Background information
Plants have an effective basal immune system that controls the infection by the majority of potentially dangerous microbes. However, some adapted pathogens secrete molecules, so-called virulence effectors, into the host tissue to facilitate their infection of the respective host plant. Most characterised effectors dampen the host’s basal immune system and promote the exhaustion of the host’s nutrient supply. This ultimately promotes the pathogen’s reproduction and disease development on the plant, ultimately leading to severe losses in yield. Resistance to such adapted pathogens is commonly mediated by specific resistance genes encoded by the plant genomes. Resistance genes often produce nucleotide-binding oligomerization domain (NOD)-like receptor proteins (NLRs). NLR proteins recognize pathogen effectors inside plant cells, thereby triggering a potent immune response that terminates pathogen proliferation. NLRs confer resistance to individual pathogen strains and can limit the host range of certain pathogens. Pathogen genes encoding recognised effectors are historically known as ‘avirulence’ (AVR) genes. NLRs either sense the AVR structure (direct recognition) or monitor AVR-mediated host target modifications (indirect recognition). A combination of direct and indirect AVR recognition in which host target decoys are integrated domains into NLR architectures represents another AVR recognition strategy. AVR-mediated activation of NLRs and subsequent initiation of immune responses is often associated with localized host cell death. The continuous use of NLR resistance genes in agriculture favours the selection of novel pathogen isolates with diversified AVR genes that avoid recognition by the NLR and thus breakdown of resistance.
The barley (Hordeum vulgare) mildew locus A NLRs (MLAs) confers resistance to the widespread mildew fungus Blumeria graminis forma specialis hordei (Bgh).
The objective of this project is to determine how a range of similar MLAs detect seemingly unrelated Bgh avirulence effectors (AVRa) to avoid powdery mildew disease development on barley lines that carry the respective Mla gene.
Results overview
Using an established transcriptome wide association study, in this project further Bgh AVRa effectors were isolated. These effectors facilitate infection of susceptible barley lines with Bgh but are recognizes by MLAs for resistance.
The initial data of the overall project could determine that a range of MLA receptors associate with AVRa effectors in yeast as assessed using yeast-2-hybrid, suggesting that the recognition of AVRa by MLA is independent of other barley host molecules.
To also determine AVR recognition by MLA in barley, an assay for transient gene expression in cereals, followed by cell death measurement as proxy for NLR/AVR recognition was developed.
Structural prediction was applied to the here studied AVRa effectors and suggests a common fold amongst the effectors recognized by MLA, even though the effectors are dissimilar on the sequence level. Because the family compromising effectors with a similar structural fold have been massively expanded in the genomes of cereal-infecting powdery mildews, the data further suggests that the recognition of this structural fold by MLA NLRs is responsible for the expansion of this effector family.
By utilising AVRa and MLA mutagenesis followed by heterologous (Nicotiana benthmiana) and homologous (barley) gene expression and subsequent cell death assays, the project could clarify which domains and individual residues of the different Bgh AVRa effectors are recognised by barley MLAs. The data showed that in the majority of cases the C-terminal part of the Bgh effectors and the C-terminal domain of the MLA NLRs seem to play a critical role in association of the matching proteins.
Cell death and interaction assays upon heterologous Mla and AVRa gene expression showed that diversified AVRa variants can also block the recognition of other AVRa effectors by MLA and as such inhibit MLA-mediated resistance. A dominant association of MLA and such a diversified AVRa variant is associated with this inhibitory effect.
