A secreted protease-like protein in Zymoseptoria tritici is responsible for avirulence on Stb9 resistance gene in wheat.

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.

The Egyptian wheat cultivar Gemmeiza-12 is a source of resistance against the fungus Zymoseptoria tritici.

BACKGROUND: Wheat is one of the world's most important cereal crops. However, the fungal pathogen Zymoseptoria tritici can cause disease epidemics, leading to reduced yields. With climate change and development of new agricultural areas with suitable environments, Z. tritici may advance into geographical areas previously unaffected by this pathogen. It is currently unknown how Egyptian wheat will perform in the face of this incoming threat. This project aimed to assess the resistance of Egyptian wheat germplasm to Z. tritici, to identify cultivars with high levels of resistance and characterise the mechanism(s) of resistance present in these cultivars. RESULTS: Eighteen Egyptian wheat cultivars were screened against two Z. tritici model isolates and exhibited a wide spectrum of responses. This ranged from resistance to complete susceptibility to one or both isolates tested. The most highly resistant cultivars from the initial screen were then tested under two environmental conditions against modern UK field isolates. Disease levels under UK-like conditions were higher, however, symptom development on the cultivar Gemmeiza-12 was noticeably slower than on other Egyptian wheats. The robustness of the resistance shown by Gemmeiza-12 was confirmed in experiments mimicking Egyptian environmental conditions, where degree of Z. tritici infection was lower. The Kompetitive allele-specific PCR (KASP) diagnostic assay suggested the presence of an Stb6 resistant allele in several Egyptian wheats including Gemmeiza-12. Infection assays using the IPO323 WT and IPO323ΔAvrStb6 mutant confirmed the presence of Stb6 in several Egyptian cultivars including Gemmeiza-12. Confocal fluorescence microscopy demonstrated that growth of the IPO323 strain is blocked at the point of stomatal penetration on Gemmeiza-12, consistent with previous reports of Stb gene mediated resistance. In addition to this R-gene mediated resistance, IPO323 spores showed lower adherence to leaves of Gemmeiza-12 compared to UK wheat varieties, suggesting other aspects of leaf physiology may also contribute to the resistance phenotype of this cultivar. CONCLUSION: These results indicate that Gemmeiza-12 will be useful in future breeding programs where improved resistance to Z. tritici is a priority.

Stomatal penetration: the cornerstone of plant resistance to the fungal pathogen Zymoseptoria tritici.

BACKGROUND: Septoria tritici blotch (STB), caused by the foliar fungus Zymoseptoria tritici, is one of the most damaging disease of wheat in Europe. Genetic resistance against this fungus relies on different types of resistance from non-host resistance (NHR) and host species specific resistance (HSSR) to host resistance mediated by quantitative trait loci (QTLs) or major resistance genes (Stb). Characterizing the diversity of theses resistances is of great importance for breeding wheat cultivars with efficient and durable resistance. While the functional mechanisms underlying these resistance types are not well understood, increasing piece of evidence suggest that fungus stomatal penetration and early establishment in the apoplast are both crucial for the outcome of some interactions between Z. tritici and plants. To validate and extend these previous observations, we conducted quantitative comparative phenotypical and cytological analyses of the infection process corresponding to 22 different interactions between plant species and Z. tritici isolates. These interactions included four major bread wheat Stb genes, four bread wheat accessions with contrasting quantitative resistance, two species resistant to Z. tritici isolates from bread wheat (HSSR) and four plant species resistant to all Z. tritici isolates (NHR). RESULTS: Infiltration of Z. tritici spores into plant leaves allowed the partial bypass of all bread wheat resistances and durum wheat resistance, but not resistances from other plants species. Quantitative comparative cytological analysis showed that in the non-grass plant Nicotiana benthamiana, Z. tritici was stopped before stomatal penetration. By contrast, in all resistant grass plants, Z. tritici was stopped, at least partly, during stomatal penetration. The intensity of this early plant control process varied depending on resistance types, quantitative resistances being the least effective. These analyses also demonstrated that Stb-mediated resistances, HSSR and NHR, but not quantitative resistances, relied on the strong growth inhibition of the few Z. tritici penetrating hyphae at their entry point in the sub-stomatal cavity. CONCLUSIONS: In addition to furnishing a robust quantitative cytological assessment system, our study uncovered three stopping patterns of Z. tritici by plant resistances. Stomatal resistance was found important for most resistances to Z. tritici, independently of its type (Stb, HSSR, NHR). These results provided a basis for the functional analysis of wheat resistance to Z. tritici and its improvement.

Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars.

Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat.

Genetics of resistance to Zymoseptoria tritici and applications to wheat breeding.

This paper reviews current knowledge about genes for resistance to Septoria tritici blotch (STB) of wheat, caused by Zymoseptoria tritici (formerly Mycosphaerella graminicola). These genes can be placed into two classes, although a few may have characteristics of both classes. Qualitative resistance is controlled by genes which control large fractions of genetic variation, 21 of which have been discovered and mapped so far. Most of them have been shown to be genotype-specific, being effective against the minority of Z. tritici isolates which are avirulent, and Stb6 has been shown to control a gene-for-gene relationship. Most qualitative resistances are unlikely to be durable and some formerly effective genes have been overcome by the evolution of pathogen virulence. Quantitative resistance is generally controlled by genes with small-to-moderate effects on STB. They have generally weaker specificity than qualitative genes and have provided more durable resistance. 89 genome regions carrying quantitative trait loci (QTL) or meta-QTL have been identified to date. Some QTL have been mapped at or near loci of qualitative genes, especially Stb6, which is present in several sources of resistance. Another gene of particular interest is Stb16q, which has been effective against all Z. tritici isolates tested so far. In addition to resistance, the susceptibility of wheat cultivars to STB can also be reduced by disease escape traits, some of which may be undesirable in breeding. The fundamental requirements for breeding for STB-resistance are genetic diversity for resistance in wheat germplasm and a field trial site at which STB epidemics occur regularly and effective selection can be conducted for resistance combined with other desirable traits. If these are in place, knowledge of resistance genes can be applied to improving control of STB.

SeptoSympto: a precise image analysis of Septoria tritici blotch disease symptoms using deep learning methods on scanned images.

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.

Variation in crossover rates across a 3-Mb contig of bread wheat (Triticum aestivum) reveals the presence of a meiotic recombination hotspot.

In bread wheat (Triticum aestivum L.), initial studies using deletion lines indicated that crossover (CO) events occur mainly in the telomeric regions of the chromosomes with a possible correlation with the presence of genes. However, little is known about the distribution of COs at the sequence level. To investigate this, we studied in detail the pattern of COs along a contig of 3.110 Mb using two F2 segregating populations (Chinese Spring × Renan (F2-CsRe) and Chinese Spring × Courtot (F2-CsCt)) each containing ~2,000 individuals. The availability of the sequence of the contig from Cs enabled the development of 318 markers among which 23 co-dominant polymorphic markers (11 SSRs and 12 SNPs) were selected for CO distribution analyses. The distribution of CO events was not homogeneous throughout the contig, ranging from 0.05 to 2.77 cM/Mb, but was conserved between the two populations despite very different contig recombination rate averages (0.82 cM/Mb in F2-CsRe vs 0.35 cM/Mb in F2-CsCt). The CO frequency was correlated with the percentage of coding sequence in Cs and with the polymorphism rate between Cs and Re or Ct in both populations, indicating an impact of these two factors on CO distribution. At a finer scale, COs were found in a region covering 2.38 kb, spanning a gene coding for a glycosyl transferase (Hga3), suggesting the presence of a CO hotspot. A non-crossover event covering at least 453 bp was also identified in the same interval. From these results, we can conclude that gene content could be one of the factors driving recombination in bread wheat.

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici.

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is among the most threatening wheat diseases in Europe. Genetic resistance remains one of the main environmentally sustainable strategies to efficiently control STB. However, the molecular and physiological mechanisms underlying resistance are still unknown, limiting the implementation of knowledge-driven management strategies. Among the 22 known major resistance genes (Stb), the recently cloned Stb16q gene encodes a cysteine-rich receptor-like kinase conferring a full broad-spectrum resistance against Z. tritici. Here, we showed that an avirulent Z. tritici inoculated on Stb16q quasi near isogenic lines (NILs) either by infiltration into leaf tissues or by brush inoculation of wounded tissues partially bypasses Stb16q-mediated resistance. To understand this bypass, we monitored the infection of GFP-labeled avirulent and virulent isolates on Stb16q NILs, from germination to pycnidia formation. This quantitative cytological analysis revealed that 95% of the penetration attempts were unsuccessful in the Stb16q incompatible interaction, while almost all succeeded in compatible interactions. Infectious hyphae resulting from the few successful penetration events in the Stb16q incompatible interaction were arrested in the sub-stomatal cavity of the primary-infected stomata. These results indicate that Stb16q-mediated resistance mainly blocks the avirulent isolate during its stomatal penetration into wheat tissue. Analyses of stomatal aperture of the Stb16q NILs during infection revealed that Stb16q triggers a temporary stomatal closure in response to an avirulent isolate. Finally, we showed that infiltrating avirulent isolates into leaves of the Stb6 and Stb9 NILs also partially bypasses resistances, suggesting that arrest during stomatal penetration might be a common major mechanism for Stb-mediated resistances.

