On the origin and propagation of the COVID-19 outbreak in the Italian Province of Trento, a tourist region of Northern Italy

15openInternationalItalian coauthor/editorBackground: Trentino is an Italian province with a tourism-based economy, bordering the regions of Lombardy and Veneto, where the two earliest and largest outbreaks of COVID-19 occurred in Italy. The earliest cases in Trentino were reported in the first week of March 2020, with most of the cases occurring in the winter sport areas in the Dolomites mountain range. The number of reported cases decreased over the summer months and was followed by a second wave in the autumn and winter of 2020. Methods: we performed high-coverage Oxford Nanopore sequencing of 253 positive SARS-CoV-2 swabs collected in Trentino between March and December 2020. Results: in this work, we analyzed genome sequences to trace the routes through which the virus entered the area, and assessed whether the autumnal resurgence could be attributed to lineages persisting undetected during summer, or as a consequence of new introductions. Conclusions: Comparing the draft genomes analyzed with a large selection of European sequences retrieved from GISAID we found that multiple introductions of the virus occurred at the early stage of the epidemics; the two epidemic waves were unrelated; the second wave was due to reintroductions of the virus in summer when traveling restrictions were upliftedopenBianco, Luca; Moser, Mirko; Silverj, Andrea; Micheletti, Diego; Lorenzin, Giovanni; Collini, Lucia; Barbareschi, Mattia; Lanzafame, Paolo; Segata, Nicola; Pindo, Massimo; Franceschi, Pietro; Rota-Stabelli, Omar; Rizzoli, Annapaola; Fontana, Paolo; Donati, ClaudioBianco, L.; Moser, M.; Silverj, A.; Micheletti, D.; Lorenzin, G.; Collini, L.; Barbareschi, M.; Lanzafame, P.; Segata, N.; Pindo, M.; Franceschi, P.; Rota-Stabelli, O.; Rizzoli, A.; Fontana, P.; Donati, C

Viral RNase3 Co-Localizes and Interacts with the Antiviral Defense Protein SGS3 in Plant Cells

Sweet potato chlorotic stunt virus (SPCSV; family Closteroviridae) encodes a Class 1 RNase III endoribonuclease (RNase3) that suppresses post-transcriptional RNA interference (RNAi) and eliminates antiviral defense in sweetpotato plants (Ipomoea batatas). For RNAi suppression, RNase3 cleaves double-stranded small interfering RNAs (ds-siRNA) and long dsRNA to fragments that are too short to be utilized in RNAi. However, RNase3 can suppress only RNAi induced by sense RNA. Sense-mediated RNAi involves host suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6). In this study, subcellular localization and host interactions of RNase3 were studied in plant cells. RNase3 was found to interact with SGS3 of sweetpotato and Arabidopsis thaliana when expressed in leaves, and it localized to SGS3/RDR6 bodies in the cytoplasm of leaf cells and protoplasts. RNase3 was also detected in the nucleus. Co-expression of RNase3 and SGS3 in leaf tissue enhanced the suppression of RNAi, as compared with expression of RNase3 alone. These results suggest additional mechanisms needed for efficient RNase3-mediated suppression of RNAi and provide new information about the subcellular context and phase of the RNAi pathway in which RNase3 realizes RNAi suppression.Peer reviewe

Dissecting the susceptibility/resistance mechanism of Vitis vinifera for the future control of downy mildew

