Biology subjects

Vallino, M.

Publications and source records attributed to Vallino, M..

4 recordsLinked to original sources

bioRxiv2024

Monitoring the directed evolution to a tripartite genome from a bipartite torradovirus genome

We have previously shown that tomato apex necrosis virus that cannot express the RNA2-ORF1 protein (P21) is not able to systemically infect plant hosts but is not affected in cell autonomous aspects of virus replication/accumulation. Here we attempted to provide P21 in trans by co-agroinfiltrating the RNA2-ORF1 null constructs (a stop codon mutant and a deletion mutant) with a P21-expressing construct under control of the 35S promoter and containing the 5 and 3 UTRs of wild type (WT) RNA2. Such construct when co-agroinfiltrated with the stop codon mutant originates a WT bipartite virus through homologous recombination. More surprisingly, when co-agroinfiltrated with the P21 deletion mutant it cannot immediately complement the mutant, but it serendipitously originates a tripartite virus with an actively replicating P21-expressing RNA3 only after this replicating RNA3 accumulates deletions in a small region inside the original 3-UTR provided by the cDNA clone. Such virus can be transmitted mechanically and by whiteflies, is competent for virion formation, and its RNA3 is encapsidated. The tripartite virus can be mechanically transferred for eleven generations without losing its infectivity or show major genomic rearrangements. Furthermore, mixing equal amounts of WT and tripartite virus inocula in the same leaf originated plants systemically infected only with the WT virus, showing that the tripartite virus has lower fitness than the WT. To our knowledge this is the first example of a stable virus evolving in vitro from bipartite to tripartite genomic structure from a synthetic construct in a plant virus.

microbiology↗

bioRxiv2022

Transcriptomics-driven characterisation of novel T7-like temperate Pseudomonas phage LUZ100

The Autographiviridae is a diverse yet distinct family of bacterial viruses marked by a strictly lytic lifestyle and a generally conserved genome organization. We here characterise Pseudomonas aeruginosa phage LUZ100, a distant relative of type phage T7. LUZ100 is a podovirus with a limited host range and identified LPS as the likely phage receptor. Interestingly, infection dynamics of LUZ100 indicated moderate adsorption rates and low virulence, hinting towards temperate behavior. This hypothesis was supported by genomic analysis, which showed that LUZ100 shares the conventional T7-like genome organization, yet encodes key genes associated with a temperate lifestyle. To unravel the peculiar characteristics of LUZ100, ONT-cappable-seq transcriptomics analysis was performed. This data generated a birds-eye view of the LUZ100 transcriptome and enabled the discovery of key regulatory elements, antisense RNA, and transcriptional unit structures. The transcriptional map of LUZ100 also allowed us to identify new RNAP-promoter pairs that can form the basis for biotechnological parts and tools for new synthetic transcription regulation circuitry. The ONT-cappable-seq data revealed that the LUZ100 integrase and a MarR-like regulator (proposed to be involved in the lytic/lysogeny decision), are actively co-transcribed in an operon. In addition, the presence of a phage-specific promoter transcribing the phage-encoded RNA polymerase, raises questions on the regulation of this polymerase, and suggests it is interwoven with the MarR-based regulation. This transcriptomics-driven characterisation of LUZ100 supports the increasing evidence that T7-like phages should not straightforwardly be marked as having a strictly lytic lifecycle. ImportanceBacteriophage T7, considered the model phage of the Autographiviridae family, is marked by a strictly lytic lifecycle and conserved genome organisation. Recently, novel phages of this clade are emerging and showing characteristics associated to a lysogenic lifecycle. Screening for temperate behaviour is of outmost importance in fields like phage therapy, where strictly lytic phages are generally required for therapeutic applications. In this study, weve used an omics-driven approach to characterise the T7-like Pseudomonas aeruginosa phage LUZ100. These results led to the identification of actively transcribed lysogeny-associated genes in the phage genome, pointing out that temperate T7-like phages are emerging more frequent than initially thought. In short, the combination of genomics and transcriptomics allowed us to obtain a better understanding of the biology of non-model Autographiviridae phages, which can be used to optimize the implementation of phages and their regulatory elements in phage therapy and biotechnological applications, respectively.

microbiology↗

bioRxiv2020

Biological characterization of Euscelidius variegatus iflavirus 1

Virus-based biocontrol technologies represent sustainable alternatives to pesticides and insecticides. Phytoplasmas are prokaryotic plant pathogens causing severe losses to crops worldwide. Novel approaches are needed since insecticides against their insect vectors and rogueing of infected plants are the only available strategies to counteract phytoplasma diseases. A new iflavirus, named EVV-1, has been described in the leafhopper phytoplasma vector Euscelidius variegatus, raising the potential to use virus-based application strategies against phytoplasma disease. Here transmission routes of EVV-1 are characterized, and localization within the host reveals the mechanism of insect tolerance to virus infection. Both vertical and horizontal transmission of EVV-1 occur and vertical transmission was more efficient. The virus is systemic and occurs in all life-stages, with the highest loads measured in ovaries and first to third instar nymphs. The basic knowledge gained here on the biology of the virus is crucial for possible future application of iflaviruses as biocontrol agents.

microbiology↗

bioRxiv2019

A complex virome that includes two distinct emaraviruses is associated to virus-like symptoms in Camellia japonica

Camellia japonica plants manifesting a complex and variable spectrum of viral symptoms like chlorotic ringspots, necrotic rings, yellowing with necrotic rings, yellow mottle, leaves and petals deformations, flower color-breaking were studied since 1940 essentially through electron microscopic analyses; however, a strong correlation between symptoms and one or more well characterized viruses was never verified. In this work samples collected from symptomatic plants were analyzed by NGS technique and a complex virome composed by viruses members of the Betaflexiviridae and Fimoviridae families was identified. In particular, the genomic fragments typical of the emaravirus group were organized in the genomes of two new emaraviruses species, tentatively named Camellia japonica associated emaravirus 1 and 2. They are the first emaraviruses described in camellia plants and were always found solely in symptomatic plants. On the contrary, in both symptomatic and asymptomatic plants, we detected five betaflexiviruses isolates that, based on aa identitiy comparisons, can be classified in two new putative species called Camellia japonica associated betaflexivirus 1 and Camellia japonica associated betaflexivirus 2. Together with other recently identified betaflexiviruses associated to Camellia japonica disease, the betaflexiviruses characterized in this study show an unusual hyper-conservation of the coat protein at aminoacidic level.

microbiology↗