Transformation and gene-disruption in the apple-pathogen, Neonectria ditissima

Background Apple production in Sweden and elsewhere is being threatened by the fungus, Neonectria ditissima, which causes a disease known as European canker. The disease can cause extensive damage and the removal of diseased wood and heavily infected trees can be laborious and expensive. Currently, there is no way to eradicate the fungus from infected trees and our knowledge of the infection process is limited. Thus, to target and modify genes efficiently, the genetic transformation technique developed for N. ditissima back in 2003 was modified. Results The original protocol from 2003 was upgraded to use enzymes currently available in the market for making protoplasts. The protoplasts were viable, able to uptake foreign DNA, and able to regenerate back into a mycelial colony, either as targeted gene-disruption mutants or as ectopic mutants expressing the green fluorescent protein (GFP). Conclusions A new genetic transformation protocol has been established and the inclusion of hydroxyurea in the buffer during the protoplast-generation step greatly increased the creation of knockout mutants via homologous recombination. Pathogenicity assays using the GFP-mutants showed that the mutants were able to infect the host and cause disease

Characterization of the Resistance to Powdery Mildew and Leaf Rust Carried by the Bread Wheat Cultivar Victo

Leaf rust and powdery mildew are two important foliar diseases in wheat. A recombinant inbred line (RIL) population, obtained by crossing two bread wheat cultivars ('Victo' and 'Spada'), was evaluated for resistance to the two pathogens at seedling stage. Upon developing a genetic map of 8726 SNP loci, linkage analysis identified three resistance Quantitative Trait Loci (QTLs), with 'Victo' contributing the resistant alleles to all loci. One major QTL (QPm.gb-7A) was detected in response to Blumeria graminis on chromosome 7A, which explained 90% of phenotypic variation (PV). The co-positional relationship with known powdery mildew (Pm) resistance loci suggested that a new source of resistance was identified in T. aestivum. Two QTLs were detected in response to Puccinia triticina: a major gene on chromosome 5D (QLr.gb-5D), explaining a total PV of about 59%, and a minor QTL on chromosome 2B (QLr.gb-2B). A positional relationship was observed between the QLr.gb-5D with the known Lr1 gene, but polymorphisms were found between the cloned Lr1 and the corresponding 'Victo' allele, suggesting that QLr.gb-5D could represent a new functional Lr1 allele. Lastly, upon anchoring the QTL on the T. aestivum reference genome, candidate genes were hypothesized on the basis of gene annotation and in silico gene expression analysis

Breeding Wheat for Powdery Mildew Resistance: Genetic Resources and Methodologies-A Review

Powdery mildew (PM) of wheat caused by Blumeria graminis f. sp. tritici is among the most important wheat diseases, causing significant yield and quality losses in many countries worldwide. Considerable progress has been made in resistance breeding to mitigate powdery mildew. Genetic host resistance employs either race-specific (qualitative) resistance, race-non-specific (quantitative), or a combination of both. Over recent decades, efforts to identify host resistance traits to powdery mildew have led to the discovery of over 240 genes and quantitative trait loci (QTLs) across all 21 wheat chromosomes. Sources of PM resistance in wheat include landraces, synthetic, cultivated, and wild species. The resistance identified in various genetic resources is transferred to the elite genetic background of a well-adapted cultivar with minimum linkage drag using advanced breeding and selection approaches. In this effort, wheat landraces have emerged as an important source of allelic and genetic diversity, which is highly valuable for developing new PM-resistant cultivars. However, most landraces have not been characterized for PM resistance, limiting their use in breeding programs. PM resistance is a polygenic trait; therefore, the degree of such resistance is mostly influenced by environmental conditions. Another challenge in breeding for PM resistance has been the lack of consistent disease pressure in multi-environment trials, which compromises phenotypic selection efficiency. It is therefore imperative to complement conventional breeding technologies with molecular breeding to improve selection efficiency. High-throughput genotyping techniques, based on chip array or sequencing, have increased the capacity to identify the genetic basis of PM resistance. However, developing PM-resistant cultivars is still challenging, and there is a need to harness the potential of new approaches to accelerate breeding progress. The main objective of this review is to describe the status of breeding for powdery mildew resistance, as well as the latest discoveries that offer novel ways to achieve durable PM resistance. Major topics discussed in the review include the genetic basis of PM resistance in wheat, available genetic resources for race-specific and adult-plant resistance to PM, important gene banks, and conventional and complimentary molecular breeding approaches, with an emphasis on marker-assisted selection (MAS)

Cross-Kingdom RNAi of Pathogen Effectors Leads to Quantitative Adult Plant Resistance in Wheat

Cross-kingdom RNA interference (RNAi) is a biological process allowing plants to transfer small regulatory RNAs to invading pathogens to trigger the silencing of target virulence genes. Transient assays in cereal powdery mildews suggest that silencing of one or two effectors could lead to near loss of virulence, but evidence from stable RNAi lines is lacking. We established transient host-induced gene silencing (HIGS) in wheat, and demonstrate that targeting an essential housekeeping gene in the wheat powdery mildew pathogen (Blumeria graminis f. sp. tritici) results in significant reduction of virulence at an early stage of infection. We generated stable transgenic RNAi wheat lines encoding a HIGS construct simultaneously silencing three B.g. tritici effectors including SvrPm3(a1/f1), a virulence factor involved in the suppression of the Pm3 powdery mildew resistance gene. We show that all targeted effectors are effectively downregulated by HIGS, resulting in reduced fungal virulence on adult wheat plants. Our findings demonstrate that stable HIGS of effector genes can lead to quantitative gain of resistance without major pleiotropic effects in wheat

