Unraveling the causes of adaptive benefits of synonymous mutations in TEM-1 ß-lactamase.

While synonymous mutations were long thought to be without phenotypic consequences, there is growing evidence they can affect gene expression, protein folding, and ultimately the fitness of an organism. In only a few cases have the mechanisms by which synonymous mutations affect the phenotype been elucidated. We previously identified 48 mutations in TEM-1 ß-lactamase that increased resistance of Escherichia coli to cefotaxime, 10 of which were synonymous. To better understand the molecular mechanisms underlying the beneficial effect of these synonymous mutations, we made a series of measurements for a panel containing the 10 synonymous together with 10 non-synonymous mutations as a reference. Whereas messenger levels were unaffected, we found that total and functional TEM protein levels were higher for 5 out of 10 synonymous mutations. These observations suggest that some of these mutations act on translation or a downstream process. Similar effects were observed for some small-benefit non-synonymous mutations, suggesting a similar causal mechanism. For the synonymous mutations, we found that the cost of resistance scales with TEM protein levels. A resistance landscape for four synonymous mutations revealed strong epistasis: none of the combinations of mutations exceeded the resistance of the largest-effect mutation and there were synthetically neutral combinations. By considering combined effects of these mutations, we could infer that functional TEM protein level is a multi-dimensional phenotype. These results suggest that synonymous mutations may have beneficial effects by increasing the expression of an enzyme with low substrate activity, which may be realized via multiple, yet unknown, post-transcriptional mechanisms.

Quantifying the adaptive potential of an antibiotic resistance enzyme.

For a quantitative understanding of the process of adaptation, we need to understand its "raw material," that is, the frequency and fitness effects of beneficial mutations. At present, most empirical evidence suggests an exponential distribution of fitness effects of beneficial mutations, as predicted for Gumbel-domain distributions by extreme value theory. Here, we study the distribution of mutation effects on cefotaxime (Ctx) resistance and fitness of 48 unique beneficial mutations in the bacterial enzyme TEM-1 ß-lactamase, which were obtained by screening the products of random mutagenesis for increased Ctx resistance. Our contributions are threefold. First, based on the frequency of unique mutations among more than 300 sequenced isolates and correcting for mutation bias, we conservatively estimate that the total number of first-step mutations that increase Ctx resistance in this enzyme is 87 [95% CI 75-189], or 3.4% of all 2,583 possible base-pair substitutions. Of the 48 mutations, 10 are synonymous and the majority of the 38 non-synonymous mutations occur in the pocket surrounding the catalytic site. Second, we estimate the effects of the mutations on Ctx resistance by determining survival at various Ctx concentrations, and we derive their fitness effects by modeling reproduction and survival as a branching process. Third, we find that the distribution of both measures follows a Fréchet-type distribution characterized by a broad tail of a few exceptionally fit mutants. Such distributions have fundamental evolutionary implications, including an increased predictability of evolution, and may provide a partial explanation for recent observations of striking parallel evolution of antibiotic resistance.

Patterns of Epistasis between beneficial mutations in an antibiotic resistance gene.

Understanding epistasis is central to biology. For instance, epistatic interactions determine the topography of the fitness landscape and affect the dynamics and determinism of adaptation. However, few empirical data are available, and comparing results is complicated by confounding variation in the system and the type of mutations used. Here, we take a systematic approach by quantifying epistasis in two sets of four beneficial mutations in the antibiotic resistance enzyme TEM-1 ß-lactamase. Mutations in these sets have either large or small effects on cefotaxime resistance when present as single mutations. By quantifying the epistasis and ruggedness in both landscapes, we find two general patterns. First, resistance is maximal for combinations of two mutations in both fitness landscapes and declines when more mutations are added due to abundant sign epistasis and a pattern of diminishing returns with genotype resistance. Second, large-effect mutations interact more strongly than small-effect mutations, suggesting that the effect size of mutations may be an organizing principle in understanding patterns of epistasis. By fitting the data to simple phenotype resistance models, we show that this pattern may be explained by the nonlinear dependence of resistance on enzyme stability and an unknown phenotype when mutations have antagonistically pleiotropic effects. The comparison to a previously published set of mutations in the same gene with a joint benefit further shows that the enzyme's fitness landscape is locally rugged but does contain adaptive pathways that lead to high resistance.

Predicting the evolution of antibiotic resistance.

Mutations causing antibiotic resistance are often associated with a cost in the absence of antibiotics. Surprisingly, a new study found that bacteria adapting to increased temperature became resistant to rifampicin. By studying the consequences of the involved mutations in different conditions and genetic backgrounds, the authors illustrate how knowledge of two fundamental genetic properties, pleiotropy and epistasis, may help to predict the evolution of antibiotic resistance.

