Resurrection of plant disease resistance proteins via helper NLR bioengineering

PLANT SCIENCES Resurrection of plant disease resistance proteins via helper NLR bioengineering

Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC two by physically preventing intramolecular rearrangements required for activation. An amino acid polymorphism at the binding interface between NRC two and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a potential strategy for resurrecting disease resistance in crop genomes.

INTRODUCTION

The nucleotide binding and leucine-rich repeat class of intracellular immune receptors is an important component of innate immunity in plants and animals. They mediate intracellular recognition of pathogens and subsequently initiate an array of immune responses to counteract infection. NLRs can be activated by pathogen-secreted virulence proteins, termed effectors, which pathogens deliver into host cells to modulate host physiology. A hallmark of plant and animal NLR activation is their oligomerization into higher-order immune complexes termed resistosomes or inflammasomes, respectively. These complexes initiate immune signaling via diverse mechanisms, often leading to a form of programmed cell death, termed hypersensitive response in plants or pyroptosis in animals. Recent studies have reported NLR-like proteins mediating antiviral immunity and programmed cell death in prokaryotes via mechanisms analogous to those found in eukaryotic NLRs, suggesting that this is a conserved defense mechanism across all three domains of life. Pathogen effectors can act both as triggers and suppressors of NLR-mediated immunity. In some cases, adapted pathogens deploy effectors that directly or indirectly interfere with NLR signaling to suppress immune activation. However, the exact biochemical mechanisms by which pathogen effectors compromise NLR-mediated immunity to promote disease remain largely unknown. Moreover, whereas multiple strategies to bioengineer effector recognition specificities in NLRs have been proposed in recent years, approaches to mitigate the impact of effector-mediated immune suppression of NLRs are lacking.

NLRs belong to the signal adenosine triphosphatases with numerous domains superfamily. They typically exhibit a tripartite domain architecture consisting of an N-terminal signaling domain, a central nucleotide binding domain, and C-terminal superstructure forming repeats. The central domain, termed NB-ARC in plant NLRs, is a hallmark of this protein family and plays a key role as a molecular switch, mediating conformational changes required for activation. NB-ARC domains consist of a nucleotide binding domain, a helical domain, and a winged helix domain. Diverse NLR activation and signaling strategies are found in nature. In some cases, one NLR protein, termed a singleton, can mediate both effector perception and subsequent immune signaling. However, some NLRs can function as receptor pairs or, in higher-order configurations, termed immune receptor networks. In these cases, one NLR acts as a pathogen sensor, requiring a second helper NLR to initiate immune signaling. Such is the case in the solanaceous NRC immune receptor network, which is composed of multiple sensor NLRs that require an array of downstream helper NLRs termed NRCs to successfully initiate immune signaling. Upon perception of their cognate effectors, sensors in this network activate oligomerization of their downstream NRC helpers into a putative NRC resistosome, without stably forming part of the mature complex. This has been termed the activation and release model. The NRC network can encompass up to half of the NLRome in some solanaceous plant species and plays a key role in mediating immunity against a variety of plant pathogens including oomycetes, bacteria, viruses, nematodes, and insects.

Plant and metazoan parasites have evolved effectors that interfere with host NLR signaling to promote disease. Parasite effectors can suppress NLR-mediated immunity indirectly by interfering with host proteins that act downstream of NLR signaling or directly by interacting with NLRs to inhibit their functions. One such example is the potato cyst nematode effector, SPRYSEC fifteen, which can suppress signaling mediated by Nicotiana benthamiana helper NLRs NRC two and NRC three and tomato helper NLR NRC one, by binding to their central NB-ARC domains.

In contrast, another Nicotiana benthamiana helper NLR NRC four, a paralog of NRC two/three, cannot be suppressed by SPRYSEC fifteen. In this study, we reasoned that mapping the binding interface between SPRYSEC fifteen and its target helper NLRs combined with leveraging NRC four resilience to inhibition would enable us to bioengineer NLR variants that evade pathogen suppression, therefore resurrecting the immune signaling activity of upstream sensors in the NRC network.