Remarkable recent changes in the genetic diversity of the avirulence gene AvrStb6 in global populations of the wheat pathogen Zymoseptoria tritici.

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is one of the most economically important diseases of wheat. Recently, both factors of a gene-for-gene interaction between Z. tritici and wheat, the wheat receptor-like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high diversity of AvrStb6 haplotypes present in earlier Z. tritici isolate collections, with up to c.18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic diversity and the incidence of haplotypes allowing evasion of Stb6-mediated resistance in more recent Z. tritici populations. Here we show, using targeted resequencing of AvrStb6, that this gene is universally present in field isolates sampled from major wheat-growing regions of the world in 2013-2017. However, in contrast to the data from previous AvrStb6 population studies, we report a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of haplotypes, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6-containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence haplotypes tend to be eliminated in subsequent populations.

A wheat cysteine-rich receptor-like kinase confers broad-spectrum resistance against Septoria tritici blotch.

The poverty of disease resistance gene reservoirs limits the breeding of crops for durable resistance against evolutionary dynamic pathogens. Zymoseptoria tritici which causes Septoria tritici blotch (STB), represents one of the most genetically diverse and devastating wheat pathogens worldwide. No fully virulent Z. tritici isolates against synthetic wheats carrying the major resistant gene Stb16q have been identified. Here, we use comparative genomics, mutagenesis and complementation to identify Stb16q, which confers broad-spectrum resistance against Z. tritici. The Stb16q gene encodes a plasma membrane cysteine-rich receptor-like kinase that was recently introduced into cultivated wheat and which considerably slows penetration and intercellular growth of the pathogen.

Detailed recombination studies along chromosome 3B provide new insights on crossover distribution in wheat (Triticum aestivum L.).

In wheat (Triticum aestivum L.), the crossover (CO) frequency increases gradually from the centromeres to the telomeres. However, little is known about the factors affecting both the distribution and the intensity of recombination along this gradient. To investigate this, we studied in detail the pattern of CO along chromosome 3B of bread wheat. A dense reference genetic map comprising 102 markers homogeneously distributed along the chromosome was compared to a physical deletion map. Most of the COs (90%) occurred in the distal subtelomeric regions that represent 40% of the chromosome. About 27% of the proximal regions surrounding the centromere showed a very weak CO frequency with only three COs found in the 752 gametes studied. Moreover, we observed a clear decrease of CO frequency on the distal region of the short arm. Finally, the intensity of interference was assessed for the first time in wheat using a Gamma model. The results showed m values of 1.2 for male recombination and 3.5 for female recombination, suggesting positive interference along wheat chromosome 3B.

Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici.

Deployment of fast-evolving disease-resistance genes is one of the most successful strategies used by plants to fend off pathogens1,2. In gene-for-gene relationships, most cloned disease-resistance genes encode intracellular nucleotide-binding leucine-rich-repeat proteins (NLRs) recognizing pathogen-secreted isolate-specific avirulence (Avr) effectors delivered to the host cytoplasm3,4. This process often triggers a localized hypersensitive response, which halts further disease development 5 . Here we report the map-based cloning of the wheat Stb6 gene and demonstrate that it encodes a conserved wall-associated receptor kinase (WAK)-like protein, which detects the presence of a matching apoplastic effector6-8 and confers pathogen resistance without a hypersensitive response 9 . This report demonstrates gene-for-gene disease resistance controlled by this class of proteins in plants. Moreover, Stb6 is, to our knowledge, the first cloned gene specifying resistance to Zymoseptoria tritici, an important foliar fungal pathogen affecting wheat and causing economically damaging septoria tritici blotch (STB) disease10-12.

