2021-BBSRC/NSF-BIO: Host Immunity as a Driver of Virulence Evolution in Cereal Rust Fungi
Lead Research Organisation:
National Institute of Agricultural Botany
Department Name: Centre for Research
Abstract
Fungi of the genus Puccinia cause rust diseases affecting wheat, barley and oat production worldwide. An effective strategy to prevent crop diseases is to breed cultivars with disease resistance (R) genes. These genes typically encode immune receptors that recognise specific pathogen proteins, called avirulence (Avr) proteins, and trigger defence responses. However, this approach is undermined by pathogen evolution to evade recognition by changing their Avr genes, so effective resistance gene deployment requires knowledge of Avr gene variation in pathogen populations.
We seek to understand how the interactions between host immunity genes and pathogen virulence genes influences the evolution of pathogen populations to overcome host resistance. We focus on three related Puccinia species of fungi that infect different cereal crop hosts: Puccinia graminis f. sp. tritici (stem rust) infecting wheat, Puccinia hordei (brown rust) infecting barley, and Puccinia coronata f. sp. avenae (crown rust) infecting oats. These present contrasting levels of sexuality versus clonality and provide an opportunity to compare and contrast between related pathogen species and their hosts and different levels of sexual versus clonal reproduction.
Critical questions to answer are: What types of genomic variation (copy number variation, single nucleotide polymorphisms, transposable element insertions) underlie virulence differences in these cereal rust populations? What are the rust Avr genes recognized by the crop R genes that are important in global breeding programs? What is the level of homozygosity/ heterozygosity at these loci and their propensity to mutate to virulence? What functions do Avr effectors perform during infection and which are most critical for pathogen fitness?
These questions will be addressed through the following four work objectives:
Objective 1: Identify Avr gene candidates through genome sequence comparisons.
Objective 2: Validate Avr gene candidates through functional assays.
Objective 3: Examine Avr gene diversity and evolution in rust populations.
Objective 4: Determine the role of Avr proteins in rust infection.
We seek to understand how the interactions between host immunity genes and pathogen virulence genes influences the evolution of pathogen populations to overcome host resistance. We focus on three related Puccinia species of fungi that infect different cereal crop hosts: Puccinia graminis f. sp. tritici (stem rust) infecting wheat, Puccinia hordei (brown rust) infecting barley, and Puccinia coronata f. sp. avenae (crown rust) infecting oats. These present contrasting levels of sexuality versus clonality and provide an opportunity to compare and contrast between related pathogen species and their hosts and different levels of sexual versus clonal reproduction.
Critical questions to answer are: What types of genomic variation (copy number variation, single nucleotide polymorphisms, transposable element insertions) underlie virulence differences in these cereal rust populations? What are the rust Avr genes recognized by the crop R genes that are important in global breeding programs? What is the level of homozygosity/ heterozygosity at these loci and their propensity to mutate to virulence? What functions do Avr effectors perform during infection and which are most critical for pathogen fitness?
These questions will be addressed through the following four work objectives:
Objective 1: Identify Avr gene candidates through genome sequence comparisons.
Objective 2: Validate Avr gene candidates through functional assays.
Objective 3: Examine Avr gene diversity and evolution in rust populations.
Objective 4: Determine the role of Avr proteins in rust infection.
Technical Summary
We seek to understand how the interactions between host immunity genes and pathogen virulence genes influences the evolution of pathogen populations to overcome host resistance. We focus on three related Puccinia species of fungi that infect different cereal crop hosts: Puccinia graminis f. sp. tritici (stem rust) infecting wheat, Puccinia hordei (brown rust) infecting barley, and Puccinia coronata f. sp. avenae (crown rust) infecting oats. These present contrasting levels of sexuality versus clonality and provide an opportunity to compare and contrast between related pathogen species and their hosts and different levels of sexual versus clonal reproduction.
The proposed work will address this under 4 objectives:
1. Identify Avr gene candidates through genome sequence comparisons. Haplotype-phased genome references will be constructed for key isolates and resequencing of additional isolates will allow for further identification of Avr loci through either genome-wide association or clonal mutation screening.
2. Validate Avr gene candidates through functional assays. Avr candidates will be expressed using cereal-infecting viral vectors and screened for their ability to infect wheat, barley or oat lines containing the corresponding resistance genes. Avr function will be verified using a protoplast transformation assay.
3. Examine Avr gene diversity and evolution in rust populations. Sequence and structural variation in Avr genes will be examined in genome assemblies and population sequence data to resolve general principles of how host resistance selects for virulence diversity in rust fungi.
4. Determine the role of Avr proteins in rust infection. Since Avr proteins are a subset of pathogen effector proteins thought to facilitate disease by targeting host cellular processes, we will use biochemical/in vivo screens to identify interacting host proteins and examine their roles by Virus-induced gene silencing.
The proposed work will address this under 4 objectives:
1. Identify Avr gene candidates through genome sequence comparisons. Haplotype-phased genome references will be constructed for key isolates and resequencing of additional isolates will allow for further identification of Avr loci through either genome-wide association or clonal mutation screening.
2. Validate Avr gene candidates through functional assays. Avr candidates will be expressed using cereal-infecting viral vectors and screened for their ability to infect wheat, barley or oat lines containing the corresponding resistance genes. Avr function will be verified using a protoplast transformation assay.
3. Examine Avr gene diversity and evolution in rust populations. Sequence and structural variation in Avr genes will be examined in genome assemblies and population sequence data to resolve general principles of how host resistance selects for virulence diversity in rust fungi.
4. Determine the role of Avr proteins in rust infection. Since Avr proteins are a subset of pathogen effector proteins thought to facilitate disease by targeting host cellular processes, we will use biochemical/in vivo screens to identify interacting host proteins and examine their roles by Virus-induced gene silencing.
Publications
Chen R
(2025)
A wheat tandem kinase activates an NLR to trigger immunity.
in Science (New York, N.Y.)
Darino M
(2022)
Apoplastic and vascular defences.
in Essays in biochemistry
Figueroa M
(2024)
Focus on the Effectors at the Interface of Plant-Microbe Interactions.
in Molecular plant-microbe interactions : MPMI
Kanyuka K
(2022)
Virus-Mediated Protein Overexpression (VOX) in Monocots to Identify and Functionally Characterize Fungal Effectors.
in Methods in molecular biology (Clifton, N.J.)
Lubega J
(2024)
Comparative Analysis of the Avirulence Effectors Produced by the Fungal Stem Rust Pathogen of Wheat.
in Molecular plant-microbe interactions : MPMI
Panwar V
(2022)
Virus-Induced Gene Silencing in Wheat and Related Monocot Species.
in Methods in molecular biology (Clifton, N.J.)
Wang D
(2023)
Pectin: a critical component in cell-wall-mediated immunity.
in Trends in plant science
| Description | This project is a collaboration between the University of Minnesota (USA), CSIRO (Australia), and NIAB (UK). So far, the project has yielded the following key findings. High-quality genome assemblies were generated for two Puccinia graminis f. sp. tritici (Pgt) isolates representative of two major races TKTTF and TTRTF using PacBio, HiFi, and Hi-C sequencing. Comparative analyses revealed genetic diversity within TKTTF, including evidence of somatic hybridisation. Variants of known Avr genes (AvrSr13, AvrSr22, AvrSr27, AvrSr35, and AvrSr50) were identified. To expand the wheat stem rust pangenome, sequencing was performed on 21 additional Pgt isolates representing global genetic diversity, with genome assemblies underway. Similarly, PacBio sequencing was completed for 11 Puccinia coronata (Pca) isolates, confirming novel mating type alleles and structural variation. Ten Puccinia hordei (Ph) isolates were also sequenced, revealing hybridisation events and transposable element expansions. These datasets will facilitate genome-wide analyses of Avr gene evolution. Virus-mediated overexpression (VOX) assays in wheat confirmed Avr activity for multiple candidate genes, including AvrSr13, AvrSr22, AvrSr26, AvrSr31, AvrSr33, and AvrSr62. A strong candidate for AvrSr31 was identified, and a study on AvrSr62 has just been accepted for publication. Additionally, novel plant virus-based vectors are being developed for gene function analysis in oat, enabling effector validation and pathogen-host interaction studies. Preliminary data suggest these vectors successfully infect three tested oat varieties, demonstrating their