the target region(s) providing both transcriptional and positional candidate genes for the QTL. Genetically mapping the differentially expressed genes relative to the QTL has the potential to discover trans-eQTL mediated regulatory relays initiated from genes within the QTL regions.

Partial resistance is generally considered to be a durable form of resistance. In barley, Rphq2, Rphq3 and Rphq4 have been identified as consistent quantitative trait loci (QTLs) for partial resistance to the barley leaf rust pathogen Puccinia hordei. These QTLs have been incorporated separately into the susceptible L94 and the partially resistant Vada barley genetic backgrounds to obtain two sets of near isogenic lines (NILs). Previous studies have shown that these QTLs are not effective at conferring disease resistance in all stages of plant development. In the present study, the two sets of QTL-NILs and the two recurrent parents, L94 and Vada, were evaluated for resistance to P. hordei isolate 1.2.1 simultaneously under greenhouse conditions from the first leaf to the flag leaf stage. Effect of the QTLs on resistance was measured by development rate of the pathogen, expressed as latency period (LP). The data show that Rphq2 prolongs LP at the seedling stage (the first and second leaf stages) but has almost no effect on disease resistance in adult plants. Rphq4 showed no effect on LP until the third leaf stage, whereas Rphq3 is consistently effective at prolonging LP from the first leaf to the flag leaf. The changes in the effectiveness of Rphq2 and Rphq4 happen at the barley tillering stage (the third to fourth leaf stages). These results indicate that multiple disease evaluations of a single plant by repeated inoculations of the fourth leaf to the flag leaf should be conducted to precisely estimate the effect of Rphq4. The present study confirms and describes in detail the plant development-dependent effectiveness of partial resistance genes and, consequently, will enable a more precise evaluation of partial resistance regulation during barley development.

Background: Higher plants possess a large multigene family encoding secreted class III peroxidase (Prx) proteins. Peroxidases appear to be associated with plant disease resistance based on observations of induction during disease challenge and the presence or absence of isozymes in resistant vs susceptible varieties. Despite these associations, there is no evidence that allelic variation of peroxidases directly determines levels of disease resistance., Methodology/principal Findings: The current study introduces a new strategy called Prx-Profiling. We showed that with this strategy a large number of peroxidase genes can be mapped on the barley genome. In order to obtain an estimate of the total number of Prx clusters we followed a re-sampling procedure, which indicated that the barley genome contains about 40 peroxidase gene clusters. We examined the association between the Prxs mapped and the QTLs for resistance of barley to homologous and heterologous rusts, and to the barley powdery mildew fungus. We report that 61% of the QTLs for partial resistance to P. hordei, 61% of the QTLs for resistance to B. graminis and 47% of the QTLs for non-host resistance to other Puccinia species co-localize with Prx based markers., Conclusions/significance: We conclude that Prx-Profiling was effective in finding the genetic location of Prx genes on the barley genome. The finding that QTLs for basal resistance to rusts and powdery mildew fungi tend to co-locate with Prx clusters provides a base for exploring the functional role of Prx-related genes in determining natural differences in levels of basal resistance.

Background: Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes., Methodology/principal Findings: We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordei-infected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis., Conclusions/significance: The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this population.

The basal resistance of barley to powdery mildew (Blumeria graminis f. sp. hordei) is a quantitatively inherited trait that is based on nonhypersensitive mechanisms of defense. A functional genomic approach indicates that many plant candidate genes are involved in the defense against formation of fungal haustoria. It is not known which of these candidate genes have allelic variation that contributes to the natural variation in powdery mildew resistance, because many of them may be highly conserved within the barley species and may act downstream of the basal resistance reaction. Twenty-two expressed sequence tag or cDNA clone sequences that are likely to play a role in the barley-Blumeria interaction based on transcriptional profiling, gene silencing, or overexpression data, as well as mlo, Ror1, and Ror2, were mapped and considered candidate genes for contribution to basal resistance. We mapped the quantitative trait loci (QTL) for powdery mildew resistance in six mapping populations of barley at seedling and adult plant stages and developed an improved high-density integrated genetic map containing 6,990 markers for comparing QTL and candidate gene positions over mapping populations. We mapped 12 QTL at seedling stage and 13 QTL at adult plant stage, of which four were in common between the two developmental stages. Six of the candidate genes showed coincidence in their map positions with the QTL identified for basal resistance to powdery mildew. This co-localization justifies giving priority to those six candidate genes to validate them as being responsible for the phenotypic effects of the QTL for basal resistance.

Nonhost resistance to plant pathogens can be constitutive or induced by microbes. Successful pathogens suppress microbe-induced plant defences by delivering appropriate effectors, which are apparently not sufficiently effective on nonhost plant species, as can be concluded from the strong host specificity of many biotroph plant pathogens. Such effectors act on particular plant targets, such as promoters or motifs in expressed sequences. Despite much progress in the elucidation of the molecular aspects of nonhost resistance to plant pathogens, very little is known about the genes that determine whether effectors can or cannot suppress the basal defence. In hosts they can, in nonhosts they cannot. The targets determining the host status of plants can be identified in inheritance studies. Recent reports have indicated that nonhost resistance is inherited polygenically, and exhibits strong similarity and association with the basal resistance of plants to adapted pathogens.

