Over-accumulation of chloroplast-nucleus located WHIRLY1 in barley leads to a decrease in growth and an enhanced stress resistance.

WHIRLY1 is a chloroplast-nucleus located DNA/RNA-binding protein with functions in development and stress tolerance. By overexpression of HvWHIRLY1 in barley, one line with a 10-fold and two lines with a 50-fold accumulation of the protein were obtained. In these lines, the relative abundance of the nuclear form exceeded that of the chloroplast form. Growth of the plants was shown to be compromised in a WHIRLY1 abundance-dependent manner. Over-accumulation of WHIRLY1 in chloroplasts had neither an evident impact on nucleoid morphology nor on the composition of the photosynthetic apparatus. Nevertheless, oeW1 plants were found to be compromised in the light reactions of photosynthesis as well as in carbon fixation. The reduction in growth and photosynthesis was shown to be accompanied by a decrease in the levels of cytokinins and an increase in the level of jasmonic acid. Gene expression analyses revealed that in nonstress conditions the oeW1 plants had enhanced levels of pathogen response (PR) gene expression indicating activation of constitutive defense. During growth in continuous light of high irradiance PR gene expression increased indicating that under stress conditions oeW1 are capable to further enhance defense.

Association mapping of wheat Fusarium head blight resistance-related regions using a candidate-gene approach and their verification in a biparental population.

KEY MESSAGE: Markers, located in Dicer1 and Ara6 genes, which are likely involved in cross-kingdom RNA trafficking, are associated with FHB resistance in GABI wheat population and were validated in biparental population. Association studies are a common approach to detect marker-trait associations for Fusarium head blight (FHB) resistance in wheat (Triticum aestivum), although verification of detected associations is exceptional. In the present study, candidate-gene association mapping (CG) of genes from silencing and secretory pathways, which may be involved in wheat resistance against FHB and cross-kingdom RNA trafficking, was performed. Fourteen markers, located in nine genes, were tested for association with FHB resistance in 356 lines from the GABI (genome analysis of the biological system of plants) wheat population. Three markers located in the genes Dicer1 and Ara6 were shown to be significantly associated with the studied trait. Verification of this finding was performed using the recombinant inbred lines (RILs) population 'Apache × Biscay', segregating for four of our 14 selected markers. We could show association of the Ara6 marker with plant height as well as association with FHB resistance for three markers located in Rab5-like GTPase gene Ara6 and Dicer1. These results confirmed the trait-marker associations detected also in the CG approach. Gene products of the associated genes are involved in response of the plant to pathogens, plant metabolism and may be involved in cross-kingdom RNA trafficking efficiency. The markers detected in the GABI wheat population, which were also validated in the biparental population, can potentially be used in wheat breeding.

Evolutionarily conserved partial gene duplication in the Triticeae tribe of grasses confers pathogen resistance.

BACKGROUND: The large and highly repetitive genomes of the cultivated species Hordeum vulgare (barley), Triticum aestivum (wheat), and Secale cereale (rye) belonging to the Triticeae tribe of grasses appear to be particularly rich in gene-like sequences including partial duplicates. Most of them have been classified as putative pseudogenes. In this study we employ transient and stable gene silencing- and over-expression systems in barley to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15). RESULTS: The partial ARM1 gene is derived from a gene-duplication event in a common ancestor of the Triticeae and contributes to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis. In barley, allelic variants of HvARM1 but not of HvPUB15 are significantly associated with levels of powdery mildew infection. Both HvPUB15 and HvARM1 proteins interact in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. A genome-wide scan for partial gene duplicates reveals further events in barley resulting in stress-regulated, potentially neo-functionalized, genes. CONCLUSION: The results suggest neo-functionalization of the partial gene copy HvARM1 increases resistance against powdery mildew infection. It further links plastid function with susceptibility to biotrophic pathogen attack. These findings shed new light on a novel mechanism to employ partial duplication of protein-protein interaction domains to facilitate the expansion of immune signaling networks.

