Data_Sheet_1_Forests influence yeast populations vectored by insects into vineyards.pdf

IntroductionIn the vineyard, yeast communities impact the ripening and fermentation of grapes and are influenced by geographical location, climate, and soil characteristics. Despite the great advancement in our knowledge of the vineyard mycobiota, a key step of the process leading to the definition of the vineyard yeast community is still poorly understood: if geography, climate, and soil influence the mycobiota, potentially through selection, where do the yeast originate from, and how can they reach the vineyard? In this perspective, it is currently acknowledged that forests host several yeast species and that insects, particularly social wasps, can vector and maintain the yeasts known to populate the vineyard. Alas, the conveyance, fostered by insects, of yeasts from the forest to the vineyard has not been proven yet. In this study, we aimed to assess the existence of links between a potential natural source of yeasts (woods), the vectors (social wasps), and the composition of the vineyard mycobiota.MethodsFor this purpose, the mycobiota of wasps caught in six Italian vineyards were analyzed over 2 years through culturomics approaches.ResultsThe results clearly indicate that the presence of wooded areas close to vineyards is associated with particular features of the mycobiota vectored by social wasps. Wasps caught in vineyards near wooded areas bear a higher number of yeast cells and higher biodiversity than insects caught in vineyards far from woods. Furthermore, insects caught in vineyards close to woods bear distinctive yeast populations, encompassing species such as Saccharomyces cerevisiae.DiscussionOverall, our work provides fundamental insights into the ecology of the vineyard mycobiota and highlights the need to maintain a vineyard-woodland mosaic landscape, thus preserving the suitable habitat for yeast species relevant to wine-making.</p

Table_5_Forests influence yeast populations vectored by insects into vineyards.XLSX

IntroductionIn the vineyard, yeast communities impact the ripening and fermentation of grapes and are influenced by geographical location, climate, and soil characteristics. Despite the great advancement in our knowledge of the vineyard mycobiota, a key step of the process leading to the definition of the vineyard yeast community is still poorly understood: if geography, climate, and soil influence the mycobiota, potentially through selection, where do the yeast originate from, and how can they reach the vineyard? In this perspective, it is currently acknowledged that forests host several yeast species and that insects, particularly social wasps, can vector and maintain the yeasts known to populate the vineyard. Alas, the conveyance, fostered by insects, of yeasts from the forest to the vineyard has not been proven yet. In this study, we aimed to assess the existence of links between a potential natural source of yeasts (woods), the vectors (social wasps), and the composition of the vineyard mycobiota.MethodsFor this purpose, the mycobiota of wasps caught in six Italian vineyards were analyzed over 2 years through culturomics approaches.ResultsThe results clearly indicate that the presence of wooded areas close to vineyards is associated with particular features of the mycobiota vectored by social wasps. Wasps caught in vineyards near wooded areas bear a higher number of yeast cells and higher biodiversity than insects caught in vineyards far from woods. Furthermore, insects caught in vineyards close to woods bear distinctive yeast populations, encompassing species such as Saccharomyces cerevisiae.DiscussionOverall, our work provides fundamental insights into the ecology of the vineyard mycobiota and highlights the need to maintain a vineyard-woodland mosaic landscape, thus preserving the suitable habitat for yeast species relevant to wine-making.</p

Table_2_Forests influence yeast populations vectored by insects into vineyards.XLSX

IntroductionIn the vineyard, yeast communities impact the ripening and fermentation of grapes and are influenced by geographical location, climate, and soil characteristics. Despite the great advancement in our knowledge of the vineyard mycobiota, a key step of the process leading to the definition of the vineyard yeast community is still poorly understood: if geography, climate, and soil influence the mycobiota, potentially through selection, where do the yeast originate from, and how can they reach the vineyard? In this perspective, it is currently acknowledged that forests host several yeast species and that insects, particularly social wasps, can vector and maintain the yeasts known to populate the vineyard. Alas, the conveyance, fostered by insects, of yeasts from the forest to the vineyard has not been proven yet. In this study, we aimed to assess the existence of links between a potential natural source of yeasts (woods), the vectors (social wasps), and the composition of the vineyard mycobiota.MethodsFor this purpose, the mycobiota of wasps caught in six Italian vineyards were analyzed over 2 years through culturomics approaches.ResultsThe results clearly indicate that the presence of wooded areas close to vineyards is associated with particular features of the mycobiota vectored by social wasps. Wasps caught in vineyards near wooded areas bear a higher number of yeast cells and higher biodiversity than insects caught in vineyards far from woods. Furthermore, insects caught in vineyards close to woods bear distinctive yeast populations, encompassing species such as Saccharomyces cerevisiae.DiscussionOverall, our work provides fundamental insights into the ecology of the vineyard mycobiota and highlights the need to maintain a vineyard-woodland mosaic landscape, thus preserving the suitable habitat for yeast species relevant to wine-making.</p