Inheritance studies on the nonhost resistance of plants would normally require interspecific crosses that suffer from sterility and abnormal segregation. Therefore, we developed the barley-Puccinia rust model system to study, using forward genetics, the specificity, number, and diversity of genes involved in nonhost resistance. We developed two mapping populations by crossing the line SusPtrit, with exceptional susceptibility to heterologous rust species, with the immune barley cultivars Vada and Cebada Capa. These two mapping populations along with the Oregon Wolfe Barley population, which showed unexpected segregation for resistance to heterologous rusts, were phenotyped with four heterologous rust fungal species. Positions of QTL conferring nonhost resistance in the three mapping populations were compared using an integrated consensus map. The results confirmed that nonhost resistance in barley to heterologous rust species is controlled by QTL with different and overlapping specificities and by an occasional contribution of an R-gene for hypersensitivity. In each population, different sets of loci were implicated in resistance. Few genes were common between the populations, suggesting a high diversity of genes conferring nonhost resistance to heterologous pathogens. These loci were significantly associated with QTL for partial resistance to the pathogen Puccinia hordei and with defense-related genes.

Partial resistance is considered race-nonspecific and durable, consistent with the concept of 'horizontal' resistance. However, detailed observations of partial resistance to leaf rust (Puccinia hordei) in barley (Hordeum vulgare) revealed small cultivar x isolate interactions, suggesting a minor-gene-for-minor-gene interaction model, similar to so-called 'vertical' resistance. Three consistent quantitative trait loci (QTLs), labelled Rphq2, Rphq3 and Rphq4, that were detected in the cross susceptible L94 x partially resistant Vada have been incorporated into the L94 background to obtain near-isogenic lines (NILs). Three isolates were used to map QTLs on seedlings of the L94 x Vada population and to evaluate the effect of each QTL on adult plants of the respective NILs under field conditions. Rphq2 had a strong effect in seedlings but almost no effect in adult plants, while Rphq3 was effective in seedlings and in adult plants against all three isolates. However, Rphq4 was effective in seedlings and in adult plants against two isolates but ineffective in both development stages against the third, demonstrating a clear and reproducible isolate-specific effect. The resistance governed by the three QTLs was not associated with a hypersensitive reaction. Those results confirm the minor-gene-for-minor-gene model suggesting specific interactions between QTLs for partial resistance and P. hordei isolates.

Partial resistance to leaf rust (Puccinia hordei G. H. Otth) in barley is a quantitative resistance that is not based on hypersensitivity. This resistance hampers haustorium formation, resulting in a long latency period in greenhouse tests. The three most consistent quantitative trait loci (QTL) uncovered in the L94 x 'Vada' mapping population were introgressed by marker-assisted backcrossing into the susceptible L94 background to obtain near-isogenic lines (NIL). We also developed the reciprocal Vada-NIL for the susceptibility alleles of those QTL. The QTL Rphq2 affected latency period of P. hordei more than the QTL Rphq3 and Rphq4. The NIL confirmed the contribution of Rphq2 to partial resistance by prolonging the latency period by 28 h on L94-Rphq2 and shortening the latency period by 23 h on Vada-rphq2. On the basis of flanking restriction fragment length polymorphism-based markers, Rphq2 appeared to be located near the telomeric end of the long arm of chromosome 2H, in a physical region of high recombination, making it the target QTL for map-based cloning. Microscopic observations on the NIL confirmed the nonhypersensitive nature of the resistance conferred by Rphq2. A high-resolution genetic map of the Rphq2 region was constructed using a population of 38 subNIL with overlapping L94 introgressions in Vada background across the region. Rphq2 mapped approximately 2 centimorgans (cM) proximal from the MlLa locus. By bulked segregant analysis and use of synteny with rice, we developed additional markers and fine-mapped Rphq2 to a genetic interval of 0.11 cM that corresponds to a stretch of sequence of, at most, 70 kb in rice. Analysis of this rice sequence revealed predicted genes encoding two proteins with unknown function, retrotransposon proteins, peroxidase proteins, and a protein similar to a mitogen-activated protein kinase kinase kinase (MAP3K). Possible homologs of those peroxidases and MAP3K in barley are candidates for the gene that contributes to partial resistance to P. hordei.