Host-induced silencing of Fusarium culmorum genes protects wheat from infection.

Plants producing antisense or double-stranded RNA molecules that target specific genes of eukaryotic pests or pathogens can become protected from their attack. This beneficial effect was also reported for plant-fungus interactions and is believed to reflect uptake of the RNAs by the fungus via an as yet unknown mechanism, followed by target gene silencing. Here we report that wheat plants pre-infected with Barley stripe mosaic virus (BSMV) strains containing antisense sequences against target genes of the Fusarium head blight (FHB) fungus F. culmorum caused a reduction of corresponding transcript levels in the pathogen and reduced disease symptoms. Stable transgenic wheat plants carrying an RNAi hairpin construct against the ß-1, 3-glucan synthase gene FcGls1 of F. culmorum or a triple combination of FcGls1 with two additional, pre-tested target genes also showed enhanced FHB resistance in leaf and spike inoculation assays under greenhouse and near-field conditions, respectively. Microscopic evaluation of F. culmorum development in plants transiently or stably expressing FcGls1 silencing constructs revealed aberrant, swollen fungal hyphae, indicating severe hyphal cell wall defects. The results lead us to propose host-induced gene silencing (HIGS) as a plant protection approach that may also be applicable to highly FHB-susceptible wheat genotypes.

Discovery of genes affecting resistance of barley to adapted and non-adapted powdery mildew fungi.

BACKGROUND: Non-host resistance, NHR, to non-adapted pathogens and quantitative host resistance, QR, confer durable protection to plants and are important for securing yield in a longer perspective. However, a more targeted exploitation of the trait usually possessing a complex mode of inheritance by many quantitative trait loci, QTLs, will require a better understanding of the most important genes and alleles. RESULTS: Here we present results from a transient-induced gene silencing, TIGS, approach of candidate genes for NHR and QR in barley against the powdery mildew fungus Blumeria graminis. Genes were selected based on transcript regulation, multigene-family membership or genetic map position. Out of 1,144 tested RNAi-target genes, 96 significantly affected resistance to the non-adapted wheat- or the compatible barley powdery mildew fungus, with an overlap of four genes. TIGS results for QR were combined with transcript regulation data, allele-trait associations, QTL co-localization and copy number variation resulting in a meta-dataset of 51 strong candidate genes with convergent evidence for a role in QR. CONCLUSIONS: This study represents an initial, functional inventory of approximately 3% of the barley transcriptome for a role in NHR or QR against the powdery mildew pathogen. The discovered candidate genes support the idea that QR in this Triticeae host is primarily based on pathogen-associated molecular pattern-triggered immunity, which is compromised by effector molecules produced by the compatible pathogen. The overlap of four genes with significant TIGS effects both in the NHR and QR screens also indicates shared components for both forms of durable pathogen resistance.

Host-induced gene silencing in barley powdery mildew reveals a class of ribonuclease-like effectors.

Obligate biotrophic pathogens of plants must circumvent or counteract defenses to guarantee accommodation inside the host. To do so, they secrete a variety of effectors that regulate host immunity and facilitate the establishment of pathogen feeding structures called haustoria. The barley powdery mildew fungus Blumeria graminis f. sp. hordei produces a large number of proteins predicted to be secreted from haustoria. Fifty of these Blumeria effector candidates (BEC) were screened by host-induced gene silencing (HIGS), and eight were identified that contribute to infection. One shows similarity to ß-1,3 glucosyltransferases, one to metallo-proteases, and two to microbial secreted ribonucleases; the remainder have no similarity to proteins of known function. Transcript abundance of all eight BEC increases dramatically in the early stages of infection and establishment of haustoria, consistent with a role in that process. Complementation analysis using silencing-insensitive synthetic cDNAs demonstrated that the ribonuclease-like BEC 1011 and 1054 are bona fide effectors that function within the plant cell. BEC1011 specifically interferes with pathogen-induced host cell death. Both are part of a gene superfamily unique to the powdery mildew fungi. Structural modeling was consistent, with BEC1054 adopting a ribonuclease-like fold, a scaffold not previously associated with effector function.

