Population genomics reveals molecular determinants of specialization to tomato in the polyphagous fungal pathogen Botrytis cinerea in France

Many fungal plant pathogens encompass multiple populations specialized on different plant species. Understanding the factors underlying pathogen adaptation to their hosts is a major challenge of evolutionary microbiology, and it should help preventing the emergence of new specialized pathogens on novel hosts. Previous studies have shown that French populations of the grey mould pathogen Botrytis cinerea parasitizing tomato and grapevine are differentiated from each other, and have higher aggressiveness on their host-of-origin than on other hosts, indicating some degree of host specialization in this polyphagous pathogen. Here, we aimed at identifying the genomic features underlying the specialization of B. cinerea populations to tomato and grapevine. Based on whole genome sequences of 32 isolates, we confirmed the subdivision of B. cinerea pathogens into two genetic clusters on grapevine and another, single cluster on tomato. Levels of genetic variation in the different clusters were similar, suggesting that the tomato-specific cluster has not recently emerged following a bottleneck. Using genome scans for selective sweeps and divergent selection, tests of positive selection based on polymorphism and divergence at synonymous and non-synonymous sites and analyses of presence/absence variation, we identified several candidate genes that represent possible determinants of host specialization in the tomato-associated population. This work deepens our understanding of the genomic changes underlying the specialization of fungal pathogen populations.

Analysis of Stop Codons within Prokaryotic Protein-Coding Genes Suggests Frequent Readthrough Events

Nonsense mutations turn a coding (sense) codon into an in-frame stop codon that is assumed to result in a truncated protein product. Thus, nonsense substitutions are the hallmark of pseudogenes and are used to identify them. Here we show that in-frame stop codons within bacterial protein-coding genes are widespread. Their evolutionary conservation suggests that many of them are not pseudogenes, since they maintain dN/dS values (ratios of substitution rates at non-synonymous and synonymous sites) significantly lower than 1 (this is a signature of purifying selection in protein-coding regions). We also found that double substitutions in codons—where an intermediate step is a nonsense substitution—show a higher rate of evolution compared to null models, indicating that a stop codon was introduced and then changed back to sense via positive selection. This further supports the notion that nonsense substitutions in bacteria are relatively common and do not necessarily cause pseudogenization. In-frame stop codons may be an important mechanism of regulation: Such codons are likely to cause a substantial decrease of protein expression levels.

Mutation rates and selection on synonymous mutations in SARS-CoV-2

The COVID-19 pandemic has seen an unprecedented response from the sequencing community. Leveraging the sequence data from more than 140,000 SARS-CoV-2 genomes, we study mutation rates and selective pressures affecting the virus. Understanding the processes and effects of mutation and selection has profound implications for the study of viral evolution, for vaccine design, and for the tracking of viral spread. We highlight and address some common genome sequence analysis pitfalls that can lead to inaccurate inference of mutation rates and selection, such as ignoring skews in the genetic code, not accounting for recurrent mutations, and assuming evolutionary equilibrium. We find that two particular mutation rates, G→U and C→U, are similarly elevated and considerably higher than all other mutation rates, causing the majority of mutations in the SARS-CoV-2 genome, and are possibly the result of APOBEC and ROS activity. These mutations also tend to occur many times at the same genome positions along the global SARS-CoV-2 phylogeny (i.e., they are very homoplasic). We observe an effect of genomic context on mutation rates, but the effect of the context is overall limited. While previous studies have suggested selection acting to decrease U content at synonymous sites, we bring forward evidence suggesting the opposite.