A microsatellite or simple sequence repeat (SSR) consensus map of barley was constructed by joining six independent genetic maps based on the mapping populations 'Igri x Franka', 'Steptoe x Morex', 'OWB(Rec) x OWB(Dom)', 'Lina x Canada Park', 'L94 x Vada' and 'SusPtrit x Vada'. Segregation data for microsatellite markers from different research groups including SCRI (Bmac, Bmag, EBmac, EBmag, HVGeneName, scsssr), IPK (GBM, GBMS), WUR (GBM), Virginia Polytechnic Institute (HVM), and MPI for Plant Breeding (HVGeneName), generated in above mapping populations, were used in the computer program RECORD to order the markers of the individual linkage data sets. Subsequently, a framework map was constructed for each chromosome by integrating the 496 "bridge markers" common to two or more individual maps with the help of the computer programme JoinMap 3.0. The final map was calculated by following a "neighbours" map approach. The integrated map contained 775 unique microsatellite loci, from 688 primer pairs, ranging from 93 (6H) to 132 (2H) and with an average of 111 markers per linkage group. The genomic DNA-derived SSR marker loci had a higher polymorphism information content value (average 0.61) as compared to the EST/gene-derived SSR loci (average 0.48). The consensus map spans 1,068 cM providing an average density of one SSR marker every 1.38 cM. Such a high-density consensus SSR map provides barley molecular breeding programmes with a better choice regarding the quality of markers and a higher probability of polymorphic markers in an important chromosomal interval. This map also offers the possibilities of thorough alignment for the (future) physical map and implementation in haplotype diversity studies of barley.

In plant breeding and germplasm evaluation, large-scale assessment of quantitative resistance is desirable, but feasible only if a simple and accurate measure is available. In several plant-pathogen systems, latent period (LP) is a parameter that is well correlated with the level of partial resistance observed in field trials. However, measuring LP or relative LP (RLP), i.e., relative to the reference accessions, is laborious. We investigated the value of relative ratio of mature pustules (RRMP, relative to the susceptible control) as a simple and rapid alternative to replace LP estimation in barley to barley leaf rust (Puccinia hordei). A set of 103 F 9 recombinant inbred lines (RILs) derived from a cross L94 × Vada was sown in a greenhouse compartment, and was inoculated at the seedling stage with isolate Uppsala or 24, and at the adult plant stage with isolate 24. In demarcated sections of leaves, the number of mature pustules was counted several times after inoculation. The ratio of mature pustules (RMP) and LP50 were calculated to assess RRMP and RLP, respectively, and to identify the quantitative trait loci (QTLs) contributing to the genetic variation. The contrasts in RRMP among accessions were highest when the susceptible reference line had developed 70 to 90% mature orange pustules, the immature infection sites being visible as pale flecks. At this optimal time of observation, the correlation between RRMP and RLP in both the seedling stage and the adult plant stage was highly significant (r = -0.82 ~-0.98). Compared with RLP, RRMP was much easier and simpler to measure and still showed good correspondence with RLP in the identification of QTLs for partial resistance. In another experiment at the seedling stage with 25 barley cultivars and lines inoculated with isolate 1.2.1, the coefficient of correlation between RRMP and RLP was -0.98. Therefore, we conclude that RRMP should have great application potential in breeding programs and germplasm screening and could be used in fundamental studies as well.

A consensus map of barley was constructed based on three reference doubled haploid (DH) populations and three recombinant inbred line (RIL) populations. Several sets of microsatellites were used as bridge markers in the integration of those populations previously genotyped with RFLP or with AFLP markers. Another set of 61 genic microsatellites was mapped for the first time using a newly developed fluorescent labelling strategy, referred to as A/T labelling. The final map contains 3,258 markers spanning 1,081 centiMorgans (cM) with an average distance between two adjacent loci of 0.33 cM. This is the highest density of markers reported for a barley genetic map to date. The consensus map was divided into 210 BINs of about 5 cM each in which were placed 19 quantitative trait loci (QTL) contributing to the partial resistance to barley leaf rust (Puccinia hordei Otth) in five of the integrated populations. Each parental barley combination segregated for different sets of QTLs, with only few QTLs shared by any pair of cultivars. Defence gene homologues (DGH) were identified by tBlastx homology to known genes involved in the defence of plants against microbial pathogens. Sixty-three DGHs were located into the 210 BINs in order to identify candidate genes responsible for the QTL effects. Eight BINs were co-occupied by a QTL and DGH(s). The positional candidates identified are receptor-like kinase, WIR1 homologues and several defence response genes like peroxidases, superoxide dismutase and thaumatin.

Tomato powdery mildew caused by Oidium neolycopersici has become a globally important disease of tomato (Lycopersicon esculentum). To study the defense responses of tomato triggered by tomato powdery mildew, we first mapped a set of resistance genes to O. neolycopersici from related Lycopersicon species. An integrated genetic map was generated showing that all the dominant resistance genes (Ol-1, Ol-3, Ol-4, Ol-5, and Ol-6) are located on tomato chromosome 6 and are organized in three genetic loci. Then, near-isogenic lines (NIL) were produced that contain the different dominant Ol genes in a L. esculentum genetic background. These NIL were used in disease tests with local isolates of O. neolycopersici in different geographic locations, demonstrating that the resistance conferred by different Ol genes was isolate-dependent and, hence, may be race-specific. In addition, the resistance mechanism was analyzed histologically. The mechanism of resistance conferred by the dominant Ol genes was associated with hypersensitive response, which varies in details depending on the Ol-gene in the NIL, while the mechanism of resistance governed by the recessive gene ol-2 on tomato chromosome 4 was associated with papillae formation.