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Genome transplantation (GT) allows the installation of purified chromosomes into recipient cells, causing the resulting organisms to adopt the genotype and the phenotype conferred by the donor cells. This key process remains a bottleneck in synthetic biology, especially for genome engineering strategies of intractable and economically important microbial species. So far, this process has only been reported using two closely related bacteria, Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma capricolum subsp. capricolum (Mcap), and the main factors driving the compatibility between a donor genome and a recipient cell are poorly understood. Here, we investigated the impact of the evolutionary distance between donor and recipient species on the efficiency of GT. Using Mcap as the recipient cell, we successfully transplanted the genome of six bacteria belonging to the Spiroplasma phylogenetic group but including species of two distinct genera. Our results demonstrate that GT efficiency is inversely correlated with the phylogenetic distance between donor and recipient bacteria but also suggest that other species-specific barriers to GT exist. This work constitutes an important step toward understanding the cellular factors governing the GT process in order to better define and eventually extend the existing genome compatibility limit.

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic. However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Flavescence dorée (FD) is a quarantine disease of grapevine, involving interactions between the plants, leafhopper vectors, and FD phytoplasma. Characterizing the susceptibility of vine varieties could limit disease propagation. After extensive surveys in vineyards, we showed that Cabernet Sauvignon (CS) is highly susceptible, with a high proportion of symptomatic branches and phytoplasma titers, in contrast to Merlot (M). Localized insect transmissions and grafting showed that phytoplasma circulate in the whole plant in the CS cultivar, but in M they are restricted to the transmission point. Insect-mediated transmission under high confinement mimicking natural conditions confirmed these phenotypes and allowed the classification of 28 Vitis accessions into three distinct categories, according to the percentage of infected plants and their phytoplasma titers. Reduced symptoms, low phytoplasma titers, and low percentages of infected plants were found to be associated in the Vitis vinifera cultivars tested. Interestingly, the low susceptibility of M was observed for one of its parents, i.e., Magdeleine Noire des Charentes. Rootstocks and their Vitis parents, although having high percentages of infected plants and intermediate to high phytoplasma titers, shared a symptomless response. This is troubling, because rootstocks can constitute a silent reservoir of contamination in mother plants or when they grow wild nearby vineyards. Altogether, data suggest distribution of genetic traits within the Vitis genus involved in insect-mediated phytoplasma transmission, multiplication, circulation, and symptom development.

207 words ; main text : 2373 words. Abstract 27 Reverse genetics has been an indispensable tool revolutionising our insights into viral 28 pathogenesis and vaccine development. Large RNA virus genomes, such as from 29Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and 30 occasional instability 1-3 . Therefore, an alternative rapid and robust reverse genetics platform 31 for RNA viruses would benefit the research community. Here we show the full functionality 32 of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA 33 viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. 34 Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical 35 samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using 36 transformation associated recombination (TAR) cloning to maintain the genome as a yeast 37 artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious 38 RNA, which was then used to rescue viable virus. Based on this platform we have been able 39 to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-2 4 40 in only a week after receipt of the synthetic DNA fragments. The technical advance we 41 describe here allows to rapidly responding to emerging viruses as it enables the generation 42 and functional characterization of evolving RNA virus variants -in real-time -during an 43 outbreak.

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects, and plants. Here, we demonstrate that highly virulent Mycoplasma mycoides subspecies capri ( Mmc ) can be fully attenuated via targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately 10% of the original Mmc genome, were successively deleted using Saccharomyces cerevisiae as an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually non-essential in the JCVI-Syn3.0 minimal cell, were excised from the genome. In vitro characterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though 10% of the genome content were removed. A novel in vivo challenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reached endpoint criteria within 6 days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of a Mycoplasma strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent in vitro or in vivo trials employing the Mycoplasma chassis will allow us to dissect the role of individual candidate Mycoplasma virulence factors and lead the way for the development of an attenuated designer vaccine.