African eggplant-associated virus: Characterization of a novel tobamovirus identified from Solanum macrocarpon and assessment of its potential impact on tomato and pepper crops.

A novel tobamovirus was identified in a fruit of Solanum macrocarpon imported into the Netherlands in 2018. This virus was further characterized in terms of host range, pathotype and genomic properties, because many tobamoviruses have the potential to cause severe damage in important crops. In the original fruit, two different genotypes of the novel virus were present. The virus was able to infect multiple plant species from the Solanaceae family after mechanical inoculation, as well as a member of the Apiaceae family. These species included economically important crops such as tomato and pepper, as well as eggplant and petunia. Both tomato and pepper germplasm were shown to harbor resistance against the novel virus. Since most commercial tomato and pepper varieties grown in European greenhouses harbor these relevant resistances, the risk of infection and subsequent impact on these crops is likely to be low in Europe. Assessment of the potential threat to eggplant, petunia, and other susceptible species needs further work. In conclusion, this study provides a first assessment of the potential phytosanitary risks of a newly discovered tobamovirus, which was tentatively named African eggplant-associated virus.

Population size mediates the contribution of high-rate and large-benefit mutations to parallel evolution.

Mutations with large fitness benefits and mutations occurring at high rates may both cause parallel evolution, but their contribution is predicted to depend on population size. Moreover, high-rate and large-benefit mutations may have different long-term adaptive consequences. We show that small and 100-fold larger bacterial populations evolve resistance to a ß-lactam antibiotic by using similar numbers, but different types of mutations. Small populations frequently substitute similar high-rate structural variants and loss-of-function point mutations, including the deletion of a low-activity ß-lactamase, and evolve modest resistance levels. Large populations more often use low-rate, large-benefit point mutations affecting the same targets, including mutations activating the ß-lactamase and other gain-of-function mutations, leading to much higher resistance levels. Our results demonstrate the separation by clonal interference of mutation classes with divergent adaptive consequences, causing a shift from high-rate to large-benefit mutations with increases in population size.

List of non-EU viruses and viroids infecting potato (Solanum tuberosum) and other tuber-forming Solanum species.

The European Commission requested a pest categorisation of the non-EU viruses and viroids of potato (hereafter referred to as viruses). As a first step, a systematic literature and database search was carried out to identify the viruses reported to naturally infect Solanum tuberosum and other tuber-forming Solanum spp (hereafter referred to as potato). Based on the global distribution and on the prevalence inside the European Union (EU), the Panel identified 40 non-EU viruses known to occur only outside the EU or with only a limited presence in the EU (reported in only one or few Member States (MSs) and/or with restricted distribution, outbreaks). Twenty-seven viruses were identified as having a significant presence in the EU (known to occur in several MSs, frequently reported in the EU, widespread in several MSs) or reported only from the EU so far, and will be excluded from further categorisation in the frame of the present mandate. Five viruses remained with an undetermined standing because the available information did not allow their allocation to one of the above groups. The viruses considered non-EU and those with undetermined standing will be further categorised if not addressed by EFSA in previous scientific opinions. Seven viruses for which non-European isolates are specifically regulated in Annex I of directive 2000/29/EC will be categorised separately. The main knowledge gaps and uncertainties of this grouping concern the natural host status of potato, the taxonomy, and/or information on the geographical distribution and prevalence of some of the analysed viruses.

Pest categorisation of non-EU viruses and viroids of potato.

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of those viruses and viroids (hereafter referred to as viruses) of Solanum tuberosum and other tuber-forming Solanum spp. (hereafter referred to as potato) which are considered to be either non-EU or of undetermined standing based on a previous EFSA opinion. These viruses belong to different families and genera and either have an established identity or produce consistent symptoms. Plants for planting is the main pathway for entry for all categorised viruses as they can all be transmitted by vegetative propagation. Several categorised viruses have a relatively wide host range and/or are vector-transmitted, increasing the potential for entry. The information currently available on geographical distribution, biology, epidemiology, impact and potential entry pathways has been evaluated with regard to the criteria to qualify as potential Union quarantine pest or as Union regulated non-quarantine pest (RNQP). Since this opinion addresses specifically the non-EU potato viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non-quarantine pests. The following viruses meet the criteria to qualify as potential Union quarantine pest: APLV, APMMV, APMoV, ChiLCV, CYSDV, PAMV, PBRSV, PVH, PVP, PVT, PYDV, PYMV, PYV, PYVV, RCVMV, SALCV, SB26/29, ToCV, ToLCNDV, ToMHaV, ToMoTV, ToSRV and ToYVSV. With the exception of the criterion regarding the potential for consequences in the EU territory, for which the Panel is unable to conclude because of lack of information, AVB, CPSbV, PaLCrV, PapMV, PVB, PVU, SB41 and TVBMV meet all the other criteria to qualify as potential Union quarantine pest. PotLV and WPMV do not qualify as potential Union quarantine pest, since they are not reported to have any impact. For most of the categorised viruses, the conclusions of the Panel have inherent uncertainties, due to the lack of quantitative data on their impact and/or absence or limited availability of information on the biology, epidemiology and geographical distribution.

