NLR immune receptors for sustainable disease resistance in cereal crops – ImmuneReceptor

Plant diseases are among the most important problems in agriculture and the use of disease resistance (R) genes is a key strategy for plant protection. ImmuneReceptor addresses fundamental questions in plant immunity to generate transferable knowledge for sustainable crop protection. It focuses on the most important class of disease resistance proteins, termed NLRs that are characterized by nucleotide-binding and leucine-rich repeat domains. NLR-coding genes are the main class of R genes employed in resistance breeding and their innovative, knowledge-based use (e.g. in pyramiding or variety mixture strategies) is a central element in sustainable crop protection. NLR proteins act as immune receptors and recognize pathogen-secreted virulence factors (effectors) termed avirulence proteins (Avrs). They are further subdivided according to their N-terminal signalling domain: CNLs, the only class of NLRs present in cereals, harbour a coiled-coil (CC) domain and TNLs possess a Toll-Interleukin-1 (TIR) domain.

Despite intense research on plant NLRs over the last 20 years, many aspects of their activity are still unknown. In particular, the molecular details of Avr recognition and the link between Avr recognition and resistance activation are poorly defined. In ImmuneReceptor, rice CNLs and their matching Avr proteins from the rice blast fungus Magnaporthe oryzae as well as selected CNLs from other cereals will be investigated by a combination of cutting edge structural biology, protein biochemistry, molecular genetics and phytopathology approaches to get a detailed structural and mechanistic understanding of Avr protein recognition and CNL function. For this, we will rely on our previous work that established the CNL pair RGA4/RGA5 from rice as a model for cereal CNLs.

Both RGA4 and RGA5 were shown to be required for the recognition of AVR1-CO39 and AVR-Pia, two sequence-unrelated M. oryzae effectors. RGA5 interacts physically with either effector via a C-terminal non-LRR sensor domain of ~200 amino acids (RGA5C-ter) and this direct binding is required for resistance and confers specificity. RGA4 is a constitutively active cell death inducer that is repressed by RGA5. Upon effector binding, RGA5 loses its repressor activity and RGA4 triggers resistance and cell death.

In the ImmuneReceptor project, molecular details of Avr-RGA5 binding will be elucidated by resolving the three-dimensional structure of RGA5C-ter and of complexes between RGA5C-ter and AVR1-CO39 or AVR-Pia. In vitro and in vivo mutant analysis will be performed to validate these structural models of Avr recognition and to investigate structure-function relations. In addition, structure guided design of new recognition specificities will be performed to serve as a first proof of concept for this type of approach on plant NLRs.

In addition, the molecular bases of RGA4-RGA5 interaction will be investigated. RGA4 and RGA5 form homo and hetero-complexes and the composition and stoichiometry of these complexes in the resting and the activated state will be determined. The CC domains are the best candidates to mediate RGA4-RGA5 complex formation since they form homo and hetero-interactions. The molecular bases of these interactions will be addressed by determining the three-dimensional structures of RGA4 and RGA5 CC domains and of their hetero complexes. These structural models will be validated by in vitro and in vivo mutant analysis to confirm the amino acids that mediate CC interactions and to determine the role of homo and hetero CC interactions in resistance activation and repression.

Detailed knowledge on RGA4/RGA5 will be extended to CNL heteropairs homologous to RGA4/RGA5 in rice and other important cereals. By a combination of structural biology and functional approaches it will be determined to what extend the mechanisms governing RGA4/RGA5 function are generic to this entire group of cereal CNL pairs and how specificity in their interactions is generated.