potential utility for functional analyses in P. coronata research. Novel sequence variants of Avr genes were identified across genome assemblies, including copy number variations and structural rearrangements at AvrSr27. Functional validation confirmed recognition for new Avr variants in Nicotiana benthamiana and wheat protoplast assays. Validation of these directly in wheat plants using VOX is under way. In contrast, the AvrSr35 variant in ITA2018-1 did not trigger recognition, explaining its virulence. This analysis establishes an evolving Avr gene atlas for Pgt lineages, providing a foundation for future surveillance and resistance breeding efforts. Transgenic wheat lines expressing known Pgt Avr effectors tagged with YFP were developed, enabling protein interaction studies. Co-immunoprecipitation and proteomic analyses identified candidate host targets, which are now undergoing validation using the split GAL4 RUBY assay. Subcellular localisation studies are in progress for AvrSr27, AvrSr35, and AvrSr50. A complementary yeast-two-hybrid screen with a quality wheat cDNA library (constructed from the total RNA from healthy and Pgt-inoculated seedlings sampled each day post-inoculation for 10 days) identified additional host interactors, with functional validation currently underway. The integration of these approaches will elucidate Avr effector functions in pathogen virulence and host defence. |
| Exploitation Route | This project is establishing an evolving Avr gene atlas for Pgt lineages, laying a foundation for future pathogen surveillance and resistance breeding efforts. |
| Sectors | Agriculture Food and Drink |
| Description | Aerobiome based genomic surveillance of fungicide resistance to track the development and spread of AMR in plant pathogens and the wider environment |
| Amount | £1,297,615 (GBP) |
| Funding ID | MR/Y034023/1 |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 02/2024 |
| End | 02/2027 |
| Description | Diagnosing and scoring diseases of crops using AI-based image analysis |
| Amount | £359,694 (GBP) |
| Funding ID | 10088620 |
| Organisation | Innovate UK |
| Sector | Public |
| Country | United Kingdom |
| Start | 01/2024 |
| End | 12/2025 |
| Description | SeptProtect: Rapid effector discovery to protect wheat from Septoria tritici blotch disease |
| Amount | £799,828 (GBP) |
| Funding ID | BB/X01617X/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 01/2024 |
| End | 12/2026 |
| Description | UK Cereal Pathogen Virulence Survey (UKCPVS) 2025-2027 |
| Amount | £258,916 (GBP) |
| Funding ID | 21120034a |
| Organisation | Agricultural and Horticulture Development Board |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 03/2025 |
| End | 03/2027 |
| Description | Wheat Genetic Improvement Network 5 (WGIN5) |
| Amount | £120,000 (GBP) |
| Funding ID | C24770 |
| Organisation | Department For Environment, Food And Rural Affairs (DEFRA) |
| Sector | Public |
| Country | United Kingdom |
| Start | 09/2024 |
| End | 09/2028 |
| Description | Peter Dodds |
| Organisation | Commonwealth Scientific and Industrial Research Organisation |
| Country | Australia |
| Sector | Public |
| PI Contribution | Expertise in stem rust disease of wheat, wheat genetics, disease resistance. |
| Collaborator Contribution | Expertise in functional genomics of cereals and associated microorganisms, virus-mediated over expression of heterologous proteins (VOX) |
| Impact | Isolation of the very fist stem rust effector protein (PubmedID 2926947) Publication (PubmedID 38170736) Groundswell 2023 Cereals 2023 NIAB Poster Day 2023 SeptProtect: Rapid effector discovery to protect wheat from Septoria tritici blotch disease Diagnosing and scoring diseases of crops using AI-based image analysis Aerobiome based genomic surveillance of fungicide resistance to track the development and spread of AMR in plant pathogens and the wider environment Session chair at the 16th International Cereal Rusts and Powdery Mildew Conference (2022) Guest editor themed issue of Essays in Biochemistry on Plant immunity (2022) Senior Editor Molecular Plant Pathology (2024) |
| Start Year | 2017 |
| Description | Prof Brian Steffenson |
| Organisation | University of Minnesota |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | Expertise in functional genomics of cereals and associated microorganisms, virus-mediated over expression of heterologous proteins (VOX). |
| Collaborator Contribution | Expertise in disease resistance in cereal crops, genetic diversity in the wild crop progenitors, host-parasite genetics, and virulence/molecular diversity in plant pathogenic fungi including cereal rusts. |
