Applying effectoromics and genomics to identify resistance against Rhynchosporium commune in barley

Rhynchosporium commune is one of the most destructive fungal pathogens of barley worldwide. It causes scald, responsible for reduced grain quality and yield losses of up to 40%. This project aimed to identify genetic resistance in barley using two different approaches: an effector approach through the identification of important pathogen virulence factors and their barley targets, and a genomics association approach. Numerous secreted effectors have been identified in many phytopathogens including R. commune. Rrs1 resistance, recognising the R. commune avirulence protein - AvrRrs1 (NIP1) has been deployed in the field to prevent infection but has soon proven ineffective. R. commune has managed to overcome this resistance by alteration or deletion of the NIP1 gene as it is not essential for pathogenicity. However, our field trial data suggests that Rrs1 remains an important component of resistance to R. commune in the field. Resistance genes recognising more essential Avr genes are likely to be more durable and as a consequence, the discovery of novel R. commune Avr genes is fundamental for the implementation of an integrated pest management approach to prevent this disease. Recent sequencing of the R. commune genome allowed identification of putative effectors. Expression of 26 potential effectors with low sequence variability in 9 sequenced R. commune strains have been analysed during barley infection. The best genes were selected for gene disruption and individual expression in barley cultivars and landraces using the Barley Stripe Mosaic Virus (BSMV) – based expression system to see if they are recognised by the plant. The work also focused on candidate effectors with putative functions. A putative protease inhibitor was chosen for functional characterisation but its function and importance for pathogenicity could not be confirmed. In addition, high amount of the candidate protein appeared to be toxic for barley and Nicotinana benthamiana. Two SA (salicylic acid)-related putative effectors were also chosen for further characterisation and revealed a direct link between the SA pathway of barley and R. commune. The results of this project suggest that R. commune might be able to manipulate the SA pathway of the host confirming the existence of a biotrophic phase of the fungus. The genomics association approach to identify resistance genes against R. commune in barley used a Genome Wide Association Scan (GWAS) using a combination of three years of disease nursery field trial data for a collection of over 500 elite spring barley cultivars. This analysis identified a number of quantitative trait loci (QTL) in barley genome regions previously shown to contain major resistance genes such as Rrs1 on chromosome 3H, Rrs2 on chromosome 7H, Rrs3 on chromosome 4H, Rrs4 on chromosome 3H, Rrs13 on chromosome 6H, Rrs14 on chromosome 1H and Rrs16 on chromosome 4H, as well as novel QTL. The work was focused on Rrs1 resistance. R. commune strains producing a type of NIP1 effector, recognised by barley lines containing Rrs1, were used to confirm the resistance in predicted Rrs1 barley cultivars. The Rrs1 interval has been narrowed down to 3 Mbp, and high resolution mapping led to the identification of 3 SNP markers which perfectly discriminated Rrs1Rh4 lines from susceptible lines. These diagnostic markers will provide a useful breeding tool for improving the design of new varieties allowing the incorporation of the Rrs1 resistance. This research takes us a step closer towards cloning the first barley major resistance (R) gene against R. commune, which is likely to be present only in Rrs1 lines and have a kinase domain very similar to the one in a putative wall associated kinase found within the Rrs1 interval in the genome assembly of susceptible cultivar Morex. It will also help us to better understand R. commune-barley pathosystem and to identify further R genes.