Periodic Reporting for period 1 - TOPPER (Targeting of host proteins by unrelated pathogen effectors and their surveillance by allelic immune receptors)
Período documentado: 2018-09-01 hasta 2020-08-31
Plant diseases are major issues in agricultural food production and human societal development. Diseases caused by destructive microbes such as fungi and bacteria result in significant yield and thus substantial economic losses.
Barley is one of the most economically important crops worldwide. However, the cultivation of cereals such as barley is threatened in particular by fungal pathogens, amongst them, powdery mildews. The success range for controlling these and other fungal phytopathogens by fungicides varies and is economically burdensome and increasingly problematic for ecological reasons. In addition, these chemical control measures need to be frequently updated and revised because of the emergence of fungicide resistance and changing regulatory constraints.
The main objective of this project is to clarify how the naturally evolved and similar barley immune receptors known as Mildew Locus A (MLA) detect diverse molecules from the powdery mildew fungus (AVRa effectors) to avoid powdery mildew disease development on barley leaves.
This defines our ability to generate disease resistance in a rational, predictable and sustainable manner, and will underpin the emergence of new biotechnological industries.
As such, the project has economic and social impact, also because the project’s results will in the long term contribute to the development of new crop varieties that are more resistant to pathogens. Such new varieties offer an alternative to chemical disease control and are more productive to growers.
Barley is one of the most economically important crops worldwide. However, the cultivation of cereals such as barley is threatened in particular by fungal pathogens, amongst them, powdery mildews. The success range for controlling these and other fungal phytopathogens by fungicides varies and is economically burdensome and increasingly problematic for ecological reasons. In addition, these chemical control measures need to be frequently updated and revised because of the emergence of fungicide resistance and changing regulatory constraints.
The main objective of this project is to clarify how the naturally evolved and similar barley immune receptors known as Mildew Locus A (MLA) detect diverse molecules from the powdery mildew fungus (AVRa effectors) to avoid powdery mildew disease development on barley leaves.
This defines our ability to generate disease resistance in a rational, predictable and sustainable manner, and will underpin the emergence of new biotechnological industries.
As such, the project has economic and social impact, also because the project’s results will in the long term contribute to the development of new crop varieties that are more resistant to pathogens. Such new varieties offer an alternative to chemical disease control and are more productive to growers.
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.
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.
Summary
As expected, this project has defined agronomically important proteins for immunity of plants to the powdery mildew fungus of the Blumeria graminis species. Explicitly, the project showed that plant NLR immune receptors can detect sequence-unrelated pathogen effectors if these effectors share a common structural fold and that this detection does not rely on other proteins specific to barley.
Beyond this state of the art, the here characterized immune related proteins may also give insight to the immune mechanisms effective against a range of diverse fungal phytopathogens because MLA immune receptors seem to be effective in controlling other fungal diseases.
In addition, the in this project established novel assays and approached now allow to molecularly study agronomically important pathogens of cereal plants.
Conclusions and socio-economic impacts
In summary, the project has significantly impacted and extended our understanding of host (receptor)/pathogen (effector) interaction.
The project’s results will in the long term support the development of new crop varieties that are more resistant to pathogens and this in turn contributes towards improving global food security.
Furthermore, the project facilitated the fellow to further re-integrate to the European Scientific community. The re-integration fellowship was crucial for the fellow to develop scientific independence. The project has further extend the fellow’s general scientific knowledge, provided the fellow with the opportunity to learn the newest techniques, enhanced the fellows communication, scientific and financial management skills and enhanced the fellows capacity to work in a collaborative framework.
As expected, this project has defined agronomically important proteins for immunity of plants to the powdery mildew fungus of the Blumeria graminis species. Explicitly, the project showed that plant NLR immune receptors can detect sequence-unrelated pathogen effectors if these effectors share a common structural fold and that this detection does not rely on other proteins specific to barley.
Beyond this state of the art, the here characterized immune related proteins may also give insight to the immune mechanisms effective against a range of diverse fungal phytopathogens because MLA immune receptors seem to be effective in controlling other fungal diseases.
In addition, the in this project established novel assays and approached now allow to molecularly study agronomically important pathogens of cereal plants.
Conclusions and socio-economic impacts
In summary, the project has significantly impacted and extended our understanding of host (receptor)/pathogen (effector) interaction.
The project’s results will in the long term support the development of new crop varieties that are more resistant to pathogens and this in turn contributes towards improving global food security.
Furthermore, the project facilitated the fellow to further re-integrate to the European Scientific community. The re-integration fellowship was crucial for the fellow to develop scientific independence. The project has further extend the fellow’s general scientific knowledge, provided the fellow with the opportunity to learn the newest techniques, enhanced the fellows communication, scientific and financial management skills and enhanced the fellows capacity to work in a collaborative framework.