Using Patterns of Signed Linkage Disequilibria to Test for Epistasis in Flies and Plants

Whether deleterious mutations affect fitness independently, or synergistically, remains an open question in evolutionary genetics. Previous work by Sohail et al. (2017) reported an abundance of negative linkage disequilibrium (LD) values among loss-of - function (LOF) mutations in several human and fruit fly datasets, a pattern the authors interpreted as evidence of negative synergistic epistasis. Here we re-visit this question in a population genomic dataset of plants (Capsella grandiflora), and a fruit fly (Drosophila melanogaster) dataset previously used by Sohail et al. When using synonymous sites as a control, as Sohail et al., we find that both species have significantly less positive LD at LOF sites than synonymous sites. However, LD is not significantly different from 0 for LOF mutations but is significantly positive for synonymous mutations in both species. We question the use of synonymous sites as an appropriate control when attempting to make inferences about LD at selected sites. We use simulations to show how admixture or mating bias towards physically proximal individuals can cause positive LD to build up among neutral mutations but has a much weaker effect on selected sites, regardless of the presence of epistasis. Finally, we use information from published biological networks to explore whether there is evidence for negative synergistic epistasis between interacting radical missense mutations. We report no significant enrichment for negative inter-network LD in C. grandiflora. However, we note a modest but significant enrichment of negative LD in D. melanogaster networks, suggestive of intra-network negative synergistic epistasis in this species.

Recombinant Fowlpox Virus Expressing gB Gene from Predominantly Epidemic Infectious Larygnotracheitis Virus Strain Demonstrates Better Immune Protection in SPF Chickens

Background: Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens. Antigenic mutation of infectious laryngotracheitis virus (ILTV) may result in a vaccination failure in the poultry industry and thus a protective vaccine against predominant ILTV strains is highly desirable. Methods: The full-length glycoprotein B (gB) gene of ILTV with the two mutated synonymous sites of fowlpox virus (FPV) transcription termination signal sequence was cloned into the insertion vector p12LS, which was co-transfected with wild-type (wt) FPV into chicken embryo fibroblast (CEF) to develop a recombinant fowlpox virus-gB (rFPV-gB) candidate vaccine strain. Furthermore, its biological and immunological characteristics were evaluated. Results: The results indicated that gB gene was expressed correctly in the rFPV by indirect immunofluorescent assay and Western blot, and the rFPV-gB provided a 100% protection in immunized chickens against the challenge of predominant ILTV strains that were screened by pathogenicity assay when compared with the commercialized rFPV vaccine, which only provided 83.3%. Conclusion: rFPV-gB can be used as a potential vaccine against predominant ILTV strains.

A putative new SARS-CoV protein, 3c, encoded in an ORF overlapping ORF3a

Identification of the full complement of genes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a crucial step towards gaining a fuller understanding of its molecular biology. However, short and/or overlapping genes can be difficult to detect using conventional computational approaches, whereas high-throughput experimental approaches – such as ribosome profiling – cannot distinguish translation of functional peptides from regulatory translation or translational noise. By studying regions showing enhanced conservation at synonymous sites in alignments of SARS-CoV-2 and related viruses (subgenus Sarbecovirus) and correlating the results with the conserved presence of an open reading frame (ORF) and a plausible translation mechanism, a putative new gene – ORF3c – was identified. ORF3c overlaps ORF3a in an alternative reading frame. A recently published ribosome profiling study confirmed that ORF3c is indeed translated during infection. ORF3c is conserved across the subgenus Sarbecovirus, and encodes a 40–41 amino acid predicted transmembrane protein.

POPULATION GENOMICS REVEALS MOLECULAR DETERMINANTS OF SPECIALIZATION TO TOMATO IN THE POLYPHAGOUS FUNGAL PATHOGEN BOTRYTIS CINEREA

Many fungal plant pathogens encompass multiple populations specialized on different plant species. Understanding the factors underlying pathogen adaptation to their hosts is a major challenge of evolutionary microbiology, and it should help preventing the emergence of new specialized pathogens on novel hosts. Previous studies have shown that French populations of the grey mould pathogen Botrytis cinerea parasitizing tomato and grapevine are differentiated from each other, and have higher aggressiveness on their host-of-origin than on other hosts, indicating some degree of host specialization in this polyphagous pathogen. Here, we aimed at identifying the genomic features underlying the specialization of B. cinerea populations to tomato and grapevine. Based on whole genome sequences of 32 isolates, we confirmed the subdivision of B. cinerea pathogens into two genetic clusters on grapevine and another, single cluster on tomato. Levels of genetic variation in the different clusters were similar, suggesting that the tomato-specific cluster has not recently emerged following a bottleneck. Using genome scans for selective sweeps and divergent selection, tests of positive selection based on polymorphism and divergence at synonymous and non-synonymous sites and analyses of presence/absence variation, we identified several candidate genes that represent possible determinants of host specialization in the tomato-associated population. This work deepens our understanding of the genomic changes underlying the specialization of fungal pathogen populations.

