Publications

Nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) proteins can form complex receptor networks to confer innate immunity. An NLR-REQUIRED FOR CELL DEATH (NRC) is a phylogenetically related node that functions downstream of a massively expanded network of disease resistance proteins that protect against multiple plant pathogens. In this study, we used phylogenomic methods to reconstruct the macroevolution of the NRC family. One of the NRCs, termed NRC0, is the only family member shared across asterid plants, leading us to investigate its evolutionary history and genetic organization. In several asterid species, NRC0 is genetically clustered with other NLRs that are phylogenetically related to NRC-dependent disease resistance genes. This prompted us to hypothesize that the ancestral state of the NRC network is an NLR helper–sensor gene cluster that was present early during asterid evolution. We provide support for this hypothesis by demonstrating that NRC0 is essential for the hypersensitive cell death that is induced by its genetically linked sensor NLR partners in 4 divergent asterid species: tomato (Solanum lycopersicum), wild sweet potato (Ipomoea trifida), coffee (Coffea canephora), and carrot (Daucus carota). In addition, activation of a sensor NLR leads to higher-order complex formation of its genetically linked NRC0, similar to other NRCs. Our findings map out contrasting evolutionary dynamics in the macroevolution of the NRC network over the last 125 million years, from a functionally conserved NLR gene cluster to a massive genetically dispersed network.

Via The Sainsbury Lab

Diseases caused by filamentous plant pathogens impact global food production, leading to severe economic and humanitarian consequences. These pathogens secrete hundreds of effectors inside the host to alter cellular processes and to promote infection and disease. Effector proteins have weak or no sequence similarity but can be grouped in structural families based on conserved protein folds. However, very few conserved effector families have been functionally characterized. We have identified a family of effectors with a shared Zinc-finger protein fold (ZiF) that is present in lineages of the blast fungus Magnaporthe oryzae that can, collectively, infect 13 different grasses. We characterized the binding of a sub-set of these proteins to putative Exo70 host targets and showed they can be recognized by the plant immune system. Furthermore, we show that other ZiF effectors do not bind Exo70 targets, suggesting functional specialization within this effector family for alternative interactors. These findings shed light on the diversity of effectors and their molecular functions, as well as potentially leading to the development of new sources of blast disease resistance.

Via The Sainsbury Lab

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant- derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TBC1D15L, to remodel the host-pathogen interface. The effector, PiE354, hijacks TBC1D15L as a susceptibility factor to usurp its GAP activity on Rab8a—a key Rab GTPase crucial for defense-related secretion. By hijacking TBC1D15L, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co- opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.

Via The Sainsbury Lab

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a prominent class of intracellular immune receptors in plants. However, our understanding of plant NLR structure and function is limited to the evolutionarily young flowering plant clade. Here, we describe an extended spectrum of NLR diversity across divergent plant lineages and demonstrate the structural and functional similarities of N-terminal domains that trigger immune responses. We show that the broadly distributed coiled-coil (CC) and toll/interleukin-1 receptor (TIR) domain families of non-flowering plants retain immune-related functions through trans-lineage activation of cell death in the angiosperm Nicotiana benthamiana. We further examined a CC subfamily specific to non-flowering lineages and uncovered an essential N-terminal MAEPL motif that is functionally comparable to motifs in resistosome-forming CC-NLRs. Consistent with a conserved role in immunity, the ectopic activation of CCMAEPL in the non-flowering liverwort Marchantia polymorpha led to profound growth inhibition, defense gene activation, and signatures of cell death. Moreover, comparative transcriptomic analyses of CCMAEPL activity delineated a common CC-mediated immune program shared across evolutionarily divergent non-flowering and flowering plants. Collectively, our findings highlight the ancestral nature of NLR-mediated immunity during plant evolution that dates its origin to at least ∼500 million years ago.