Table_1_Forests influence yeast populations vectored by insects into vineyards.XLSX

IntroductionIn the vineyard, yeast communities impact the ripening and fermentation of grapes and are influenced by geographical location, climate, and soil characteristics. Despite the great advancement in our knowledge of the vineyard mycobiota, a key step of the process leading to the definition of the vineyard yeast community is still poorly understood: if geography, climate, and soil influence the mycobiota, potentially through selection, where do the yeast originate from, and how can they reach the vineyard? In this perspective, it is currently acknowledged that forests host several yeast species and that insects, particularly social wasps, can vector and maintain the yeasts known to populate the vineyard. Alas, the conveyance, fostered by insects, of yeasts from the forest to the vineyard has not been proven yet. In this study, we aimed to assess the existence of links between a potential natural source of yeasts (woods), the vectors (social wasps), and the composition of the vineyard mycobiota.MethodsFor this purpose, the mycobiota of wasps caught in six Italian vineyards were analyzed over 2 years through culturomics approaches.ResultsThe results clearly indicate that the presence of wooded areas close to vineyards is associated with particular features of the mycobiota vectored by social wasps. Wasps caught in vineyards near wooded areas bear a higher number of yeast cells and higher biodiversity than insects caught in vineyards far from woods. Furthermore, insects caught in vineyards close to woods bear distinctive yeast populations, encompassing species such as Saccharomyces cerevisiae.DiscussionOverall, our work provides fundamental insights into the ecology of the vineyard mycobiota and highlights the need to maintain a vineyard-woodland mosaic landscape, thus preserving the suitable habitat for yeast species relevant to wine-making.</p

Table_3_Forests influence yeast populations vectored by insects into vineyards.XLSX

IntroductionIn the vineyard, yeast communities impact the ripening and fermentation of grapes and are influenced by geographical location, climate, and soil characteristics. Despite the great advancement in our knowledge of the vineyard mycobiota, a key step of the process leading to the definition of the vineyard yeast community is still poorly understood: if geography, climate, and soil influence the mycobiota, potentially through selection, where do the yeast originate from, and how can they reach the vineyard? In this perspective, it is currently acknowledged that forests host several yeast species and that insects, particularly social wasps, can vector and maintain the yeasts known to populate the vineyard. Alas, the conveyance, fostered by insects, of yeasts from the forest to the vineyard has not been proven yet. In this study, we aimed to assess the existence of links between a potential natural source of yeasts (woods), the vectors (social wasps), and the composition of the vineyard mycobiota.MethodsFor this purpose, the mycobiota of wasps caught in six Italian vineyards were analyzed over 2 years through culturomics approaches.ResultsThe results clearly indicate that the presence of wooded areas close to vineyards is associated with particular features of the mycobiota vectored by social wasps. Wasps caught in vineyards near wooded areas bear a higher number of yeast cells and higher biodiversity than insects caught in vineyards far from woods. Furthermore, insects caught in vineyards close to woods bear distinctive yeast populations, encompassing species such as Saccharomyces cerevisiae.DiscussionOverall, our work provides fundamental insights into the ecology of the vineyard mycobiota and highlights the need to maintain a vineyard-woodland mosaic landscape, thus preserving the suitable habitat for yeast species relevant to wine-making.</p

Folded Structure and Insertion Depth of the Frog-Skin Antimicrobial Peptide Esculentin-1b(1–18) in the Presence of Differently Charged Membrane-Mimicking Micelles

Antimicrobial peptides (AMPs) are effectors of the innate immunity of most organisms. Their role in the defense against pathogen attack and their high selectivity for bacterial cells make them attractive for the development of a new class of antimicrobial drugs. The N-terminal fragment of the frog-skin peptide esculentin-1b (Esc(1–18)) has shown broad-spectrum antimicrobial activity. Similarly to most cationic AMPs, it is supposed to act by binding to and damaging the negatively charged plasma membrane of bacteria. Differently from many other AMPs, Esc(1–18) activity is preserved in biological fluids such as serum. In this work, a structural investigation was performed through NMR spectroscopy. The 3D structure was obtained in the presence of either zwitterionic or negatively charged micelles as membrane models for eukaryotic and prokaryotic membranes, respectively. Esc(1–18) showed a higher affinity for and deeper insertion into the latter and adopted an amphipathic helical structure characterized by a kink at the residue G8. These findings were confirmed by measuring penetration into lipid monolayers. The presence of negatively charged lipids in the bilayer appears to be necessary for Esc(1–18) to bind, to fold in the right three-dimensional structure, and, ultimately, to exert its biological role as an AMP

IFI16 protein is a negative regulator of Herpesvirus replication.