Sequence-based mapping of the polyploid wheat genome.

The emergence of new sequencing technologies has provided fast and cost-efficient strategies for high-resolution mapping of complex genomes. Although these approaches hold great promise to accelerate genome analysis, their application in studying genetic variation in wheat has been hindered by the complexity of its polyploid genome. Here, we applied the next-generation sequencing of a wheat doubled-haploid mapping population for high-resolution gene mapping and tested its utility for ordering shotgun sequence contigs of a flow-sorted wheat chromosome. A bioinformatical pipeline was developed for reliable variant analysis of sequence data generated for polyploid wheat mapping populations. The results of variant mapping were consistent with the results obtained using the wheat 9000 SNP iSelect assay. A reference map of the wheat genome integrating 2740 gene-associated single-nucleotide polymorphisms from the wheat iSelect assay, 1351 diversity array technology, 118 simple sequence repeat/sequence-tagged sites, and 416,856 genotyping-by-sequencing markers was developed. By analyzing the sequenced megabase-size regions of the wheat genome we showed that mapped markers are located within 40-100 kb from genes providing a possibility for high-resolution mapping at the level of a single gene. In our population, gene loci controlling a seed color phenotype cosegregated with 2459 markers including one that was located within the red seed color gene. We demonstrate that the high-density reference map presented here is a useful resource for gene mapping and linking physical and genetic maps of the wheat genome.

Identification of wheat gene Sr35 that confers resistance to Ug99 stem rust race group.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating disease that can cause severe yield losses. A previously uncharacterized Pgt race, designated Ug99, has overcome most of the widely used resistance genes and is threatening major wheat production areas. Here, we demonstrate that the Sr35 gene from Triticum monococcum is a coiled-coil, nucleotide-binding, leucine-rich repeat gene that confers near immunity to Ug99 and related races. This gene is absent in the A-genome diploid donor and in polyploid wheat but is effective when transferred from T. monococcum to polyploid wheat. The cloning of Sr35 opens the door to the use of biotechnological approaches to control this devastating disease and to analyses of the molecular interactions that define the wheat-rust pathosystem.

Targeted analysis of nucleotide and copy number variation by exon capture in allotetraploid wheat genome.

BACKGROUND: The ability of grass species to adapt to various habitats is attributed to the dynamic nature of their genomes, which have been shaped by multiple rounds of ancient and recent polyploidization. To gain a better understanding of the nature and extent of variation in functionally relevant regions of a polyploid genome, we developed a sequence capture assay to compare exonic sequences of allotetraploid wheat accessions. RESULTS: A sequence capture assay was designed for the targeted re-sequencing of 3.5 Mb exon regions that surveyed a total of 3,497 genes from allotetraploid wheat. These data were used to describe SNPs, copy number variation and homoeologous sequence divergence in coding regions. A procedure for variant discovery in the polyploid genome was developed and experimentally validated. About 1% and 24% of discovered SNPs were loss-of-function and non-synonymous mutations, respectively. Under-representation of replacement mutations was identified in several groups of genes involved in translation and metabolism. Gene duplications were predominant in a cultivated wheat accession, while more gene deletions than duplications were identified in wild wheat. CONCLUSIONS: We demonstrate that, even though the level of sequence similarity between targeted polyploid genomes and capture baits can bias enrichment efficiency, exon capture is a powerful approach for variant discovery in polyploids. Our results suggest that allopolyploid wheat can accumulate new variation in coding regions at a high rate. This process has the potential to broaden functional diversity and generate new phenotypic variation that eventually can play a critical role in the origin of new adaptations and important agronomic traits.

A physical map of the 1-gigabase bread wheat chromosome 3B.

As the staple food for 35% of the world's population, wheat is one of the most important crop species. To date, sequence-based tools to accelerate wheat improvement are lacking. As part of the international effort to sequence the 17-billion-base-pair hexaploid bread wheat genome (2n = 6x = 42 chromosomes), we constructed a bacterial artificial chromosome (BAC)-based integrated physical map of the largest chromosome, 3B, that alone is 995 megabases. A chromosome-specific BAC library was used to assemble 82% of the chromosome into 1036 contigs that were anchored with 1443 molecular markers, providing a major resource for genetic and genomic studies. This physical map establishes a template for the remaining wheat chromosomes and demonstrates the feasibility of constructing physical maps in large, complex, polyploid genomes with a chromosome-based approach.