Evidence for Strong Mutation Bias toward, and Selection against, U Content in SARS-CoV-2: Implications for Vaccine Design

Large-scale re-engineering of synonymous sites is a promising strategy to generate vaccines either through synthesis of attenuated viruses or via codon-optimized genes in DNA vaccines. Attenuation typically relies on deoptimization of codon pairs and maximization of CpG dinucleotide frequencies. So as to formulate evolutionarily informed attenuation strategies that aim to force nucleotide usage against the direction favored by selection, here, we examine available whole-genome sequences of SARS-CoV-2 to infer patterns of mutation and selection on synonymous sites. Analysis of mutational profiles indicates a strong mutation bias toward U. In turn, analysis of observed synonymous site composition implicates selection against U. Accounting for dinucleotide effects reinforces this conclusion, observed UU content being a quarter of that expected under neutrality. Possible mechanisms of selection against U mutations include selection for higher expression, for high mRNA stability or lower immunogenicity of viral genes. Consistent with gene-specific selection against CpG dinucleotides, we observe systematic differences of CpG content between SARS-CoV-2 genes. We propose an evolutionarily informed approach to attenuation that, unusually, seeks to increase usage of the already most common synonymous codons. Comparable analysis of H1N1 and Ebola finds that GC3 deviated from neutral equilibrium is not a universal feature, cautioning against generalization of results.

Excessive Parallelism in Protein Evolution of Lake Baikal Amphipod Species Flock

Repeated emergence of similar adaptations is often explained by parallel evolution of underlying genes. However, evidence of parallel evolution at amino acid level is limited. When the analyzed species are highly divergent, this can be due to epistatic interactions underlying the dynamic nature of the amino acid preferences: The same amino acid substitution may have different phenotypic effects on different genetic backgrounds. Distantly related species also often inhabit radically different environments, which makes the emergence of parallel adaptations less likely. Here, we hypothesize that parallel molecular adaptations are more prevalent between closely related species. We analyze the rate of parallel evolution in genome-size sets of orthologous genes in three groups of species with widely ranging levels of divergence: 46 species of the relatively recent lake Baikal amphipod radiation, a species flock of very closely related cichlids, and a set of significantly more divergent vertebrates. Strikingly, in genes of amphipods, the rate of parallel substitutions at nonsynonymous sites exceeded that at synonymous sites, suggesting rampant selection driving parallel adaptation. At sites of parallel substitutions, the intraspecies polymorphism is low, suggesting that parallelism has been driven by positive selection and is therefore adaptive. By contrast, in cichlids, the rate of nonsynonymous parallel evolution was similar to that at synonymous sites, whereas in vertebrates, this rate was lower than that at synonymous sites, indicating that in these groups of species, parallel substitutions are mainly fixed by drift.

The divergence between SARS-CoV-2 and RaTG13 might be overestimated due to the extensive RNA modification

Aim: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the world. There is urgent need to understand the phylogeny, divergence and origin of SARS-CoV-2. Materials & methods: A recent study claimed that there was 17% divergence between SARS-CoV-2 and RaTG13 (a SARS-related coronaviruses) on synonymous sites by using sequence alignment. We re-analyzed the sequences of the two coronaviruses with the same methodology. Results: We found that 87% of the synonymous substitutions between the two coronaviruses could be potentially explained by the RNA modification system in hosts, with 65% contributed by deamination on cytidines (C-T mismatches) and 22% contributed by deamination on adenosines (A-G mismatches). Conclusion: Our results demonstrate that the divergence between SARS-CoV-2 and RaTG13 has been overestimated.