Biology subjects

Madhuprakash, J.

Publications and source records attributed to Madhuprakash, J..

4 recordsLinked to original sources

bioRxiv2025

A plant pathogen effector blocks stepwise assembly of a helper NLR resistosome

Helper NLRs function as central nodes in plant immune networks. Upon activation, they oligomerize into inflammasome-like resistosomes to initiate immune signaling, yet the dynamics of resistosome assembly remain poorly understood. Here, we show that the virulence effector AVRcap1b from the Irish potato famine pathogen Phytophthora infestans suppresses immune activation by directly engaging oligomerization intermediates of the tomato helper NLR SlNRC3. Cryo-EM structures of SlNRC3 in AVRcap1b-bound and unbound states reveal that AVRcap1b bridges multiple protomers, stabilizing a stalled intermediate and preventing formation of a functional resistosome. Leveraging AVRcap1b as a molecular tool, we also capture an additional SlNRC3 resistosome intermediate showing that assembly proceeds in a stepwise manner from dissociated monomers. These findings uncover a previously unrecognized vulnerability in NLR activation and reveal a pathogen strategy that disrupts immune complex assembly. This work advances mechanistic understanding of resistosome formation and uncovers a previously unrecognized facet of pathogen-plant coevolution.

plant biology↗

bioRxiv2025

An effector from the potato late blight pathogen bridges ENTH-domain protein TOL9a to an activated helper NLR to suppress immunity

Pathogens counteract central nodes of NLR immune receptor networks to suppress immunity. However, the mechanisms by which pathogens hijack helper NLR pathways are poorly understood. Here, we show that an effector from the potato late blight pathogen Phytophthora infestans bridges the host protein NbTOL9a, a putative member of the host ESCRT pathway, to a helper NLR to suppress immunity. In this work, we solved the crystal structure of the RXLR-LWY effector AVRcap1b in complex with the ENTH domain of NbTOL9a. The structure revealed that unlike other RXLR-LWY effectors, AVRcap1b has a novel L-shaped fold that defines a new structural family of effectors in the Phytophthora genus. Moreover, we defined the AVRcap1b/NbTOL9a binding interface and designed effector mutants that dont bind NbTOL9a, impairing immune suppression. This indicates that ENTH binding is required for full virulence activity of this effector. Lastly, we show that AVRcap1b associates specifically with activated NbNRC2 independently of NbTOL9a binding. This suggests that the effector functions as a bridge that interconnects NbNRC2 with the NbTOL9a pathway. These results illustrate an unprecedented pathogen mechanism to hijack helper NLR pathways and suppress immunity.

plant biology↗

bioRxiv2025

Functional insights into a peculiar tetra-modular LPMO from the human pathogen Enterobacter cloacae

Enterobacter cloacae is a Gram-negative nosocomial human pathogen that inhabits diverse ecological niches. Its genome encodes a conserved set of putative chitin-active enzymes, including a peculiar lytic polysaccharide monooxygenase (LPMO), termed EcLPMO, which we functionally characterized in this study. EcLPMO is a tetra-modular protein consisting of an auxiliary activity family 10 (AA10) catalytic domain, two central domains of unknown function (DUF-A and DUF-B), and a C-terminal carbohydrate-binding module (CBM73). Functional assays using full-length EcLPMO and its truncated variants demonstrated that the AA10 domain oxidatively cleaves chitin at the C1 position. The CBM73 module enhances chitin binding and promotes synergy with endogenous chitinases. Notably, EcLPMO displayed a particularly strong synergistic effect with the unimodular chitinase EcChiA, leading to up to 14-fold and 60-fold increases in GlcNAc release from - and {beta}-chitin, respectively. Deletion of both DUFs reduced EcLPMO activity. While DUF-A alone and the association of DUF-A and DUF-B showed limited chitin binding, DUF-B alone exhibited no binding, suggesting a distinct role. Unexpectedly, using state-of-the-art structural modelling (AlphaFold3), we observed that the DUF-B domain contains two highly conserved histidines that coordinate the AA10-bound copper, forming a previously unreported inter-domain tetra-histidine copper coordination center. These findings highlight the structural and functional complexity of EcLPMO and suggest that its accessory domains, particularly DUF-B, may contribute to enzyme stability and substrate interaction. We speculate that DUF-B may protect the LPMO active site from oxidative damage, a feature that could prove crucial in its ecological and pathogenic contexts.

biochemistry↗

bioRxiv2024

A disease resistance protein triggers oligomerization of its NLR helper into a hexameric resistosome to mediate innate immunity

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 1-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.

plant biology↗