Jochen Kumlehn

HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminis

This work examines the effects of RNA interference constructs expressed in host cells on target RNAs in Blumeria graminis, an obligate biotrophic fungal pathogen of barley, and finds that RNAs in the host can affect pathogen transcript levels and pathogen development, thereby providing both a useful research tool and a potentially important means for engineering plant disease resistance. 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.

Uniparental Chromosome Elimination at Mitosis and Interphase in Wheat and Pearl Millet Crosses Involves Micronucleus Formation, Progressive Heterochromatinization, and DNA Fragmentation

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.

A Set of Modular Binary Vectors for Transformation of Cereals

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.

Apomictic and Sexual Ovules of Boechera Display Heterochronic Global Gene Expression Patterns

It is difficult for a purely mutational model to explain the evolution of asexuality in plants and animals. This work finds that expression patterns of many reproduction genes, including an overabundance of regulatory factors, differ during sexual and asexual ovule development, thus providing a possible mechanism for inducing the complex reproductive changes required to generate clonal offspring. We have compared the transcriptomic profiles of microdissected live ovules at four developmental stages between a diploid sexual and diploid apomictic Boechera. We sequenced >2 million SuperSAGE tags and identified (1) heterochronic tags (n = 595) that demonstrated significantly different patterns of expression between sexual and apomictic ovules across all developmental stages, (2) stage-specific tags (n = 577) that were found in a single developmental stage and differentially expressed between the sexual and apomictic ovules, and (3) sex-specific (n = 237) and apomixis-specific (n = 1106) tags that were found in all four developmental stages but in only one reproductive mode. Most heterochronic and stage-specific tags were significantly downregulated during early apomictic ovule development, and 110 were associated with reproduction. By contrast, most late stage-specific tags were upregulated in the apomictic ovules, likely the result of increased gene copy number in apomictic (hexaploid) versus sexual (triploid) endosperm or of parthenogenesis. Finally, we show that apomixis-specific gene expression is characterized by a significant overrepresentation of transcription factor activity. We hypothesize that apomeiosis is associated with global downregulation at the megaspore mother cell stage. As the diploid apomict analyzed here is an ancient hybrid, these data are consistent with the postulated link between hybridization and asexuality and provide a hypothesis for multiple evolutionary origins of apomixis in the genus Boechera.

Agrobacterium-Mediated Gene Transfer to Cereal Crop Plants: Current Protocols for Barley, Wheat, Triticale, and Maize

The development of powerful “omics” technologies has enabled researchers to identify many genes of interest for which comprehensive functional analyses are highly desirable. However, the production of lines which ectopically express recombinant genes, or those in which endogenous genes are knocked down via stable transformation, remains a major bottleneck for the association between genetics and gene function in monocotyledonous crops. Methods of effective DNA transfer into regenerable cells of immature embryos from cereals by means of Agrobacterium tumefaciens have been modified in a stepwise manner. The effect of particular improvement measures has often not been significantly evident, whereas their combined implementation has resulted in meaningful advances. Here, we provide updated protocols for the Agrobacterium-mediated generation of stably transgenic barley, wheat, triticale and maize. Based upon these methods, several hundred independent transgenic lines have been delivered, with efficiencies of inoculated embryos leading to stably transgenic plants reaching 86% in barley, 10% in wheat, 4% in triticale, and 24% in maize.

A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells

Little is known about the role the cytoskeleton plays in signaling with regard to the outcome of plant–microbe interactions. This work describes a regulator of cytoskeleton organization and of disease susceptibility shuttling between microtubules and the monomeric G-protein RACB at the cell periphery. Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.

Increasing Sucrose Uptake Capacity of Wheat Grains Stimulates Storage Protein Synthesis

Increasing grain sink strength by improving assimilate uptake capacity could be a promising approach toward getting higher yield. The barley (Hordeum vulgare) sucrose transporter HvSUT1 (SUT) was expressed under control of the endosperm-specific Hordein B1 promoter (HO). Compared with the wild type, transgenic HOSUT grains take up more sucrose (Suc) in vitro, showing that the transgene is functional. Grain Suc levels are not altered, indicating that Suc fluxes are influenced rather than steady-state levels. HOSUT grains have increased percentages of total nitrogen and prolamins, which is reflected in increased levels of phenylalanine, tyrosine, tryptophan, isoleucine, and leucine at late grain development. Transcript profiling indicates specific stimulation of prolamin gene expression at the onset of storage phase. Changes in gene expression and metabolite levels related to carbon metabolism and amino acid biosynthesis suggest deregulated carbon-nitrogen balance, which together indicate carbon sufficiency and relative depletion of nitrogen. Genes, deregulated together with prolamin genes, might represent candidates, which respond positively to assimilate supply and are related to sugar-starch metabolism, cytokinin and brassinosteroid functions, cell proliferation, and sugar/abscisic acid signaling. Genes showing inverse expression patterns represent potential negative regulators. It is concluded that HvSUT1 overexpression increases grain protein content but also deregulates the metabolic status of wheat (Triticum aestivum) grains, accompanied by up-regulated gene expression of positive and negative regulators related to sugar signaling and assimilate supply. In HOSUT grains, alternating stimulation of positive and negative regulators causes oscillatory patterns of gene expression and highlights the capacity and great flexibility to adjust wheat grain storage metabolism in response to metabolic alterations.

A Maize Cystatin Suppresses Host Immunity by Inhibiting Apoplastic Cysteine Proteases

This work identifies the maize cystatin CC9 as a compatibility factor in the biotrophic interaction with the fungal pathogen Ustilago maydis. The results show that CC9 functions as a suppressor of host defense responses by inhibiting defense-related apoplastic Cys proteases in the maize apoplast. Ustilago maydis is a biotrophic pathogen causing maize (Zea mays) smut disease. Transcriptome profiling of infected maize plants indicated that a gene encoding a putative cystatin (CC9) is induced upon penetration by U. maydis wild type. By contrast, cc9 is not induced after infection with the U. maydis effector mutant Δpep1, which elicits massive plant defenses. Silencing of cc9 resulted in a strongly induced maize defense gene expression and a hypersensitive response to U. maydis wild-type infection. Consequently, fungal colonization was strongly reduced in cc9-silenced plants, while recombinant CC9 prevented salicylic acid (SA)–induced defenses. Protease activity profiling revealed a strong induction of maize Cys proteases in SA-treated leaves, which could be inhibited by addition of CC9. Transgenic maize plants overexpressing cc9-mCherry showed an apoplastic localization of CC9. The transgenic plants showed a block in Cys protease activity and SA-dependent gene expression. Moreover, activated apoplastic Cys proteases induced SA-associated defense gene expression in naïve plants, which could be suppressed by CC9. We show that apoplastic Cys proteases play a pivotal role in maize defense signaling. Moreover, we identified cystatin CC9 as a novel compatibility factor that suppresses Cys protease activity to allow biotrophic interaction of maize with the fungal pathogen U. maydis.

BAX INHIBITOR-1 is required for full susceptibility of barley to powdery mildew.

BAX INHIBITOR-1 (BI-1) is one of the few proteins known to have cross-kingdom conserved functions in negative control of programmed cell death. Additionally, barley BI-1 (HvBI-1) suppresses defense responses and basal resistance to the powdery mildew fungus Blumeria graminis f. sp. hordei and enhances resistance to cell death-provoking fungi when overexpressed in barley. Downregulation of HvBI-1 by transient-induced gene silencing or virus-induced gene silencing limited susceptibility to B. graminis f. sp. hordei, suggesting that HvBI-1 is a susceptibility factor toward powdery mildew. Transient silencing of BI-1 did not limit supersusceptibility induced by overexpression of MLO. Transgenic barley plants harboring an HvBI-1 RNA interference (RNAi) construct displayed lower levels of HvBI-1 transcripts and were less susceptible to powdery mildew than wild-type plants. At the cellular level, HvBI-1 RNAi plants had enhanced resistance to penetration by B. graminis f. sp. hordei. These data support a function of BI-1 in modulating cell-wall-associated defense and in establishing full compatibility of B. graminis f. sp. hordei with barley.

Systemic virus‐induced gene silencing allows functional characterization of maize genes during biotrophic interaction with Ustilago maydis

Infection of maize (Zea mays) plants with the corn smut fungus Ustilago maydis leads to the formation of large tumors on the stem, leaves and inflorescences. In this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed massive and stage-specific changes in host gene expression during disease progression. To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a virus-induced gene silencing (VIGS) system based on the brome mosaic virus (BMV) for maize. Conditions were established that allowed successful U. maydis infection of BMV-preinfected maize plants. This set-up enabled quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (qRT-PCR)-based readout. In proof-of-principle experiments, an U. maydis-induced terpene synthase was shown to negatively regulate disease development while a protein involved in cell death inhibition was required for full virulence of U. maydis. The results suggest that this system is a versatile tool for the rapid identification of maize genes that determine compatibility with U. maydis.