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Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are found throughout several plant-associated microbial taxa and are typically considered to possess cytolytic activity exclusively on dicot plant species. However, cytolytic NLPs are also produced by pathogens of monocot plants such as the onion (Allium cepa) pathogen Botrytis squamosa. We determined the cytotoxic activity of B. squamosa BsNep1, as well as other previously characterized NLPs, on various monocot plant species and assessed the plant plasma membrane components required for NLP sensitivity. Leaf infiltration of NLPs showed that onion cultivars are differentially sensitive to NLPs, and analysis of their sphingolipid content revealed that the GIPC series A : series B ratio did not correlate to NLP sensitivity. A tri-hybrid population derived from a cross between onion and two wild relatives showed variation in NLP sensitivity within the population. We identified a quantitative trait locus (QTL) for NLP insensitivity that colocalized with a previously identified QTL for B. squamosa resistance and the segregating trait of NLP insensitivity correlated with the sphingolipid content. Our results demonstrate the cytotoxic activity of NLPs on several monocot plant species and legitimize their presence in monocot-specific plant pathogens.

The structural characterization of Glycosyl-Inositol-Phospho-Ceramides (GIPCs), which are the main sphingolipids of plant tissues, is a critical step towards the understanding of their physiological function. After optimization of their extraction, numerous plant GIPCs have been characterized by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) full scan analysis of negative ions provides a quick overview of GIPC distribution. Clear differences were observed for the two plant models studied: six GIPC series bearing from two to seven saccharide units were detected in tobacco BY-2 cell extracts, whereas GIPCs extracted from A. thaliana cell cultures and leaves were less diverse, with a dominance of species containing only two saccharide units. The number of GIPC species was around 50 in A. thaliana and 120 in tobacco BY-2 cells. MALDI-MS/MS spectra gave access to detailed structural information relative to the ceramide moiety, the polar head, as well as the number and types of saccharide units. Once released from GIPCs, fatty acid chains and long-chain bases were analyzed by GC/MS to verify that all GIPC series were taken into account by the MALDI-MS/MS approach. ESI-MS/MS provided complementary information for the identification of isobaric species and fatty acid chains. Such a methodology, mostly relying on MALDI-MS/MS, should open new avenues to determine structure-function relationships between glycosphingolipids and membrane organization.

A strain-specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant-based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high-molecular-weight structures. H5-containing structures were purified and examination by transmission electron microscopy confirmed virus-like particle (VLP) assembly. High-performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP-producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 microg of purified influenza H5-VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 microg of H5-VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.

Ubiquitination plays important roles in plant development, including programmed cell death. Here, we characterize a novel membrane-bound RING motif protein, encoded by RING1, that is expressed at a low level in all Arabidopsis tissues but can be upregulated by fumonisin B1 (FB1) treatment and pathogen infection. RING1 displays E3 ubiquitin ligase activity in vitro, which is dependent on the integrity of the RING motif. GFP fusion protein localization and cell fractionation experiments show that this E3 ligase is associated with the lipid rafts of plasma membranes. Knock-down of RING1 transcripts using artificial microRNA (amiR-R1(159)) leads to FB1 hyposensitivity, but overexpression of RING1 confers hypersensitivity. Additionally, expression of the pathogenesis-related 1 (PR-1) gene is lower and delayed in amiR-R1(159) plants compared with wild-type and RING1-overexpressing plants. The FB1 hyposensitivity of amiR-R1(159) plants can be rescued by expression of cleavage-resistant RING1mut transcripts. Our results suggest that RING1 acts as a signal from the plasma membrane lipid rafts to trigger the FB1-induced plant programmed cell death pathway.

International audience; Recognition of lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, by microglia occurs through its binding to specific receptors, cluster of differentiation 14 and toll-like receptor-4. LPS binding to these receptors triggers the synthesis of proinflammatory cytokines that coordinate the brain innate immune response to protect the CNS of the infection. Docosahexaenoic acid (DHA), a n-3 polyunsaturated fatty acid highly incorporated in the brain, is a potent immunomodulator. In this study, we investigated whether DHA modulates LPS receptor localization and, as a consequence, LPS-induced signaling pathway and proinflammatory cytokine production. We demonstrated that DHA, when added exogenously, is specifically enriched in membrane phospholipids, but not in raft lipids of microglial cells. DHA incorporation in membrane impaired surface presentation of LPS receptors cluster of differentiation 14 and toll-like receptor-4, but not their membrane subdomain localization. LPS-induced nuclear factor kappa B activation was inhibited by DHA, hence, LPS-induced proinflammatory cytokine synthesis of interleukin-1β and tumor necrosis factor α was strongly attenuated. We suggest that DHA is highly anti-inflammatory by targeting LPS receptor surface location, therefore reducing LPS action on microglia. This effect represents a new insight by which DHA modulates in the brain the expression of proinflammatory cytokines in response to bacterial product.

In the presence of oleoyl-CoA, purified and intact mitochondria from potato tuber formed phosphatidylcholine from labeled lysophosphatidylcholine. The labeled oleoyl moiety of the acyl-CoA was also incorporated in the absence of exogenous lysolipids, such incorporation being largely increased by the addition of exogenous lysophosphatidylcholine. In the presence of various other lysophospholipids, no synthesis of the corresponding phospholipids was observed, suggesting a high specificity of the acyltransferase towards the acyl acceptor. This enzyme was chiefly located in the outer membrane of mitochondria. These results indicate that any acylglycerophosphorylcholine transferred from the endoplasmic reticulum to mitochondria may be acylated to phosphatidylcholine.