Pest categorisation of potato virus M (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus M (PVM). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact compared to the current situation in the EU and availability of control measures of non-EU isolates of PVM has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PVM are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. Populations of PVM can be subdivided into two strains: the ordinary strain (PVM-O) is present in the EU, while the divergent strain (PVM-D) is absent from the EU or considered to have at most a limited distribution in the EU. Non-EU isolates of PVM-O are not expected to have an additional impact in the EU compared to EU isolates and therefore do not meet the corresponding criterion to qualify as a potential Union quarantine pest. The Panel is unable to conclude on the potential impact of non-EU PVM-D isolates in the EU territory, but PVM-D isolates meet all the other criteria to qualify as a potential Union quarantine pest.

Pest categorisation of potato virus S (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus S (PVS). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact compared to the current situation in the EU, and availability of control measures of non-EU isolates of PVS has been evaluated with regard to the criteria to qualify as potential Union quarantine pest. Because non-EU isolates of PVS are absent from the EU, they do not meet one of the requirements to be regulated as an RNQP (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. Populations of PVS can be subdivided into two strains: the ordinary strain (PVS-O) with a worldwide distribution (including the EU), and the Andean strain (PVS-A) which is absent from the EU or considered to have at most a limited distribution in the EU. Two additional divergent isolates (PVS-A/PVS-O recombinants and PVS-arracacha) have also been categorised. Non-EU isolates of PVS-A are expected to have an additional impact as compared to the PVS isolates currently present in the EU, and therefore meet all the criteria to qualify as potential Union quarantine pests; the magnitude of the additional impact is, however, unknown. Non-EU isolates of PVS-A/PVS-O recombinants and of PVS-arracacha also meet these criteria, with the exception of the criterion regarding the potential additional consequences in the EU territory for which the Panel was unable to conclude. Non-EU PVS-O isolates are not expected to have an additional impact in the EU as compared to EU isolates and therefore do not meet the corresponding criterion.

Pest categorisation of potato virus A (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus A (PVA). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact over the current situation and availability of control measures of non-EU isolates of PVA has been evaluated with regard to the criteria to qualify as potential Union quarantine pest. Because non-EU isolates of PVA are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. This categorisation was performed considering two groups of isolates: those reported in Solanum betaceum (PVA-TamMV, not reported from the EU) and all other isolates (hereafter referred to as PVA, worldwide distribution). Non-EU isolates of PVA and of PVA-TamMV do not meet one of the criteria evaluated by EFSA to be regarded as a potential Union quarantine pest, since they are not expected to have an additional impact in the EU.

Pest categorisation of potato virus V (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus V (PVV). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact and availability of control measures of non-EU isolates of PVV has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PVV are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. This categorisation was performed considering two lineages, PVV-I (present in and outside the EU) and PVV-II (not reported in the EU), and isolate PVV-PA4 (unknown distribution). Non-EU isolates of PVV-I and PVV-PA4 do not meet one of the criteria evaluated by EFSA to be regarded as a potential Union quarantine pest, since they are not expected to have an additional impact in the EU. With the exception of the criterion regarding the potential consequences in the EU territory, for which the Panel is unable to conclude, non-EU isolates of PVV-II meet all the other criteria to qualify as a potential Union quarantine pest.

Pest categorisation of potato virus X (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus X (PVX). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact and availability of control measures of non-EU isolates of PVX has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PVX are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. On the basis of their ability to overcome potato resistance genes, PVX isolates can be divided into several pathotypes. PVX isolates that are not able to overcome resistance genes and PVX isolates that are able to overcome the Nb and/or Nx resistance genes are already present in the EU. Isolates able to overcome the Rx resistance gene have only been reported from South America. These Rx breaking isolates could potentially have an additional impact over the current situation in the EU and therefore meet all the criteria to qualify as a potential Union quarantine pest. All other non-EU isolates, should they be introduced, are not expected to have additional impact and therefore do not meet this criterion to qualify as a potential Union quarantine pest.