23CO.NA.VI. 2020 – 8° Convegno Nazionale di Viticoltura, Udine, Italy, July 5-7, 2021openInternationalBothThe Eurasian grapevine (Vitis vinifera), a species cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. Nevertheless, germplasm from Georgia (Southern Caucasus, the first grapevine domestication centre), characterized by a high genetic variability, showed resistance traits to P. viticola. The cultivar Mgaloblishvili exhibited the most promising phenotype in terms of resistance against P. viticola. Its defence response results in: i) low disease intensity; ii) low sporulation; iii) damaged mycelium; iv) production of antimicrobial compounds such as volatile organic compounds (VOCs), whose effectiveness on the pathogen was evaluated by leafdisc assays. At the transcriptomic level, its resistance mechanism is determined by the differential expression of both resistance and susceptible genes. The resistance genes are related to: i) pathogen recognition through PAMP, DAMP and effector receptors; ii) ethylene signalling pathway; iii) synthesis of antimicrobial compounds (VOCs) and fungal wall degrading enzymes; iv) development of structural barriers (cell wall reinforcement). The first putative susceptible gene was the transcription factor VviLBDIf7 gene, whose validation was carried out by dsRNA (double-stranded RNA) assay. In this work, these unique results on plant-pathogen interaction are reviewed with the aim of developing new strategies to control the disease.openRicciardi, Valentina; Marcianò, Demetrio; Sargolzaei, Maryam; Marrone Fassolo, Elena; Fracassetti, Daniela; Brilli, Matteo; Moser, Mirko; Vahid, Shariati J.; Tavakole, Elahe; Maddalena, Giuliana; Passera, Alessandro; Casati, Paola; Pindo, Massimo; Cestaro, Alessandro; Costa, Alex; Bonza, Maria Cristina; Maghradze, David; Tirelli, Antonio; Failla, Osvaldo; Bianco, Piero Attilio; Quaglino, Fabio; Toffolatti, Silvia Laura; De Lorenzis, GabriellaRicciardi, V.; Marcianò, D.; Sargolzaei, M.; Marrone Fassolo, E.; Fracassetti, D.; Brilli, M.; Moser, M.; Vahid, S.J.; Tavakole, E.; Maddalena, G.; Passera, A.; Casati, P.; Pindo, M.; Cestaro, A.; Costa, A.; Bonza, M.C.; Maghradze, D.; Tirelli, A.; Failla, O.; Bianco, P.A.; Quaglino, F.; Toffolatti, S.L.; De Lorenzis, G

Validation platform implementation description – D5.2

Deliverable D5.2 del projecte OneFITPostprint (published version

Results analysis and validation - D5.3

Deliverable D5.3 del projecte OneFITPostprint (author’s final draft

The Vinculin-ΔIn20/21 Mouse: Characteristics of a Constitutive, Actin-Binding Deficient Splice Variant of Vinculin

BACKGROUND: The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigate vinculin-DeltaEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-DeltaEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-DeltaIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-DeltaIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-DeltaEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-DeltaEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-DeltaEx20 variant unveils vinculin's dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites. CONCLUSIONS/SIGNIFICANCE: Vinculin-DeltaEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development

Gene expression analysis in ‘Candidatus Phytoplasma mali’-resistant and -susceptible Malus genotypes

