Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases. These results open therapeutic opportunities by providing a route of entry and a repair mechanism for lysosomes in pathological situations. This work is the result of our search for the mechanism of action of Apolipoprotein D (ApoD), a neuroprotective lipid-binding protein that confers cell resistance to oxidative stress. ApoD is one of the few genes consistently over-expressed in the aging brain of all vertebrate species, and no nervous system disease has been found concurring without ApoD over-expression. All evidence supports ApoD as an endogenous mechanism of protection. We demonstrate here that this extracellular lipid binding protein is endocytosed and targeted in a finely controlled way to subsets of lysosomes in need of protection, those most sensitive to oxidative stress. ApoD reveals the existence of biologically relevant lysosomal heterogeneity that conditions the oxidation state of cells, their phagocytic or autophagic capacity, and the final output in neurodegenerative conditions. The stable presence of ApoD in lysosomes is sufficient and necessary for lysosomes to recover from oxidation-induced membrane permeabilization and loss of proton gradients. ApoD-mediated control of lysosomal membrane integrity represents a new cell-protection mechanism at the hub of many cellular functions, and is critical for the outcome of a wide variety of neurodegenerative diseases. Therapeutic opportunities open, by providing a route of entry and a repair mechanism for lysosomes in pathological situations.

The fruitfly compound eye has been broadly used as a model for neurodegenerative diseases. Classical quantitative techniques to estimate the degeneration level of an eye under certain experimental conditions rely either on time consuming histological techniques to measure retinal thickness, or pseudopupil visualization and manual counting. Alternatively, visual examination of the eye surface appearance gives only a qualitative approximation provided the observer is well-trained. Therefore, there is a need for a simplified and standardized analysis of fruitfly eye degeneration extent for both routine laboratory use and for automated high-throughput analysis. We have designed the freely available ImageJ plugin FLEYE, a novel and user-friendly method for quantitative unbiased evaluation of neurodegeneration levels based on the acquisition of fly eye surface pictures. The incorporation of automated image analysis tools and a classification algorithm sustained on a built-in statistical model allow the user to quickly analyze large sample size data with reliability and robustness. Pharmacological screenings or genetic studies using the Drosophila retina as a model system may benefit from our method, because it can be easily implemented in a fully automated environment. In addition, FLEYE can be trained to optimize the image detection capabilities, resulting in a versatile approach to evaluate the pattern regularity of other biological or non-biological samples and their experimental or pathological disruption.

RNA interference (RNAi)‐based control technologies are gaining popularity as potential alternatives to synthetic fungicides in the ongoing effort to manage plant pathogenic fungi. Among these methods, spray‐induced gene silencing (SIGS) emerges as particularly promising due to its convenience and feasibility for development. This approach is a new technology for plant disease management, in which double‐stranded RNAs (dsRNAs) targeting essential or virulence genes are applied to plants or plant products and subsequently absorbed by plant pathogens, triggering a gene silencing effect and the inhibition of the infection process. Spray‐induced gene silencing has demonstrated efficacy in laboratory settings against various fungal pathogens. However, as research progressed from the laboratory to the greenhouse and field environments, novel challenges arose, such as ensuring the stability of dsRNAs and their effective delivery to fungal targets. Here, we provide a practical guide to SIGS for the control of plant pathogenic fungi. This guide outlines the essential steps and considerations needed for designing and assessing dsRNA molecules. It also addresses key challenges inherent to SIGS, including delivery and stability of dsRNA molecules, and how nanoencapsulation of dsRNAs can aid in overcoming these obstacles. Additionally, the guide underscores existing knowledge gaps that warrant further research and aims to provide assistance to researchers, especially those new to the field, encouraging the advancement of SIGS for the control of a broad range of fungal pathogens.

Global change is exacerbating the prevalence of plant diseases caused by pathogenic fungi in forests worldwide. The conventional use of chemical fungicides, which is commonplace in agricultural settings, is not sanctioned for application in forest ecosystems, so novel control strategies are imperative. Spray-induced gene silencing (SIGS) is a promising approach that can modulate the expression of target genes in eukaryotes in response to double-stranded RNA (dsRNA) present in the environment that triggers the RNA interference mechanism. SIGS exhibited notable success in reducing virulence when deployed against some crop fungal pathogens, such as Fusarium graminearum, Botrytis cinerea, and Sclerotinia sclerotiorum, among others. However, there is a conspicuous dearth of studies evaluating the applicability of SIGS for managing forest pathogens. This research aimed to determine whether SIGS could be used to control F. circinatum, a widely impactful forest pathogen that causes pine pitch canker disease. Through a bacterial synthesis, we produced dsRNA molecules to target fungal essential genes involved in vesicle trafficking (Vps51, DCTN1, and SAC1), signal transduction (Pp2a, Sit4, Ppg1, and Tap42), and cell wall biogenesis (Chs1, Chs2, Chs3b, and Gls1) metabolic pathways. We confirmed that F. circinatum is able to uptake externally applied dsRNA, triggering an inhibition of the pathogen’s virulence. Furthermore, this study pioneers the demonstration that recurrent applications of dsRNAs in SIGS are more effective in protecting plants than single applications. Therefore, SIGS emerges as an effective and sustainable approach for managing plant pathogens, showcasing its efficacy in controlling a globally significant forest pathogen subject to quarantine measures.

Introduction: Mediterranean forests are currently facing a surge in abiotic stressors such as droughts and massive fires as a result of climate crisis and human pressure. Susceptibility to biotic stressors has also increased, including a variety of pests and pathogens capable of weakening and potentially killing forest flora. Biodiversity of microbiome protects forests against declines as it increases trees’ resilience and adaptability.Objectives: With the objective of analyzing the relationship between health status and fungal diversity, in the present work the mycobiota of declined and healthy specimens of keystone Mediterranean tree species is described and compared.Methods: To this end, bark and wood from declining Spanish forests of Castanea sativa (chestnut), Quercus ilex (holm oak), Quercus suber (cork oak), and Quercus pyrenaica (pyrenean oak) were sampled and the Internal Transcribed Spacer (ITS1) genomic region was sequenced.Results: Results showed a predominance of Ascomycota, Basidiomycota, and Mucoromycota in all samples. Alpha diversity at genus level was not affected by health status and was characterized by uneven, poorly distributed fungal communities dominated by a few genera. Differentially abundant (DA) genera between healthy and declined samples were found in chestnut (15), holm oak (6), and pyrenean oak (4) trees, but not in cork oak. Some genera known for their plant protection potential, such as Trichoderma, were found exclusively in healthy trees. Functional profiles revealed a predominance of phytopathogens and saprobes in all tree species, irrespective of their health status.Discussion: This study emphasizes the importance of Mediterranean forests as biodiversity refuges and highlights the value of above-ground tissues as a valid approach to assess shifts in forests’ microbiome diversity in response to biotic and abiotic stressors.