Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding domain and leucine-rich repeat (NLR) proteins. Autoactive NLRs, some cases a specific domain of NLR, induce plant cell death in the absence of pathogen infection. In this study, we identified a group of NLRs (ANLs; ancient and autonomous NLRs) carrying autoactive coiled-coil (CC A) domains in pepper (Capsicum annuum L.) by genome-wide transient expression analysis. The CC A-mediated cell death mimics hypersensitive cell death triggered by interaction between NLR and pathogen effectors. Sequence alignment and mutagenesis analyses revealed that the intact α1 helix of CC A is critical for both CC A-and ANL-mediated cell death. The cell death induced by CC A does not require NRG1/ADR1 or NRC type helper NLRs, suggesting ANLs may function as singleton NLRs. We also found that CC A localize in the plasma membrane as Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive CC A s are well conserved in other Solanaceae plants as well as a monocot plant, rice. Further phylogenetic analyses revealed that ANLs are present in all tested seed plants (spermatophytes). Our studies not only uncover the autonomous NLR clade in plants but also provide powerful resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in plant.
The hypersensitive response (HR) is a robust immune response mediated by nucleotidebinding, leucine-rich repeat receptors (NLRs). However, the early molecular event that links activated NLRs to cell death is unclear.Here, we demonstrate that NLRs target plasma membrane H + -ATPases (PMAs) that generate electrochemical potential, an essential component of living cells, across the plasma membrane. CC A 309, an autoactive N-terminal domain of a coiled-coil NLR (CNL) in pepper, is associated with PMAs. Silencing or overexpression of PMAs reversibly affects cell death induced by CC A 309 in Nicotiana benthamiana.CC A 309-induced extracellular alkalization causes plasma membrane depolarization, followed by cell death. Coimmunoprecipitation analyses suggest that CC A 309 inhibits PMA activation by preoccupying the dephosphorylated penultimate threonine residue of PMA. Moreover, pharmacological experiments using fusicoccin, an irreversible PMA activator, showed that inhibition of PMAs contributes to CNL-type (but not Toll interleukin-1 receptor NLR-type) resistance protein-induced cell death.We suggest PMAs as primary targets of plasma membrane-associated CNLs leading to HRassociated cell death by disturbing the electrochemical gradient across the membrane. These results provide new insight into NLR-mediated cell death in plants, as well as innate immunity in higher eukaryotes.
1 Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding 2 domain and leucine-rich repeat (NLR) proteins. Autoactive NLRs, in some cases a specific 3 NLR domain, induce plant cell death in the absence of pathogen infection. In this study, we 4 identified a group of NLRs (G10) carrying autoactive coiled-coil (CC) domains in pepper 5 (Capsicum annuum L. cv. CM334) by genome-wide transient expression analysis. The G10-6 CC-mediated cell death mimics hypersensitive response (HR) cell death triggered by 7 interaction between NLR and effectors derived from pathogens. Sequence alignment and 8 mutagenesis analyses revealed that the intact α 1 helix of G10-CCs is critical for both G10-9 CC-and R gene-mediated HR cell death. The cell death induced by G10-CCs does not 10 require known helper NLRs, suggesting G10-NLRs are novel singleton NLRs. We also found 11 that G10-CCs localize in the plasma membrane as Arabidopsis singleton NLR ZAR1. 12 Extended studies revealed that autoactive G10-CCs are well conserved in other Solanaceae 13 plants, including tomato, potato, and tobacco, as well as a monocot plant, rice. Furthermore, 14 G10-NLR is an ancient form of NLR that present in all tested seed plants (spermatophytes). 15Our studies not only uncover the autonomous NLR cluster in plants but also provide powerful 16 resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in 17 plants.18 1 1 131 CNL clades (34). Taken together, these data suggest that G10-NLRs have features specific to 132 other CNLs and they may play unique a role(s) in plant immunity.
Hypersensitive response (HR) is a robust immune response mediated by plant nucleotide-binding and leucine-rich repeat receptor (NLR). However, the early molecular event linking NLR to cell death is obscure. Here we demonstrate that NLR targets plasma membrane H+-ATPases (PMA) generating electrochemical potential across the membrane. CCA309, an autoactive N-terminal domain of pepper coiled-coil NLR (CNL), associates with PMAs and its autoactivity is affected by silencing or overexpression of PMA. CCA309-induced extracellular alkalization accompanied with membrane depolarization is followed by calcium influx and cell death. CCA309 interacts with C-terminal regulatory domain of PMA and 14-3-3 negatively affects CCA309-induced cell death. Moreover, pharmacological experiments with fusicoccin, an irreversible PMA activator, confirmed that CC- and CNL-mediated cell death occurred through inhibiting PMA. We propose PMAs as the primary target of plasma membrane-associated CNL to disrupt electrochemical homeostasis leading to HR cell death.
Pathogen effectors can suppress various plant immune responses, suggesting their multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we performed Phytophthora capsici CRISIS screening. In Nicotiana benthamiana, cell death induced by the RxLR effector CRISIS2 is inhibited by irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene silencing analyses revealed that CRISIS2 physically and functionally associates with PMAs and induces cell death independent of immune receptors. CRISIS2 induces apoplastic alkalization by suppressing PMA activity by association with the C-terminal regulatory domain of PMA. In planta expression of CRISIS2 significantly enhanced virulence of P. capsici, whereas host-induced gene silencing of CRISIS2 compromised disease symptom and biomass of P. capsici. Furthermore, co-immunoprecipitation assays revealed CRISIS2 associates with BAK1, the co-receptor of PRRs. CRISIS2 interferes with the FLS2-BAK1 complex induced by flagellin perception and impairs downstream signaling from PRR complex. Proteomics and gene silencing assays identified putative PRRs negatively regulate the virulence of P. capsici in N. benthamiana as interactors of CRISIS2 and BAK1. Our study identified a novel RxLR effector playing multiple roles in suppression of plant defense and induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.