Convergent evidence for a role of WIR1 proteins during the interaction of barley with the powdery mildew fungus Blumeria graminis.

Pathogen attack triggers a multifaceted defence response in plants that includes the accumulation of pathogenesis-related proteins and their corresponding transcripts. One of these transcripts encodes for WIR1, a small glycine- and proline-rich protein of unknown function that appears to be specific to grass species. Here we describe members of the HvWIR1 multigene family of barley with respect to phylogenetic relationship, transcript regulation, co-localization with quantitative trait loci for resistance to the barley powdery mildew fungus Blumeria graminis (DC.) E.O. Speer f.sp. hordei, the association of single nucleotide polymorphisms or gene haplotypes with resistance, as well as phenotypic effects of gene silencing by RNAi. HvWIR1 is encoded by a multigene family of moderate complexity that splits up into two major clades, one of those being also represented by previously described cDNA sequences from wheat. All analysed WIR1 transcripts accumulated in response to powdery mildew attack in leaves and all mapped WIR1 genes were associated with quantitative trait loci for resistance to B. graminis. Moreover, single nucleotide polymorphisms or haplotypes of WIR1 members were associated with quantitative resistance of barley to B. graminis, and transient WIR1 gene silencing affected the interaction of epidermal cells with the pathogen. The presented data provide convergent evidence for a role of the HvWIR1a gene and possibly other family members, during the interaction of barley with B. graminis.

HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis.

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.

Protein polyubiquitination plays a role in basal host resistance of barley.

To study protein ubiquitination pathways in the interaction of barley (Hordeum vulgare) with the powdery mildew fungus (Blumeria graminis), we measured protein turnover and performed transient-induced gene silencing (TIGS) of ubiquitin and 26S proteasome subunit encoding genes in epidermal cells. Attack by B. graminis hyperdestabilized a novel unstable green fluorescent protein fusion that contains a destabilization domain of a putative barley 1-aminocyclopropane-1-carboxylate synthase, suggesting enhanced protein turnover. Partial depletion of cellular ubiquitin levels by TIGS induced extreme susceptibility of transformed cells toward the appropriate host pathogen B. graminis f. sp hordei, whereas papilla-based resistance to the nonhost pathogen B. graminis f. sp tritici and host resistance mediated by the mlo gene (for mildew resistance locus O) remained unaffected. Cells were rescued from TIGS-induced ubiquitin depletion by synthetic genes encoding wild-type or mutant barley monoubiquitin proteins. The strongest rescue was from a gene encoding a K63R mutant form of ubiquitin blocked in several ubiquitination pathways while still allowing Lys-48-dependent polyubiquitination required for proteasomal protein degradation. Systematic RNA interference of 40 genes encoding all 17 subunits of the proteasome 19S regulatory particle failed to induce hypersusceptibility against B. graminis f. sp hordei. This suggests a role for Lys-48-linked protein polyubiquitination, which is independent from the proteasome pathway, in basal host defense of barley.

Cloning, functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k).

The nitrate transporter from Chlorella sorokiniana (accession number AY026523) has been cloned by screening a cDNA library based on mRNA isolated after 30 min treatment of Chlorella with 5 mM nitrate and with a RT-PCR product (730 bp) as a probe. The Chlorella sequence has similarity to known nitrate transporters of the NRT2 family (high-affinity nitrate transporters). The cDNA clone was used for functional expression in Xenopus oocytes and a nitrate-dependent current was measured at pH 5.5 but not at pH 7.4. A second algal gene or a second gene product was not needed for functional expression in Xenopus. Inhibitor studies in Chlorella indicated that protein phosphorylation/dephosphorylation is involved in nitrate induction of ChNRT2.1. In addition to nitrate, ChNRT2.1 expression is induced by nitroprusside, a NO donor, and is affected by glucose.