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

Genome Biology Discovery of genes affecting resistance of barley to adapted and non-adapted powdery mildew fungi Dimitar Douchkov Stefanie Lck Annika Johrde Daniela Nowara Axel Himmelbach Jeyaraman Rajaraman Nils Stein Rajiv Sharma Benjamin Kilian Patrick Schweizer 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. - Background Plant-pathogen co-evolution has shaped a multifaceted innate immunity system triggered by the recognition of non-self-molecules via pathogen recognition receptors (PRRs) belonging to the family of receptor-like kinases (RLKs) [1]. These non-self-molecules known as pathogenassociated molecular patterns (PAMPs) or, more generally, microbe-associated molecular patterns (MAMPs) include conserved domains of proteins such as bacterial flagellin (flg22) or chitin fragments from fungal cell walls [2]. PAMP-triggered immunity (PTI) has been recognized as the most ancient type of plant defense sharing also components with the innate immunity system of vertebrate * Correspondence: 1Leibniz-Institut fr Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben, Corrensstrasse 3, 06466 Stadt Seeland, Germany Full list of author information is available at the end of the article and invertebrate animals. Downstream of PRRs its molecular components include MAP kinases, WRKY transcription factors as well as an arsenal of downstream-responsive, (WRKY-regulated) genes encoding proteins that generate reactive oxygen species, reinforce, and break down plant and pathogen cell-walls, respectively, or catalyze the synthesis of pathogen-toxic compounds such as phytoalexins. On top of PTI plants can activate an effectortriggered immunity (ETI) response that is based on the direct or indirect recognition of avirulenve (Avr) effector molecules of some pathogen races by major R-genes encoding nucleotide-binding leucine-rich repeat (NB-LRR) proteins, and on the initiation of a very strong local defense response often culminating in host-cell death. One of the preferred targets of effectors are PRRs, which have been found to be guarded by several NB-LRR type or PRR-like proteins therefore also being involved in ETI [3]. Durable and broad-range non-host resistance (NHR) to virtually all races of non-adapted pathogens appears to be an important manifestation of PTI in many cases [4,5] although there is also experimental evidence that NHR can - at least in grass species - be mediated by as little as one major R gene recognizing an indispensable Avr effector [6]. Race-specificity of NHR QTL to non- or only partially adapted fungal pathogens has also been described, similar to QTL for host quantitative resistance (QR) that is another manifestation of PTI [7] and QR is also referred to as race-non-specific or horizontal resistance [8-10]. However, in contrast to the very robust NHR response, QR is often not very efficient suffering from effector-triggered susceptibility (ETS) brought about by small secreted proteins or peptides from adapted pathogens that are active in the plant apoplast or inside host cells [11]. The introgression of single major R genes usually confers strong protection against specific adapted pathogen races carrying the matching avirulence (Avr) effector genes, but the trait is often overcome by rapidly evolving new pathogen races with mutated Avr effectors acting in concert with other functionally redundant effectors. In principle, QR could also be mediated by partially functional (defeated) major R-genes weakly recognizing ubiquitous Avr effectors such as ECP1 or ECP2 [12], but molecular evidence for this type of interactions is scarce [13,14]. Barley (Hordeum vulgare ssp. vulgare) is an important crop plant and exhibits genetic variability determining to which extent it is successfully colonized by a given pathogen. This opens up the possibility to improve QR as a quantitative trait by introgressing and/or combining resistance-related alleles. Often, however, QR was found to be inherited by many QTLs making the trait difficult to handle in breeding practice due to complex crossing schemes, phenotype scoring ambiguities and linkage drag problems [9]. Knowing the genes that encode important QR components in crop plants would render targeted QR improvement by allele mining and gene marker-assisted as well as pathway-oriented introgression more efficient. One of the major diseases of barley is powdery mildew caused by the obligate biotrophic ascomycete fungus Blumeria graminis f.sp. hordei (Bgh) [15] that also fulfills several criteria of a model plantpathogen interaction due to a large body of physiological, cellular, biochemical, and molecular information on changes in the host during compatible or resistant interactions [16-18]. Transient expression and genesilencing assays such as transient-induced gene silencing (TIGS) in bombarded epidermal cells have been developed over the years and proven to be valuable tools for a better understanding of barley/powdery mildew interactions [19-21]. NHR of barley against non-adapted formae speciales or species of powdery mildew such as the wheat pathogen B. graminis f.sp. tritici (Bgt) efficiently blocks fungal penetration attempts at the epidermal cell wall and shares a number of genes with the QR pathway triggered by the adapted barley powdery mildew fungus [22-24]. Therefore, th (...truncated)