Ancient variation of the AvrPm17 gene in powdery mildew limits the effectiveness of the introgressed rye Pm17 resistance gene in wheat

Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable-a phenomenon that remains poorly understood, as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used quantitative trait locus (QTL) mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of reoccurring gene conversion events and are members of a mildew sublineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sublineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangling complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations become an integral part of introgression breeding to ensure effective and durable resistance in wheat

Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade

The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, we study its spread and evolution by analyzing a global sample of 172 mildew genomes. Our analyses show that B.g. tritici emerged in the Fertile Crescent during wheat domestication. After it spread throughout Eurasia, colonization brought it to America, where it hybridized with unknown grass mildew species. Recent trade brought USA strains to Japan, and European strains to China. In both places, they hybridized with local ancestral strains. Thus, although mildew spreads by wind regionally, our results indicate that humans drove its global spread throughout history and that mildew rapidly evolved through hybridization.Centro de Investigaciones AgropecuariasFil: Sotiropoulos, Alexandros G. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Arango-Isaza, Epifanía. University of Zurich. Department of Evolutionary Biology and Environmental Studies; SuizaFil: Ban, Tomohiro. Yokohama City University. Kihara Institute for Biological Research; JapónFil: Barbieri, Chiara. University of Zurich. Department of Evolutionary Biology and Environmental Studies; SuizaFil: Barbieri, Chiara. Max Planck Institute for Evolutionary Anthropology. Department of Linguistic and Cultural Evolution; AlemaniaFil: Bourras, Salim. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Bourras, Salim. University of Agricultural Sciences. Department of Forest Mycology and Plant Pathology; SueciaFil: Cowger, Christina. North Carolina State University; Estados Unidos. USDA-ARS Department of Plant Pathology; Estados UnidosFil: Czembor, Paweł C. National Research Institute. Plant Breeding and Acclimatization Institute; PoloniaFil: Ben-David, Roi. ARO-Volcani Center. Institute of Plant Sciences. Department of Vegetables and Field Crops; IsraelFil: Dinoor, Amos. University of Jerusalem. The Robert H. Smith Faculty of Agriculture, Food & Environment. Department of Plant Pathology and Microbiology; IsraelFil: Ellwood, Simon R. Curtin University. School of Molecular and Life Sciences. Centre for Crop and Disease Management; AustraliaFil: Graf, Johannes. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Hatta, Koichi. National Agricultural Research Organization. Hokkaido Agricultural Research Center Field Crop Research and Development; JapónFil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Centro de Investigaciones Agropecuarias; ArgentinaFil: Wicker, Thomas. University of Zurich. Department of Plant and Microbial Biology; Suiz

A chromosome-scale genome assembly reveals a highly dynamic effector repertoire of wheat powdery mildew

Blumeria graminis f. sp. tritici (B.g. tritici) is the causal agent of the wheat powdery mildew disease. The highly fragmented B.g. tritici genome available so far has prevented a systematic analysis of effector genes that are known to be involved in host adaptation. To study the diversity and evolution of effector genes we produced a chromosome‐scale assembly of the B.g. tritici genome. The genome assembly and annotation was achieved by combining long‐read sequencing with high‐density genetic mapping, bacterial artificial chromosome fingerprinting and transcriptomics. We found that the 166.6 Mb B.g. tritici genome encodes 844 candidate effector genes, over 40% more than previously reported. Candidate effector genes have characteristic local genomic organization such as gene clustering and enrichment for recombination‐active regions and certain transposable element families. A large group of 412 candidate effector genes shows high plasticity in terms of copy number variation in a global set of 36 isolates and of transcription levels. Our data suggest that copy number variation and transcriptional flexibility are the main drivers for adaptation in B.g. tritici. The high repeat content may play a role in providing a genomic environment that allows rapid evolution of effector genes with selection as the driving force

Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade

The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, Sotiropoulos et al. analyze a global sample of 172 mildew genomes, providing evidence that humans drove global spread of the pathogen throughout history and that mildew rapidly evolved through hybridization with local fungal strains.The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, we study its spread and evolution by analyzing a global sample of 172 mildew genomes. Our analyses show that B.g. tritici emerged in the Fertile Crescent during wheat domestication. After it spread throughout Eurasia, colonization brought it to America, where it hybridized with unknown grass mildew species. Recent trade brought USA strains to Japan, and European strains to China. In both places, they hybridized with local ancestral strains. Thus, although mildew spreads by wind regionally, our results indicate that humans drove its global spread throughout history and that mildew rapidly evolved through hybridization

Hybridization of powdery mildew strains gives rise to pathogens on novel agricultural crop species

Throughout the history of agriculture, many new crop species (polyploids or artificial hybrids) have been introduced to diversify products or to increase yield. However, little is known about how these new crops influence the evolution of new pathogens and diseases. Triticale is an artificial hybrid of wheat and rye, and it was resistant to the fungal pathogen powdery mildew (Blumeria graminis) until 2001 (refs. 1,2,3). We sequenced and compared the genomes of 46 powdery mildew isolates covering several formae speciales. We found that B. graminis f. sp. triticale, which grows on triticale and wheat, is a hybrid between wheat powdery mildew (B. graminis f. sp. tritici) and mildew specialized on rye (B. graminis f. sp. secalis). Our data show that the hybrid of the two mildews specialized on two different hosts can infect the hybrid plant species originating from those two hosts. We conclude that hybridization between mildews specialized on different species is a mechanism of adaptation to new crops introduced by agriculture