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.

Summary: Virus diseases are widespread threats for crop production, which can, in many cases, be controlled efficiently by exploiting naturally occurring resistance. Barley, an important cereal species of the Triticeae, carries two genes, rym4 and rym5, which are located in the telomeric region of chromosome 3HL and confer recessive resistance to various strains of the Barley yellow mosaic virus complex. The barley ‘eukaryotic translation initiation factor 4E’ (Hv‐eIF4E) was identified as a candidate for resistance gene function by physical mapping on a 650 kb contig. It is located in a chromosomal region characterized by suppressed recombination, in a position collinear to its homologue on rice chromosome 1L. Sequence diversity in the coding region of Hv‐eIF4E, as calculated from a collection of unrelated barley accessions, revealed non‐silent single nucleotide polymorphisms (SNPs) in four of its five exons. Stable transformation of a resistant barley genotype with a genomic fragment or a full‐length cDNA of Hv‐eIF4E derived from susceptible cultivars induced susceptibility to Barley mild mosaic virus. Moreover, the identification of SNPs diagnostic for rym4 and rym5 provides evidence that these are two alleles, which confer different resistance specificities. These findings demonstrate that variants of Hv‐eIF4E confer multiallelic recessive virus resistance in a monocot species. The identification of eIF4E as the causal host factor for bymovirus resistance illustrates that mutations in this basic component of the eukaryotic translation complex form a seminal mechanism for recessive virus resistance in both dicot and monocot plants.

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.

Summary: A novel genetic transformation method for barley (Hordeum vulgare L.), based on infection of androgenetic pollen cultures with Agrobacterium tumefaciens, is presented. Winter‐type barley cv. ‘Igri’ was amenable to stable integration of transgenes mediated by A. tumefaciens strain LBA4404 harbouring a vector system that confers hypervirulence, or by the non‐hypervirulent strain GV3101 with a standard binary vector. The efficacy of gene transfer was substantially influenced by pollen pre‐culture time, choice of Agrobacterium strain and vector system, Agrobacterium population density, medium pH and the concentrations of acetosyringone, CaCl₂ and glutamine. After co‐culture, rapid removal of viable agrobacteria was crucial for subsequent development of the pollen culture. To this end, the growth of agrobacteria was suppressed by the concerted effects of appropriate antibiotics, low pH, reduced level of glutamine and high concentrations of CaCl₂ and acetosyringone. Following infection with LBA4404 and GV3101, about 31% and 69%, respectively, of the primary transgenic (T₀) plants carried a single copy of the sequence integrated. The use of hypervirulent A. tumefaciens and hygromycin resistance as a selectable marker resulted in 3.7 T₀ plants per donor spike. About 60% of the primary transgenic plants set seed, indicating spontaneous genome doubling. An analysis of 20 T₁ populations revealed that four progenies did not segregate for reporter gene expression. This indicates that the approach pursued enables the generation of instantly homozygous primary transgenic plants. The method established will be a valuable tool in functional genomics as well as for the biotechnological improvement of barley.

Complete uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanisms underlying selective elimination of the paternal chromosomes during the development of wheat (Triticum aestivum) × pearl millet (Pennisetum glaucum) hybrid embryos. All pearl millet chromosomes were eliminated in a random sequence between 6 and 23 d after pollination. Parental genomes were spatially separated within the hybrid nucleus, and pearl millet chromatin destined for elimination occupied peripheral interphase positions. Structural reorganization of the paternal chromosomes occurred, and mitotic behavior differed between the parental chromosomes. We provide evidence for a novel chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei is different, and heterochromatinization and DNA fragmentation of micronucleated pearl millet chromatin is the final step during haploidization.