2021-BBSRC/NSF-BIO: Host Immunity as a Driver of Virulence Evolution in Cereal Rust Fungi

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.

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.