Apple proliferation (AP) disease is the most important graft-transmissible and vector-borne disease of apple in Europe. ‘Candidatus Phytoplasma mali’ (Ca. P. mali) is the causal agent of AP. Apple (Malus x domestica) and other Malus species are the only known woody hosts. In European apple orchards, the cultivars are mainly grafted on one rootstock, M. x domestica cv. M9. M9 like all other M. x domestica cultivars is susceptible to ‘Ca. P. mali’. Resistance to AP was found in the wild genotype Malus sieboldii (MS) and in MS-derived hybrids but they were characterised by poor agronomic value. The breeding of a new rootstock carrying the resistant and the agronomic traits was the major aim of a project of which this work is a part. The objective was to shed light into the unknown resistance mechanism. The plant-phytoplasma interaction was studied by analysing differences between the ‘Ca. P. mali’-resistant and -susceptible genotypes related to constitutively expressed genes or to induced genes during infection. The cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was employed in both approaches. Differences related to constitutively expressed genes were identified between two ‘Ca. P. mali’-resistant hybrid genotypes (4551 and H0909) and the ‘Ca. P. mali’-susceptible M9. 232 cDNA-AFLP bands present in the two resistant genotypes but absent in the susceptible one were isolated but several different products associated to each band were found. Therefore, two different macroarray hybridisation experiments were performed with the cDNA-AFLP fragments yielding 40 sequences encoding for genes of unknown function or a wide array of functions including plant defence. In the second approach, individuation and analysis of the induced genes was carried out exploiting an in vitro system in which healthy and ‘Ca. P. mali’-infected micropropagated plants were maintained under controlled conditions. Infection trials using in vitro grafting of ‘Ca. P. mali’ showed that the resistance phenotype could be reproduced in this system. In addition, ex vitro plants were generated as an independent control of the genes differentially expressed in the in vitro plants. The cDNA-AFLP analysis in in vitro plants yielded 63 bands characterised by over-expression in the infected state of both the H0909 and MS genotypes. The major part (37 %) of the associated sequences showed homology with products of unknown function. The other genes were involved in plant defence, energy transport/oxidative stress response, protein metabolism and cellular growth. Real-time qPCR analysis was employed to validate the differential expression of the genes individuated in the cDNA-AFLP analysis. Since no internal controls were available for the study of the gene expression in Malus, an analysis on housekeeping genes was performed. The most stably expressed genes were the elongation factor-1 α (EF1) and the eukaryotic translation initiation factor 4-A (eIF4A). Twelve out of 20 genes investigated through qPCR were significantly differentially expressed in at least one genotype either in in vitro plants or in ex vitro plants. Overall, about 20% of the genes confirmed their cDNA-AFLP expression pattern in M. sieboldii or H0909. On the contrary, 30 % of the genes showed down-regulation or were not differentially expressed. For the remaining 50 % of the genes a contrasting behaviour was observed. The qPCR data could be interpreted as follows: the phytoplasma infection unbalance photosynthetic activity and photorespiration down-regulating genes involved in photosynthesis and in the electron transfer chain. As result, and in contrast to M. x domestica genotypes, an up-regulation of genes of the general response against pathogens was found in MS. These genes involved the pathway of H2O2 and the production of secondary metabolites leading to the hypothesis that a response based on the accumulation of H2O2 in MS would be at the base of its resistance. This resembles a phenomenon known as “recovery” where the spontaneous remission of the symptoms is observed in old susceptible plants but occurring in a stochastic way while the resistance in MS is an inducible but stable feature. As additional product of this work three cDNA-AFLP-derived markers were developed which showed independent distribution among the seedlings of two breeding progenies and were associated to a genomic region characteristic of MS. These markers will contribute to the development of molecular markers for the resistance as well as to map the resistance on the Malus genome.Apfeltriebsucht (AT) Krankheit ist die wichtigste pfropf- und vektorübertragbare Krankheit von Apfel in Europa. "Candidatus Phytoplasma mali '(Ca. P. mali) ist der