Via The Sainsbury Lab

Secreted immune proteases Rcr3 and Pip1 of tomato are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvum but only Rcr3 act as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial Rcr3 residues for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2 dependent immunity. Despite substantial divergences in Rcr3 orthologs from eggplant and tobacco, only minimal alterations were sufficient to trigger Avr2/Cf-2-triggered immune signalling. Tomato Pip1, by contrast, was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side next to the substrate binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both proteases are suboptimal for binding Avr2. This study advances our understanding of the evolution of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

Via The Sainsbury Lab

Secreted immune proteases Rcr3 (Required for Cladosporium resistance-3) and Pip1 (Phytophthora- inhibited protease-1) of tomato (Solanum lycopersicum) are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signalling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues, and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

Via The Sainsbury Lab

NRCs are essential helper NLR (nucleotide-binding domain and leucine-rich repeat) proteins that execute the immune response triggered by disease resistance proteins, also known as sensor NLRs. The structure of the resting state of NbNRC2 was recently revealed to be a homodimer. However, the sensor-activated state has not yet been elucidated. In this study, we used cryo-EM to determine the structure of sensor-activated NbNRC2, which forms a hexameric inflammasome-like structure known as resistosome. To confirm the functional significance of the hexamer, we mutagenized the interfaces involved in oligomerization and found that mutations in three nucleotide-binding domain interface residues abolish oligomerization and immune signalling. Comparative structural analyses between the resting state NbNRC2 homodimer and the sensor-activated homohexamer revealed significant structural rearrangements before and after activation, providing insights into NLR activation mechanisms. Furthermore, structural comparisons between the NbNRC2 hexamer and previously reported CC-NLR pentameric assemblies revealed features in NbNRC2 that allow for the integration of an additional protomer. We also used the NbNRC2 hexamer structure to assess the recently released AlphaFold 3 for the prediction of activated CC-NLR oligomers. This revealed that AlphaFold 3 allows for high-confidence modelling of the N-terminal α1-helices of NbNRC2 and other CC-NLRs, a region that has proven difficult to fully resolve using structural approaches. Overall, our work sheds light on the structural and biochemical mechanisms underpinning NLR activation and expands our understanding of NLR structural diversity.

Via The Sainsbury Lab

Via The Sainsbury Lab

• Nucleotide-binding domain and leucine-rich repeat (NLR) proteins with pathogen sensor activities have evolved to initiate immune signaling by activating helper NLRs. However, the mechanisms underpinning helper NLR activation by sensor NLRs remain poorly understood. Although coiled coil (CC) type sensor NLRs such as the Potato virus X disease resistance protein Rx have been shown to activate the oligomerization of their downstream helpers NRC2, NRC3 and NRC4, the domains involved in sensor–helper signaling are not known.
• Here, we used Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana to show that the nucleotide-binding (NB) domain within the NB-ARC of Rx is necessary and sufficient for oligomerization and immune signaling of downstream helper NLRs. In addition, the NB domains of the disease resistance proteins Gpa2 (cyst nematode resistance), Rpi-amr1, Rpi-amr3 (oomycete resistance) and Sw-5b (virus resistance) are also sufficient to activate their respective downstream NRC helpers.
• Using transient expression in the lettuce (Lactuca sativa), we show that Rx (both as full length or as NB domain truncation) and its helper NRC2 form a minimal functional unit that can be transferred from solanaceous plants (lamiids) to Campanulid species.
• Our results challenge the prevailing paradigm that NLR proteins exclusively signal via their N-terminal domains and reveal a signaling activity for the NB domain of NRC-dependent sensor NLRs. We propose a model in which helper NLRs can perceive the status of the NB domain of their upstream sensors.

Via The Sainsbury Lab

Crop production often faces challenges from plant diseases, and biological control emerges as an effective, environmentally friendly, cost-effective, and sustainable alternative to chemical control. Wheat blast disease caused by fungal pathogen Magnaporthe oryzae Triticum (MoT), is a potential catastrophic threat to global food security. This study aimed to identify potential bacterial isolates from rice and wheat seeds with inhibitory effects against MoT. In dual culture and seedling assays, three bacterial isolates (BTS-3, BTS-4, and BTLK6A) demonstrated effective suppression of MoT growth and reduced wheat blast severity when artificially inoculated at the seedling stage. Genome phylogeny identified these isolates as Bacillus subtilis (BTS-3) and B. velezensis (BTS-4 and BTLK6A). Whole-genome analysis revealed the presence of genes responsible for controlling MoT through antimicrobial defense, antioxidant defense, cell wall degradation, and induced systemic resistance (ISR). Taken together, our results suggest that the suppression of wheat blast disease by seed endophytic B. subtilis (BTS-3) and B. velezensis (BTS-4 and BTLK6A) is liked with antibiosis and induced systemic resistance to wheat plants. A further field validation is needed before recommending these endophytic bacteria for biological control of wheat blast.

Via The Sainsbury Lab