Oomycete effector AVRblb2 targets cyclic nucleotide-gated channels through calcium sensors to suppress pattern-triggered immunity.

Transient and rapid increase in cytosolic Ca2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection.

Single and mixture toxicity evaluation of avobenzone and homosalate to male zebrafish and H295R cells.

Avobenzone and homosalate are widely used in sunscreens to provide ultraviolet (UV) protection, either as single compounds or in combination. Some UV filters exhibit estrogenic or anti-androgenic activities, however, studies regarding their interactions and toxicity in mixtures are limited. In this study, the effect of the toxicity of a binary mixture comprising avobenzone (0.72 µg L-1) and homosalate (1.02 and 103 µg L-1) on steroid hormone biosynthesis were investigated using male zebrafish and human adrenocortical carcinoma (H295R) cells. In fish exposed to homosalate, a significant decrease in the gonadosomatic index, testosterone level, and transcription of several genes (e.g, hsd3b2, cyp17a1, and hsd17b1) and a significant increase in the hepatosomatic index, liver steatosis, 17ß-estradiol level, and transcription of vtg gene were observed. These results suggest that estrogenic and anti-androgenic effects of homosalate were mediated by the steroidogenic pathway. The presence of 0.72 µg L-1 of avobenzone augmented the anti-androgenic responses in male fish. The testosterone level in the H295R cells were significantly decreased after they were exposed to homosalate alone or in combination with avobenzone, which is consistent with observations in male zebrafish. Further studies need to be conducted to understand the endocrine disrupting properties of long-term exposure to substances typically used in sunscreens.

The Phytophthora capsici RxLR effector CRISIS2 triggers cell death via suppressing plasma membrane H+-ATPase in the host plant.

Pathogen effectors can suppress various plant immune responses, suggesting that they have multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we screened the Phytophthora capsici RxLR cell death-inducer suppressing immune system (CRISIS). We found that the cell death induced by the CRISIS2 effector in Nicotiana benthamiana was inhibited by the irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene-silencing analyses revealed that CRISIS2 physically and functionally associated with PMAs and induced host cell death independent of immune receptors. CRISIS2 induced apoplastic alkalization by suppressing PMA activity via its association with the C-terminal regulatory domain. In planta expression of CRISIS2 significantly enhanced the virulence of P. capsici, whereas host-induced gene-silencing of CRISIS2 compromised the disease symptoms and the biomass of the pathogen. Thus, our study has identified a novel RxLR effector that plays multiple roles in the suppression of plant defense and in the induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant.

Phytophthora infestans RxLR Effector PITG06478 Hijacks 14-3-3 to Suppress PMA Activity Leading to Necrotrophic Cell Death.

Pathogens often induce cell death for their successful proliferation in the host plant. Plasma membrane H+-ATPases (PMAs) are targeted by either pathogens or plant immune receptors in immune response regulation. Although PMAs play pivotal roles in host cell death, the molecular mechanism of effector-mediated regulation of PMA activity has not been described. Here, we report that the Phytophthora infestans RxLR effector PITG06478 can induce cell death in Nicotiana benthamiana but the induced cell death is inhibited by fusicoccin (FC), an irreversible PMA activator. PITG06478, which is localized at the plasma membrane, is not directly associated with the PMA but is associated with Nb14-3-3s, a PMA activator. Immunoblot analyses revealed that the interaction between PITG06478 and Nb14-3-3s was disrupted by FC. PMA activity in PITG06478-expressing plants was eventually inhibited, and cell death likely occurred because the 14-3-3 protein was hijacked. Our results further confirm the significance of PMA activity in host cell death and provide new insight into how pathogens utilize essential host components to sustain their life cycle. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plasma membrane-localized plant immune receptor targets H+ -ATPase for membrane depolarization to regulate cell death.

The hypersensitive response (HR) is a robust immune response mediated by nucleotide-binding, 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. CCA 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 CCA 309 in Nicotiana benthamiana. CCA 309-induced extracellular alkalization causes plasma membrane depolarization, followed by cell death. Coimmunoprecipitation analyses suggest that CCA 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 HR-associated 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.

Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR clade in seed plants.

Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding domain leucine-rich repeat (NLR) proteins. Full-length NLRs or a specific domain of NLRs often induce plant cell death in the absence of pathogen infection. In this study we used genome-wide transient expression analysis to identify a group of NLRs (ANLs; ancient and autonomous NLRs) carrying autoactive coiled-coil (CCA ) domains in pepper (Capsicum annuum). CCA -mediated cell death mimics hypersensitive cell death triggered by the interaction between NLRs and pathogen effectors. Sequence alignment and mutagenesis analyses revealed that the intact α1 helix of CCA s is critical for both CCA - and ANL-mediated cell death. Cell death induced by CCA s does not require NRG1/ADR1 or NRC type helper NLRs, suggesting ANLs may function as singleton NLRs. We also found that CCA s localize to the plasma membrane, as demonstrated for Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive CCA s are well conserved in other Solanaceae plants as well as in rice, a monocot plant. Further phylogenetic analyses revealed that ANLs are present in all tested seed plants (spermatophytes). Our study not only uncovers the autonomous NLR clade in plants but also provides powerful resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in plants.