Erreger der AT. Apfel (Malus x domestica) und andere Malus Arten sind die einzigen bekannten holzigen Wirte. In europäischen Apfelanlagen sind die Sorten vor allem auf einer Unterlage, M. x domestica cv. M9, veredelt. M9 ist wie alle anderen M. x domestica Sorten anfällig für "Ca. P. mali". Resistenz gegen AT wurde im wilden Genotyp Malus sieboldii (MS) und in MS-Hybriden gefunden, welche aber nur geringen agronomischen Wert haben. Die Züchtung einer neuen Unterlage, die Resistenz und agronomischen Eigenschaften verbindet, war das Hauptziel eines Projekts, von dem diese Arbeit ein Teil ist. Das Ziel war, etwas über den unbekannten Resistenzmechanismus zu erfahren. Hierzu wurde die Phytoplasma-Pflanzen-Interaktion durch die Analyse der Unterschiede zwischen den 'Ca. P. mali'-resistenten und -anfälligen Genotypen sowohl im Bezug auf konstitutiv exprimierte Gene als auch in Bezug auf nach Infektion induzierte Gene untersucht. Die cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) Technik wurde in beiden Ansätzen verwendet. Unterschiede von konstitutiv exprimierten Genen wurden zwischen zwei "Ca. P. mali'-resistenten Hybrid-Genotypen (4551 und H0909) und dem "Ca. P. mali'-anfälligen M9 identifiziert. 232 cDNA-AFLP-Banden anwesend in den beiden resistenten Genotypen und fehlend in dem anfälligen Genotyp wurden isoliert, aber mehrere verschiedene Produkte waren mit jeder Bande assoziiert. Deshalb wurden zwei verschiedene Makroarray-Hybridisierungsexperimente mit den cDNA- AFLP-Fragmenten durchgeführt. 40 kodierenden Sequenzen für Gene mit unbekannter Funktion sowie eine breite Palette von Funktionen, einschließlich pflanzlicher Abwehr, wurden gefunden. Im zweiten Ansatz wurde die Identifizierung und Analyse induzierter Gene unter Ausnutzung eines in vitro-Systems durchgeführt, in dem gesunde und 'Ca. P. mali'-infizierte Gewebekultur-vermehrte Pflanzen unter kontrollierten Bedingungen gehalten wurden. Infektionsversuche mittels in vitro-Pfropfung von "Ca. P. mali "zeigten, dass der Resistenz-Phänotyp in diesem System reproduziert werden konnte. Darüber hinaus wurden ex vitro-Pflanzen erzeugt und dienten als unabhängige Kontrolle für die in vitro differentiell exprimierten Gene. Die cDNA-AFLP-Analyse in in vitro-Pflanzen ergab 63 Banden, die in infizierten Pflanzen von H0909 und MS überexprimiert waren. Der größte Teil (37%) der zugehörigen Sequenzen zeigte Homologie mit Produkten unbekannter Funktion. Die anderen Gene waren in pflanzlicher Abwehr, Energietransport bzw. Reaktion auf oxydativen Stress, Protein-Stoffwechsel und Zellwachstum involviert. Eine Real-time qPCR Analyse wurde durchgeführt, um die differentielle Expression der Gene zu validieren, welche in der cDNA-AFLP-Analyse gefunden wurde. Da keine internen Kontrollen für die Analyse der Genexpression in Malus zur Verfügung standen, wurde eine Analyse von Genen des Grundstoffwechsels durchgeführt. Die am stabilsten exprimierten Gene waren der Elongationsfaktor-1 α (EF1) und der eukaryotische Translationsfaktor 4A (eIF4A). Zwölf der 20 mittels qPCR untersuchten Gene wurden signifikant differentiell exprimiert, und zwar in mindestens einem Genotyp in in vitro- oder in ex vitro-Pflanzen. Für etwa 20% der Gene konnte ihr cDNA-AFLP Expressionsmuster in MS oder H0909 in in vitro- oder ex vitro- Pflanzen bestätigt werden. Dagegen zeigten 30% der Gene Unterexpression oder waren nicht differentiell exprimiert. Für die restlichen 50% der Gene wurde ein kontrastierendes Verhalten beobachtet. Die qPCR-Daten sind wie folgt interpretierbar: die Phytoplasma-Infektion führt zu einem Ungleichgewicht in der photosynthetischen Aktivität und der Photorespiration, in dem Gene, die in Prozessen wie Photosynthese und Elektronentransfer-Kette beteiligt sind, unterexprimiert werden. Im Ergebnis, und im Gegensatz zu M. x domestica Genotypen, wurde eine Überexpression von Genen der allgemeinen Reaktion auf Pathogene in MS gefunden. Die Identifizierung von Genen für die Produktion von H2O2 und von sekundären Pflanzeninhaltsstoffen führte zu der Hypothese, dass die Resistenz auf der Akkumulation von H2O2 in MS basiert. Dies ähnelt einem als "recovery" beschriebenen Phänomen, bei dem eine spontane Remission der Symptome auf stochastische Weise in alten anfälligen Pflanzen beobachtet wird. Hingegen ist die Resistenz in MS eine induzierbare, aber stabile Reaktion. Als weiteres Produkt dieser Arbeit wurden drei cDNA-AFLP-Marker entwickelt, die eine unabhängige Verteilung in der Nachkommenschaft von zwei Kreuzungspopulationen zeigten und die mit einer genomischen Region von MS assoziiert wurden. Diese Marker werden zur Entwicklung molekularer Marker für die Resistenz sowie zur Kartierung der Resistenz im Malus-Genom beitrage