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RNA interference (RNAi) is a technique used for transgene-mediated gene silencing based on the mechanism of posttranscriptional gene silencing (PTGS). PTGS is an ubiquitous basic biological phenomenon involved in the regulation of transcript abundance and plants' immune response to viruses. PTGS also mediates genomic stability by silencing of retroelements. RNAi has become an important research tool for studying gene function by strong and selective suppression of target genes. Here, we present , a software tool for design optimization of RNAi constructs necessary for specific target gene knock-down. It offers efficiency prediction of RNAi sequences and off-target search, required for the practical application of RNAi. is an open-source (CC BY-SA license) desktop software that works in Microsoft Windows environment and can use custom sequence databases in standard FASTA format.

The increasing availability of genetic and genomic resources has underscored the need for automated microscopic phenotyping in plant-pathogen interactions to identify genes involved in disease resistance. Building on accumulated experience and leveraging automated microscopy and software, we developed BluVision Micro , a modular, machine learning-aided system designed for high-throughput microscopic phenotyping. This system is adaptable to various image data types and extendable with modules for additional phenotypes and pathogens. BluVision Micro was applied to screen 196 genetically diverse barley genotypes for interactions with powdery mildew fungi, delivering accurate, sensitive, and reproducible results. This enabled the identification of novel genetic loci and marker-trait associations in the barley genome. The system also facilitated high-throughput studies of labor-intensive phenotypes, such as precise colony area measurement. Additionally, BluVision ’s open-source software supports the development of specific modules for various microscopic phenotypes, including high-throughput transfection assays for disease resistance-related genes.

Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host’s cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

Genetic pathogen control is an economical and sustainable alternative to the use of chemicals. In order to breed resistant varieties, information about potentially unused genetic resistance mechanisms is of high value. We phenotyped 8,316 genotypes of the winter wheat collection of the German Federal ex situ gene bank for Agricultural and Horticultural Crops, Germany , for resistance to powdery mildew (PM), Blumeria graminis f. sp. tritici , one of the most important biotrophic pathogens in wheat. To achieve this, we used a semi-automatic phenotyping facility to perform high-throughput detached leaf assays. This data set, combined with genotyping-by-sequencing (GBS) marker data, was used to perform a genome-wide association study (GWAS). Alleles of significantly associated markers were compared with SNP profiles of 171 widely grown wheat varieties in Germany to identify currently unexploited resistance conferring genes. We also used the Chinese Spring reference genome annotation and various domain prediction algorithms to perform a domain enrichment analysis and produced a list of candidate genes for further investigation. We identified 51 significantly associated regions. In most of these, the susceptible allele was fixed in the tested commonly grown wheat varieties. Eleven of these were located on chromosomes for which no resistance conferring genes have been previously reported. In addition to enrichment of leucine-rich repeats (LRR), we saw enrichment of several domain types so far not reported as relevant to PM resistance, thus, indicating potentially novel candidate genes for the disease resistance research and prebreeding in wheat.

Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.

Powdery mildew is an important foliar disease of barley (Hordeum vulgare L.) caused by the biotrophic fungus Blumeria graminis f. sp. hordei (Bgh). The understanding of the resistance mechanism is essential for future resistance breeding. In particular, the identification of race-nonspecific resistance genes is important because of their regarded durability and broad-spectrum activity. We assessed the severity of powdery mildew infection on detached seedling leaves of 267 barley accessions using two poly-virulent isolates and performed a genome-wide association study exploiting 201 of these accessions. Two-hundred and fourteen markers, located on six barley chromosomes are associated with potential race-nonspecific Bgh resistance or susceptibility. Initial steps for the functional validation of four promising candidates were performed based on phenotype and transcription data. Specific candidate alleles were analyzed via transient gene silencing as well as transient overexpression. Microarray data of the four selected candidates indicate differential regulation of the transcription in response to Bgh infection. Based on our results, all four candidate genes seem to be involved in the responses to powdery mildew attack. In particular, the transient overexpression of specific alleles of two candidate genes, a potential arabinogalactan protein and the barley homolog of Arabidopsis thaliana’s Light-Response Bric-a-Brac/-Tramtrack/-Broad Complex/-POxvirus and Zinc finger (AtLRB1) or AtLRB2, were top candidates of novel powdery mildew susceptibility genes.

The recent characterization of the polysaccharide composition of papillae deposited at the barley cell wall during infection by the powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh), has provided new targets for the generation of enhanced disease resistance. The role of callose in papilla-based penetration resistance of crop species is largely unknown because the genes involved in the observed callose accumulation have not been identified unequivocally. We have employed both comparative and functional genomics approaches to identify the functional orthologue of AtGsl5 in the barley genome. HvGsl6 (the barley glucan synthase-like gene), which has the highest sequence identity to AtGsl5, is the only Bgh-induced gene among the HvGsls examined in this study. Through double-stranded RNA interference (dsRNAi)-mediated silencing of HvGsl6, we have shown that the down-regulation of HvGsl6 is associated with a lower accumulation of papillary and wound callose and a higher susceptibility to penetration of the papillae by Bgh, compared with control lines. The results indicate that the HvGsl6 gene is a functional orthologue of AtGsl5 and is involved in papillary callose accumulation in barley. The increased susceptibility of HvGsl6 dsRNAi transgenic lines to infection indicates that callose positively contributes to the barley fungal penetration resistance mechanism.

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).

Pattern recognition receptors (PRRs) belonging to the multigene family of receptor-like kinases (RLKs) are the sensing devices of plants for microbe- or pathogen-associated molecular patterns released from microbial organisms. Here we describe (for ) encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates non-host resistance of barley to the non-adapted wheat powdery mildew fungus f.sp. . Transgenic barley lines with silenced allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus f.sp. . Transient expression of the barley or wheat genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that is a factor mediating non-host resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus.