Götz Hensel

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.

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.

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.

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.

Promoters of the Barley Germin-Like GER4 Gene Cluster Enable Strong Transgene Expression in Response to Pathogen Attack

The GER4 transcript encoding a germin-like protein of barley is derived from a dense cluster of tandemly duplicated, pathogen-induced genes that are subject to purifying selection. The GER4c promoter was found to regulate strong and pathogen-specific expression of the GUS reporter in barley, and multiple redundant WRKY factor binding sites were required for promoter activation. Immunity of plants triggered by pathogen-associated molecular patterns (PAMPs) is based on the execution of an evolutionarily conserved defense response that includes the accumulation of pathogenesis-related (PR) proteins as well as multiple other defenses. The most abundant PR transcript of barley (Hordeum vulgare) leaf epidermis attacked by the powdery mildew fungus Blumeria graminis f. sp hordei encodes the germin-like protein GER4, which has superoxide dismutase activity and functions in PAMP-triggered immunity. Here, we show that barley GER4 is encoded by a dense cluster of tandemly duplicated genes (GER4a-h) that underwent several cycles of duplication. The genomic organization of the GER4 locus also provides evidence for repeated gene birth and death cycles. The GER4 promoters contain multiple WRKY factor binding sites (W-boxes) preferentially located in promoter fragments that were exchanged between subfamily members by gene conversion. Mutational analysis of TATA-box proximal W-boxes used GER4c promoter-β-glucuronidase fusions to reveal their enhancing effects and functional redundancy on pathogen-induced promoter activity. The data suggest enhanced transcript dosage as an evolutionary driving force for the local expansion and functional redundancy of the GER4 locus. In addition, the GER4c promoter provides a tool to study signal transduction of PAMP-triggered immunity and to engineer strictly localized and pathogen-regulated disease resistance in transgenic cereal crops.

Ectopic Expression of Constitutively Activated RACB in Barley Enhances Susceptibility to Powdery Mildew and Abiotic Stress

Small RAC/ROP-family G proteins regulate development and stress responses in plants. Transient overexpression and RNA interference experiments suggested that the barley (Hordeum vulgare) RAC/ROP protein RACB is involved in susceptibility to the powdery mildew fungus Blumeria graminis f. sp. hordei. We created transgenic barley plants expressing the constitutively activated RACB mutant racb-G15V under control of the maize (Zea mays) ubiquitin 1 promoter. Individuals of the T1 generation expressing racb-G15V were significantly more susceptible to B. graminis when compared to segregating individuals that did not express racb-G15V. Additionally, racb-G15V-expressing plants showed delayed shoot development from the third leaf stage on, downward rolled leaves, and stunted roots. Expression of racb-G15V decreased photosynthetic CO2-assimilation rates and transpiration of nonstressed leaves. In contrast, racb-G15V-expressing barley leaves, when detached from water supply, showed increased water loss and enhanced transpiration. Water loss was associated with reduced responsiveness to abscisic acid in regard to transpiration when compared to segregants not expressing racb-G15V. Hence, RACB might be a common signaling element in response to both biotic and abiotic stress.

Whirly1 in chloroplasts associates with intron containing RNAs and rarely co-localizes with nucleoids

The nucleic acid binding protein Whirly1 of barley has been located to both chloroplasts and the nucleus of the same cell. Immunogold labelling furthermore showed that in vivo Whirly1 does not strictly co-localize with DNA in chloroplasts, while it is closely associated with DNA in the nucleus. High-resolution imaging of Whirly1-GFP and PEND-RFP fusion proteins revealed that only a minor part of Whirly1 co-localizes with nucleoids. The co-localization with nucleoids is in accordance with the detection of Whirly1 in a conventionally prepared fraction of the transcriptionally active chromosome (TAC). By further purification and enrichment of transcriptional activity Whirly1, however, was lost from the TAC fraction. Knockdown of Whirly1 in transgenic barley plants had neither impact on transcription of selected protein coding genes nor on genes coding for ribosomal RNAs or tRNAs. The results of RIP-chip experiments showed that barley Whirly1 as its maize orthologue associates with a set of intron containing plastid RNAs. Taken together, the results suggest that plastid-located Whirly1 functions primarily in RNA metabolism rather than as a DNA binding protein.

RBOHF2 of Barley Is Required for Normal Development of Penetration Resistance to the Parasitic Fungus Blumeria graminis f. sp. hordei

Plant respiratory burst oxidase homologs are prominent sources of reactive oxygen species (ROS) in signal transduction and in interaction with microbes. However, the function of respiratory burst oxidase homologue (RBOH) genes in interaction with microbes might differ for certain plant and pathogen species. We produced transgenic barley knock down (KD) for the HvRBOHF2 isoform of NADPH oxidases. Young HvRBOHF2 KD shoots did not show obvious morphological alterations from the wild type but adult HvRBOHF2 KD plants developed fewer tillers, were less fertile, and showed spontaneous cell death in leaf mesophyll. Additionally, HvRBOHF2 KD plants were unable to contain wound-induced cell death. Before developmental failure became obvious, young HvRBOHF2 KD seedlings were much more susceptible to penetration by the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei. Strikingly, the B. graminis f. sp. hordei-induced cell-wall-associated oxidative burst was not substantially attenuated in HvRBOHF2 KD plants but enhanced susceptibility apparently influenced the subcellular site of hydrogen peroxide accumulation. Taken together, misexpression of HvRBOHF2 caused failure of barley to normally develop penetration resistance to B. graminis f. sp. hordei and to control leaf cell death.

Constitutively activated barley ROPs modulate epidermal cell size, defense reactions and interactions with fungal leaf pathogens

RHO-like monomeric G-proteins of plants (ROPs, also called RACs), are involved in plant development and interaction with the environment. The barley (Hordeum vulgare) ROP protein HvRACB has been shown to be required for entry of the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) into living host cells. To get a deeper insight into evolutionarily conserved functions of ROPs in cell polarity and pathogen responses, we stably expressed constitutively activated (CA) mutant variants of different barley ROPs (HvRACB, HvRAC1, HvRAC3) in barley. CA HvROPs induced epidermal cell expansion and/or abolished polarity in tip growing root hairs. All three CA HvROPs enhanced susceptibility of barley to penetration by Bgh whereas only CA HvRAC1 supported whole cell H2O2 production in non-penetrated cells. Despite increasing penetration by Bgh, CA HvRAC1 promoted callose deposition at sites of fungal attack and resistance to penetration by Magnaporthe oryzae. The data show an involvement of ROPs in polar growth processes of the monocot barley and in responses to fungal pathogens with different life style.

Immature pollen-derived doubled haploid formation in barley cv. Golden Promise as a tool for transgene recombination

Barley transformation mediated by Agrobacterium tumefaciens is routinely performed in a number of laboratories. However, elimination of selectable marker genes and formation of plants homozygous for the transgene via conventional segregation is laborious and time-consuming. Here we suggest a concept that includes the production of primary transgenic plants via infection of immature embryos with A. tumefaciens followed by androgenetic generation of a segregating population of entirely homozygous plants. Selectable marker-free, truebreeding plants carrying a single-opy transgene integrant may thus be efficiently and rapidly obtained. However, amenability to Agrobacterium-mediated transformation as well as androgenetic potential is genotype-dependent. Efficient genetic transformation by infection of immature embryos is so far confined to the spring type cultivar ‘Golden Promise’ which, however, turned out to be recalcitrant in pollen embryogenesis. To facilitate androgenetic generation of homozygous segregants from primary transformants, we have established a method for embryogenic pollen culture in cv. Golden Promise that includes conventional cold-treatment and subsequent preculture of immature pollen under starvation conditions prior to transfer to complete nutrient medium. Further we show that conditioning of the pollen culture medium by co-culture of immature wheat pistils as well as addition of pistil-preconditioned medium considerably support androgenetic development. Employment of the established method using immature pollen of primary transgenic plants demonstrates that selectable marker-free, true-breeding transgenic progeny can be rapidly obtained pursuing the concept proposed. The protocol presented will be useful in functional genomics as well as in molecular breeding approaches.