Race-Specific Resistance Mechanisms and Evasion from Recognition: Insights from the Wheat-Powdery Mildew Pathosystem

Abstract

Wheat powdery mildew disease is caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt). To achieve successful infection, Bgt produces many different effector proteins that modify host cell processes. Resistant wheat lines possess resistance (R) genes, whose encoded R proteins can detect specific effectors (then called avirulence [Avr] effectors) and subsequently activate the immune response. This stops pathogen spread, mostly by inducing cell death. One of the most extensively researched R genes in wheat encodes for the the nucleotide-binding leucine-rich repeat (NLR) receptor Pm3, which exists in various protein variants that can recognise sequence-unrelated Avr effectors from Bgt (i.e. AvrPm3a2/f2, AvrPm3b2/c2, AvrPm3d). Recently, it has been shown that non-NLR genes, such as tandem kinase genes, also contribute to race-specific resistance in cereal crops. To understand the molecular basis of resistance, it is critical to identify and characterise R genes in wheat, as well as their corresponding Avr effectors in Bgt.

In the first part of this thesis, I developed AvrXpose, a novel approach for identifying Avr effectors in Bgt. Using UV mutagenesis, I was able to identify Bgt mutants with gain of virulence on wheat lines containing specific R genes. I showed that gain of virulence on the R genes Pm3b and Pm3c co-occurs in Bgt mutants with mutations in the corresponding Avr effector, AvrPm3b2/c2. I also showed that Bgt mutants virulent on Pm3a have mutations in an ankyrin-repeat-containing gene, Bgt-646. Interestingly, mutants in Bgt-646 also exhibited virulence on WTK4, a tandem kinase R gene unrelated to Pm3a. This finding, together with expression analysis of different Bgt effector genes, suggested that Bgt-646 is a regulator of a set of effector genes in Bgt, thereby affecting avirulence to different wheat R genes.

In the second part, I aimed to identify the Avr effector recognised by WTK4. I combined a bi-parental mapping approach with AvrXpose, and both methods led to the identification of the same Avr candidate, encoding an RNase-like effector, the typical structure of all Bgt Avrs identified to date. Remarkably, WTK4-virulent mutants had either mutations in AvrWTK4, or a reduced expression of it, which, in two cases, was the result of mutations in Bgt-646. I discovered that WTK4, besides a kinase and a pseudokinase domain, also possesses a heavy metal associated (HMA)-like domain and showed that it may act as a decoy for pathogen perception by directly interacting specifically with the avirulent variant of AvrWTK4.

In the last part of this thesis, I aimed to characterize the introgression landscape in a global wheat panel, and, at the same time, identify novel sources of powdery mildew resistance. I produced a cross between the resistant wheat cultivar Pamukale and the susceptible cultivar Frisal and used the F2 progeny to map a novel R gene locus on the long arm of chromosome 7D. Interestingly, the F2 progeny segregated for resistance in a recessive manner. Genomic approaches revealed that the R gene locus, named PmPam, resides in an introgression from an unknown wild crop relative, unique to the cultivar Pamukale.

The development of AvrXpose has enabled a range of exciting new projects and discoveries that would not have been possible with other approaches. AvrXpose has allowed the identification of Bgt-646, a regulator of Avr factors, further demonstrating the relevance of expression reduction as a gain-of-virulence mechanism in Bgt. It also supported the identification of the first Avr effector from Bgt interacting with a non-NLR resistance protein, WTK4, providing valuable knowledge for understanding non-NLR-based race-specific resistance in wheat. Finally, the genetic mapping of a novel R gene locus further confirmed that introgressions are valuable sources of agronomically interesting traits.