<p>A) HELFs were electroporated with a mixture of four different small interfering RNAs (siRNA IFI16), scrambled control siRNA (siRNA ctrl), or left not electroporated (NE), and then infected with the indicated viruses at a multiplicity of 0.05 PFU/cell. Cell-free supernatants were harvested on the indicated hours post infection (hpi) and virus amounts determined by plaque assays. The data shown are the average of three experiments ± SD (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA followed by Bonferroni's post test). B) HELFs were transfected with siRNA IFI16 or siRNA ctrl or left not electroporated (NE) and IFI16 expression assayed by Western blotting on the indicated days (d) with anti-IFI16 polyclonal Abs. β-actin was included as a loading control. C) HELFs were treated with siRNA as described for panel A and then infected with HCMV at an MOI of 0.05 (left panel) or 1 (right panel) PFU/cell. Cell-free supernatants were harvested on the indicated hours post infection and the virus amounts determined by plaque assays. The data shown are the average of three experiments ± SD.</p

IFI16 impairs IR-1 binding.

<p>A) Nuclear protein extracts from HELFs infected with AdV IFI16 or AdV LacZ at an MOI of 200 for 24 hours and then with HCMV at an MOI of 2 for 24 hours were incubated with a radiolabeled oligonucleotide containing the consensus IR-1 binding site. Competition experiments were performed with 100-fold excess of cold specific oligonucleotide in either the wild type or the mutated form. B) Super-shift experiments were performed by adding polyclonal antibodies against Sp1, control antobody (ctrl). Experiments were repeated at least three times and one representative result is shown.</p

Interplay between Sp1 and IFI16 down-regulates HCMV replication.

<p>A) Nuclear cell protein extracts from HELFs mock infected (lanes 1, 4, 7), infected with HCMV at an MOI of 2 for 24 hours (lanes 2, 5, 8), or infected with AdV IFI16 or AdV LacZ (data not shown) at an MOI of 200 for 24 hours and then with HCMV (lanes 3, 6, 9) were immunoprecipitated with polyclonal antibodies against IFI16 or control antibody (ctrl). Samples were then immunoblotted with polyclonal antibodies against Sp1 or IFI16. B) Infected cell lysates were immunoprecipitated with anti-IFI16 polyclonal antibodies in the absence (lane 2 or 4) or presence of benzonase (lanes 3 and 5) or with control antibody (ctrl). Proteins from the IP were analyzed by Western blotting using polyclonal antibody recognizing Sp1. C) Nuclear extracts from HELFs mock infected or infected with AdV IFI16 or AdV LacZ at an MOI of 200 for 24 hours and then with HCMV at an MOI of 2 for 24 hours were analyzed by ChIP assay using anti-Sp1 rabbit polyclonal antibody. Sp1-coprecipitating DNA was analyzed by quantitative real-time PCR with IR-1 sequence specific primers. An unrelated rabbit polyclonal anti-serum was used as control (data not shown). Non-immunoprecipitated whole cell extract (input) obtained from AdV LacZ- or AdV IFI16-infected cells was employed to normalize the IR-1 viral DNA subjected to immunoprecipitation. Experiments were repeated at least three times and one representative result is shown (mean ± SD) (***p<0.001, unpaired t test). D) Nuclear cell protein extracts from HELFs stably transduced with the recombinant Lentivirus carrying the full-length IFI16 protein (IFI16wt), or dnIFI16 ORFs (ΔDIFI16 or ΔBIFI16), were infected with HCMV at an MOI of 2 for 24 hours, immunoprecipitated with monoclonal antibodies against flag V5 or control antibodies (ctrl), and immunoblotted with polyclonal antibodies against Sp1 (top panel) or flag V5 (bottom panel).</p

Effects of IFI16 overexpression on the activity of HCMV UL54, UL44, and MIEP promoters.

<p>A) Luciferase reporter plasmids containing HCMV UL54 promoter segments progressively deleted from the 5′ end and mutated in the IR-1 or DR-ATF elements were transfected into HELFs that were subsequently infected with either AdV LacZ or AdV IFI16 at an MOI of 200. 24 hours later, cells were infected with HCMV (MOI of 0.5) and luciferase activity assessed after a further 24 hpi. B) Luciferase reporter plasmids containing the MIEP (major immediate-early promoter<b>)</b> segment were transfected into HELFs subsequently infected as described for panel A. C) Luciferase reporter plasmids containing HCMV UL44 promoter segments progressively deleted from the 5′ end and the 3′ end were transfected into HELFs subsequently infected as described for panel A. Sp1, putative binding sites for Sp1 transcription factor; oval circles indicates TATA boxes. Experiments were repeated at least three times and one representative result is shown (mean ± SD) (*p<0.05, **p<0.01, ***p<0.001, unpaired t test for comparison of AdV LacZ <i>vs</i> AdV IFI16).</p