Pest categorisation of potato virus Y (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus Y (PVY). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways and potential additional impact of non-EU isolates of PVY, has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PVY are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. Populations of PVY can be subdivided into several strains and groups of isolates: strain C (PVY-C), strain N (PVY-N), strain O (PVY-O) and a wide range of recombinant isolates (PVY-recombinants) which have a worldwide distribution (including the EU). Two groups of isolates, i.e. the Brazilian (PVY-Br) and Chilean (PVY-Ch) isolates, are considered absent from the EU. Non-EU isolates of PVY-C, PVY-N, PVY-O and PVY-recombinants identified so far are not expected to have an additional impact in the EU compared to the PVY isolates already present and, therefore, do not meet the corresponding criterion to qualify as a potential Union quarantine pest. The Panel is unable to conclude on the potential additional impact of isolates of PVY-Br and PVY-Ch in the EU territory, but these isolates meet all the other criteria to qualify as potential Union quarantine pests.

Pest categorisation of potato leafroll virus (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato leafroll virus (PLRV). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact and availability of control measures of non-EU isolates of PLRV has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PLRV are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. This categorisation was performed considering two groups of PLRV isolates: those associated with the tomato yellow top disease (PLRV-TYTV), not reported from the EU, and all other isolates (hereafter referred to as PLRV), with a worldwide distribution. Isolates of PLRV-TYTV could potentially have an additional impact over the current situation in the EU and therefore meet all the criteria to qualify as a potential Union quarantine pest. All other non-EU PLRV isolates, should they be introduced, are not expected to have additional impact and therefore do not meet this criterion to qualify as a potential Union quarantine pest.

Publisher Correction: Science diplomacy for plant health.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen.

BACKGROUND: Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MSE was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome. RESULTS: All examined birch species contained several PR-10 genes. In total, 134 unique sequences were recovered. Sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. Five subfamilies were common to all birch species. Genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. Isoforms that were similar to isoforms with a high IgE-reactivity (Bet v 1a = PR-10.01A01) were abundant in all species except B. lenta, while the hypoallergenic isoform Bet v 1d (= PR-10.01B01) was only found in B. pendula and its closest relatives. CONCLUSION: Q-TOF LC-MSE allows efficient screening of Bet v 1 isoforms by determining the presence and relative abundance of these isoforms in pollen. B. pendula contains a Bet v 1-mixture in which isoforms with a high and low IgE-reactivity are both abundant. With the possible exception of B. lenta, isoforms identical or very similar to those with a high IgE-reactivity were found in the pollen proteome of all examined birch species. Consequently, these species are also predicted to be allergenic with regard to Bet v 1 related allergies.

Seven different genes encode a diverse mixture of isoforms of Bet v 1, the major birch pollen allergen.

BACKGROUND: Pollen of the European white birch (Betula pendula, syn. B. verrucosa) is an important cause of hay fever. The main allergen is Bet v 1, member of the pathogenesis-related class 10 (PR-10) multigene family. To establish the number of PR-10/Bet v 1 genes and the isoform diversity within a single tree, PCR amplification, cloning and sequencing of PR-10 genes was performed on two diploid B. pendula cultivars and one interspecific tetraploid Betula hybrid. Sequences were attributed to putative genes based on sequence identity and intron length. Information on transcription was derived by comparison with homologous cDNA sequences available in GenBank/EMBL/DDJB. PCR-cloning of multigene families is accompanied by a high risk for the occurrence of PCR recombination artifacts. We screened for and excluded these artifacts, and also detected putative artifact sequences among database sequences. RESULTS: Forty-four different PR-10 sequences were recovered from B. pendula and assigned to thirteen putative genes. Sequence homology suggests that three genes were transcribed in somatic tissue and seven genes in pollen. The transcription of three other genes remains unknown. In total, fourteen different Bet v 1-type isoforms were identified in the three cultivars, of which nine isoforms were entirely new. Isoforms with high and low IgE-reactivity are encoded by different genes and one birch pollen grain has the genetic background to produce a mixture of isoforms with varying IgE-reactivity. Allergen diversity is even higher in the interspecific tetraploid hybrid, consistent with the presence of two genomes. CONCLUSION: Isoforms of the major birch allergen Bet v 1 are encoded by multiple genes, and we propose to name them accordingly. The present characterization of the Bet v 1 genes provides a framework for the screening of specific Bet v 1 genes among other B. pendula cultivars or Betula species, and for future breeding for trees with a reduced allergenicity. Investigations towards sensitization and immunotherapy should anticipate that patients are exposed to a mixture of Bet v 1 isoforms of different IgE-reactivity, even if pollen originates from a single birch tree.

Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes.

BACKGROUND: Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the alpha-gliadins contain several peptides that are associated to the disease. RESULTS: We obtained 230 distinct alpha-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All alpha-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, alpha-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-alpha9 and glia-alpha20, but never the intact epitopes glia-alpha and glia-alpha2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific. CONCLUSION: Our analysis shows that alpha-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxicity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.