| Impact | Publication (PMID 38170736) Groundswell 2023 Cereals 2023 NIAB Poster Day 2023 SeptProtect: Rapid effector discovery to protect wheat from Septoria tritici blotch disease Diagnosing and scoring diseases of crops using AI-based image analysis Aerobiome based genomic surveillance of fungicide resistance to track the development and spread of AMR in plant pathogens and the wider environment Session chair at the 16th International Cereal Rusts and Powdery Mildew Conference (2022) Guest editor themed issue of Essays in Biochemistry on Plant immunity (2022) Senior Editor Molecular Plant Pathology (2024) |
| Start Year | 2022 |
| Description | British Crop Production Council Diseases Review meeting |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Agriculture practitioners including growers attended to hear about the cereal diseases of importance during the 2024 season. |
| Year(s) Of Engagement Activity | 2024 |
| Description | Cereals 2023 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster boards and live wheat and barley plot exhibits with recommended list varieties and several variety mixtures displaying a range of rust resistance scores. A two-days event full of one-on-one conversations with growers, farmers, agricultural advisors, breeders, and fellows academics about fungal diseases of cereal crops, disease resistance, evolution of resistance to fungicides in cereal pathogens and opportunistic human pathogens such as Aspergillus fumigatus which are wide spread in the environment. |
| Year(s) Of Engagement Activity | 2023 |
| Description | Cereals 2024 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster boards and live wheat and barley plot exhibits with recommended list varieties and several variety mixtures displaying a range of rust resistance scores. A two-days event full of one-on-one conversations with growers, farmers, agricultural advisors, breeders, and fellows academics about fungal diseases of cereal crops, disease resistance, evolution of resistance to fungicides in cereal pathogens and opportunistic human pathogens such as Aspergillus fumigatus which are wide spread in the environment. |
| Year(s) Of Engagement Activity | 2024 |
| Description | Ceres Rural Agronomy Technical Day |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | A group of farming consultants and agronomists attended a seminar series focused on management of crop diseases, including cereal rusts and septoria. |
| Year(s) Of Engagement Activity | 2024 |
| Description | Eastern Professional Development group |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Professional Practitioners |
| Results and Impact | Framers, agronomists, and growers attended a series of educational seminars on the various aspects of agriculture, including management of cereal diseases. |
| Year(s) Of Engagement Activity | 2025 |
| Description | Groundswell 2023 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster boards and live crop plot exhibits. A two-days event full of one-on-one conversations with growers, farmers, agricultural advisors, breeders, and fellows academics about fungal diseases of cereal crops, disease resistance, evolution of resistance to fungicides in cereal pathogens and opportunistic human pathogens such as Aspergillus fumigatus which are wide spread in the environment. |
| Year(s) Of Engagement Activity | 2023 |
| Description | NIAB Poster Day 2023 |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Interactive displays and exhibits from across NIAB's research teams including Plant Pathology, projects, departments, sites and services. The farm team also gave us a demonstration of satellite-guided tractors tackling an obstacle course in the car park. It was great to see so many staff getting together, discussing and learning about the work NIAB does. |
| Year(s) Of Engagement Activity | 2023 |
| Description | Visit to our research facilities by Frontier Agriculture |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Professional Practitioners |
| Results and Impact | A regional sales team of agronomy products visited Niab to learn about different areas of our work, including in the area of integrated pest management (IPM). They were given a tour our research facilities, and a series of mini-presentations to describe our main research areas. |
| Year(s) Of Engagement Activity | 2025 |
| Description | Visit to our research facilities by the Agricultural Industries Confederation (AIC) |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Industry/Business |
| Results and Impact | A delegation from the Agricultural Industries Confederation (AIC) - the UK agricultural supply industry's leading trade association, visited Niab Cambridge to learn about our research and facilities. |
| Year(s) Of Engagement Activity | 2025 |