Biology subjects

D'Ambrosio, J. M.

Publications and source records attributed to D'Ambrosio, J. M..

4 recordsLinked to original sources

bioRxiv2025

Phosphoinositide-specific Phospholipase C 2 (SlPLC2) Facilitates Vesicle Formation and Modulates Immune Signaling in Tomato Phytophthora infestans Interactions

Phosphoinositide-specific phospholipase C (PI-PLC) is a signaling enzyme that hydrolyzes membrane phosphoinositide to generate lipid- and lipid-derived second messengers. In plants, PI-PLCs have been implicated in various physiological processes, including immunity. Tomato SlPLC2 was previously shown to be implicated in susceptibility to the necrotrophic fungus Botrytis cinerea. However, whether this observation extends to pathogens with different lifestyles and evolutionary origins, remains unknown. Here, we investigated SlPLC2 function during infection with Phytophthora infestans, a globally devastating oomycete and causal agent of potato and tomato late blight. CRISPR-Cas9 knockout SlPLC2 plants showed significantly reduced disease symptoms, lower pathogen biomass, and decreased sporangia production, indicating impaired colonization. Upon infection, SlPLC2 knockout lines displayed attenuated expression of salicylic acid (SA)- and jasmonic acid (JA)-responsive genes suggesting disrupted hormone signaling. Consistent with PLC role during plant defense, early immune responses such as hydrogen peroxide accumulation and callose deposition were reduced in knockout plants. At the cellular level, these plants allow fewer infection vesicles formation by P. infestans, accompanied by an increase in expression of a biotrophy-associated effector gene PiAvrblb2, suggesting impaired establishment of infection. In Nicotiana benthamiana SlPLC2-GFP localizes predominantly at the plasma membrane. Upon P. infestans inoculations, SlPLC2-GFP localizes to the membrane surrounding infection vesicles, where we also detected phosphoinositides PI4P and PI(4,5)P2. Overexpression of SlPLC2 in Nicotiana benthamiana enhanced susceptibility to P. infestans, reinforcing its positive role in colonization. Together, these findings identify SlPLC2 as a susceptibility factor associated with enhanced P. infestans colonization. StatementThis study identifies SlPLC2 as a key susceptibility factor that supports Phytophthora infestans infection by coordinating vesicle formation and early immune responses in tomato.

plant biology↗

bioRxiv2025

Ist2, a protein involved in phosphatidylserine transport, is an ER lipid scramblase

Lipid scramblases allow passive flip-flop of phospholipids between bilayer leaflets, thereby promoting membrane symmetry. At the endoplasmic reticulum (ER), where phospholipid synthesis is restricted to one of the two leaflets, scramblase activity should be essential for equilibrated membrane growth. However, phospholipid scramblases at the ER are poorly understood. The yeast protein Ist2 contains an ER domain and a cytosolic tail that binds the plasma membrane (PM) and participates in the transfer of phosphatidylserine (PS). Here, we show both in vitro and in silico that the ER- domain of Ist2, which bears homology to the TMEM16 proteins, possesses a lipid scramblase activity. Ist2 activity is not regulated by Ca2+, in contrast to TMEM16 proteins, but is affected by the lipid composition of the bilayer used in simulations. In cells, we do not find a strong impact of the scramblase domain of Ist2 in on PS distribution; however, its over-expression or deletion affects processes at the ER such as vesicular transport, lipid droplet biogenesis and general phospholipid transport, with a specific contribution of residues important for lipid scrambling. Our study therefore identifies the first dedicated phospholipid scramblase in yeast and demonstrates that membrane asymmetry can impact diverse membrane-remodeling processes at the ER.

cell biology↗

bioRxiv2020

A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterization in different cell types

Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g. Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. To facilitate further cellular investigations, notably by imaging techniques, we present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterization in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localization of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localization of Nsp13 to the centrosome, Orf3a to late endosomes, and Orf9b to mitochondria.

cell biology↗

bioRxiv2020

Osh6 requires Ist2 for localization to the ER-PM contacts and efficient phosphatidylserine transport

Osh6 and Osh7 are lipid transfer proteins (LTPs) that move phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM). High PS level at the PM is key for many cellular functions. Intriguingly, Osh6/7 localize to ER-PM contact sites, although they lack membrane-targeting motifs, in contrast to multidomain LTPs that both bridge membranes and convey lipids. We show that Osh6 localization to contact sites depends on its interaction with the cytosolic tail of the ER-PM tether Ist2, a homologue of TMEM16 proteins. We identify a motif in the Ist2 tail, conserved in yeasts, as the Osh6-binding region, and we map an Ist2-binding surface on Osh6. Mutations in the Ist2 tail phenocopy osh6{Delta} osh7{Delta} deletion: they decrease cellular PS levels, and block PS transport to the PM. Our study unveils an unexpected partnership between a TMEM16-like protein and a soluble LTP, which together mediate lipid transport at contact sites.

cell biology↗