I’m therefore incredibly thankful to all co-authors that contributed to this project. Special thanks to @marioncmueller.bsky.social for contributing many crucial analyses and brilliant ideas, and to Beat Keller for his continuous support of the project over so many years.

In hindsight, the many setbacks and project-related headaches were compensated by these fascinating insights into the promiscuity of NLR proteins towards effector targets. The project also sparked multiple innovations, large and small, that proved useful for various other research questions.

Personal note: When I joined the (already ongoing) search for ominous AvrPm3e in the Keller lab in 2016, I didn’t know what I signed up for. Approach after approach was unsuccessful until we found an isolate effectively breaking Pm3e resistance in 2022, allowing us to finally map both AVR genes.

In conclusion, our findings demonstrate the potential of Pm3 immune receptors for broad-spectrum resistance against wheat powdery mildew through recognition of structurally diverse effectors and highlight their amenability for NLR engineering.

Subsequently we used this knowledge to generate engineered Pm3 NLRs that fully incorporate Pm3d- and Pm3e specificities and verified their further extended recognition spectra, now including three mildew AVRs, in Nicotiana and wheat.

Using chimeric NLRs between Pm3d, Pm3e and the ancestral Pm3CS (completely overcome by powdery mildew) we then identified individual amino acid polymorphisms defining the recognition specificity of Pm3d and Pm3e towards their various effector targets.

We concluded that Pm3d and Pm3e provide broad-spectrum resistance by each recognizing two AVR effectors. While both NLRs converge on a common RNAse-like AVR, they each recognize a unique second AVR factor. In the case of Pm3e that’s a structurally unrelated effector protein.

Intriguingly, AvrPm3e_1 belongs to a group of structurally related effectors with >100 members in wheat powdery mildew (~13% of the entire effector complement) that, so far, has not been implicated as AVR factors neither for Pm3 nor for other resistance sources in wheat or barley.

We found that Pm3e, like Pm3d, has the ability to recognize variants of the BgtE-20069b effector (now AvrPm3d_1/AvrPm3e_2). However, its second AVR, BgtE-5754 (AvrPm3e_1), encodes a much larger effector protein predicted to exhibit a completely different structure!

But what about Pm3e? Here our mapping approach identified two genetically unlinked AVR loci on chromosomes 4 and 9, with the later one perfectly overlapping with the AvrPm3d locus. Identifying the underlying AVRs held an interesting surprise!

Similar to all other known AVRs in cereal powdery mildews, both AvrPm3d genes encode small effectors with an RNAse-like fold. We concluded that the broad-spectrum resistance of Pm3d is likely due to its ability to recognize two effectors, thereby reducing the likelihood of resistance breakdown.

However, the locus contained two AVR genes: the previously described BgtE-20069b effector (AvrPm3d_1) as well as its neighboring gene BgtE-20069a (AvrPm3d_2), a close paralog that was missed in our previous study. Only isolates compromised in both AVRs can break the Pm3d resistance.

Using our recently developed avirulence depletion mapping strategy (doi.org/10.1371/jour...), we found that there is a single AvrPm3d locus in wheat powdery mildew.

Interestingly the Pm3d and Pm3e NLRs differ by only three and two amino acids, respectively, from the ancestral Pm3CS which has been completely overcome by the powdery mildew pathogen. So what makes Pm3d and Pm3e so successful? To answer this we aimed at identifying the corresponding AVR effectors.

The wheat Pm3 NLR locus is highly diverse with at least 17 alleles providing resistance against wheat powdery mildew. We challenged transgenic Pm3 wheat lines with >90 mildew isolates collected worldwide and identified two Pm3 alleles, Pm3d and Pm3e, that confer broad-spectrum resistance.