Vanessa Ferreira

Peroxisomes are dynamic organelles that play important roles in cellular metabolic and signaling pathways and are implicated in several disease conditions. Although a detailed comprehension of peroxisome dynamics is crucial in various contexts of health and disease, its accurate assessment still presents significant challenges, mainly due to their morphological heterogeneity. Here, we discuss different strategies to study peroxisome dynamics and present a semi-automated macro, PeroxiDynA, that enables the analysis of peroxisome dynamics from single-plane confocal images of mammalian cells in ImageJ. Our findings confirm that PeroxiDynA can be successfully employed to determine relative peroxisome number and spatial distribution. A comparison between PeroxiDynA and other image analysis strategies reveals that it results in increased reliability in the analysis of peroxisome abundance and morphology. Although future improvements are still required to minimize the time and limitations imposed during analysis of peroxisomal features, we propose that PeroxiDynA is a useful tool for biomolecular studies, not only to analyze peroxisomes but also the dynamics of small biological structures in cells from mammals and other organisms.

The effect of γ-aminobutyric acid (GABA) on the metabolome of two strains of Lasiodiplodia theobromae isolated from grapevine that hold a different degree of virulence to the host plant (LA-SOL3 (more virulent), LA-SV1 (less virulent)) was investigated. The culture filtrates and crude extracts from the two strains grown in the presence and absence of 10 mM of GABA were tested for phytotoxicity on tomato plant cuttings and leaves, respectively. Considering the opportunistic nature of this fungus for humans, crude extracts were also tested for cytotoxicity on mammalian cell lines. We found that culture filtrates and crude extracts have a decreased toxicity in the presence of GABA. Metabolomic analysis, conducted on both strains at both growth conditions, revealed the production of several compounds, such as indole-3-carboxylic acid (ICA, which is the main compound produced by L. theobromae), 3-indolecarboxyaldehyde, (3R,4S)-botryodiplodin, (R)-mellein. Finally, data demonstrate that GABA both induces a decrease in the amount of ICA, and a diversification of the metabolites produced by L. theobromae.

Neofusicoccum parvum is a fungal pathogen associated with a wide range of plant hosts. Despite being widely studied, the molecular mechanism of infection of N. parvum is still far from being understood. Analysis of N. parvum genome lead to the identification of six putative genes encoding necrosis and ethylene-inducing proteins (NLPs). The sequence of NLPs genes (NprvNep 1-6) were analyzed and four of the six NLP genes were successfully cloned, expressed in E. coli and purified by affinity chromatography. Pure recombinant proteins were characterized according to their phytotoxic and cytotoxic effects to tomato leaves and to mammalian Vero cells, respectively. These assays revealed that all NprvNeps tested are cytotoxic to Vero cells and also induce cell death in tomato leaves. NprvNep2 was the most toxic to Vero cells, followed by NprvNep1 and 3. NprvNep4 induced weaker, but, nevertheless, still significant toxic effects to Vero cells. A similar trend of toxicity was observed in tomato leaves: the most toxic was NprvNep 2 and the least toxic NprvNep 4. This study describes for the first time an overview of the NLP gene family of N. parvum and provides additional insights into its pathogenicity mechanism.

In the course of investigations on the role of secondary metabolites in plant-microbe interactions, the production of secondary metabolites by Macrophomina phaseolina isolates from Eucalyptus globulus, was studied. This fungus is responsible for several plant diseases which affect crop productivity and industry. Although secondary metabolites may play a role in disease development, there are very few reports on M. phaseolina metabolomics and, as far as we know, isolates from eucalypts have not been investigated for secondary metabolites production. In the present paper, metabolites typical of fungi, from the family Botryosphaeriaceae, were identified for the first time as products of M. phaseolina. Furthermore, the isolate under examination was grown in the presence and absence of host stem tissue, and metabolite profiles were compared. Five products are reported for the first time in this species and azelaic acid was exclusively produced in the presence of eucalypt stem. Finally, phytotoxicity and cytotoxicity tests of culture filtrates and crude organic extracts were also performed. Key Contribution: Lipophilic metabolites produced by M. phaseolina might play a role in the plant-fungus interactions responsible for serious diseases of E. globulus.

Lasiodiplodia theobromae is a fungal plant pathogen that has been associated with Botryosphaeria dieback of grapevine. Despite several studies on L. theobromae, until now the production of secondary metabolites by strains isolated from grapevines has not been reported. The ability of two strains of L. theobromae isolated from grapevine to produce lipophilic metabolites was studied. Although many typical compounds of low molecular weight were identified from the crude extracts of both strains (e.g., lasiolactols, substituted 2-dihydrofuranones, melleins, jasmonic acid, 3-indolcarboxylic acid, botryodiplodins), (2R/2S,3S,4S)-3-epi-botryodiplodin was isolated for the first time as a natural compound. Furthermore, a comparative study of metabolite production was conducted at 25 and 37 C to understand temperature effects on metabolite profiles. Some metabolites were produced only by one strain (e.g., (3S,4S)-4-acetyl-3-methyl-2-dihydrofuranone produced by LA-SOL3) and others only at a specific temperature (e.g., jasmonic acid at 25 C, botryodiplodins at 37 C). Phytotoxicity and cytotoxicity of pure compounds were evaluated to clarify the influence of lipophilic metabolites on the biological activities of culture filtrates of both strains. The most toxic compound for Vero and 3T3 cells was (2R/2S,3S,4S)-3-epi-botryodiplodin.