Within-host dynamics of the emergence of tomato yellow leaf curl virus recombinants

Tomato yellow leaf curl virus (TYLCV) is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other begomoviruses. Recent analysis of mixed infections between TYLCV and Tomato leaf curl Comoros begomovirus (ToLCKMV) has shown that, although natural selection preserves certain co-evolved intra-genomic interactions, numerous and diverse recombinants are produced at 120 days post-inoculation (dpi), and recombinant populations from different tomato plants are very divergent. Here, we investigate the population dynamics that lead to such patterns in tomato plants co-infected with TYLCV and ToLCKMV either by agro-inoculation or using the natural whitefly vector Bemisia tabaci . We monitored the frequency of parental and recombinant genotypes independently in 35 plants between 18 and 330 dpi and identified 177 recombinants isolated at different times. Recombinants were detected from 18 dpi and their frequency increased over time to reach about 50% at 150 dpi regardless of the inoculation method. The distribution of breakpoints detected on 96 fully sequenced recombinants was consistent with a continuous generation of new recombinants as well as random and deterministic effects in their maintenance. A severe population bottleneck of around 10 genomes was estimated during early systemic infection-a phenomenon that could account partially for the heterogeneity in recombinant patterns observed among plants. The detection of the same recombinant genome in six of the thirteen plants analysed beyond 30 dpi supported the influence of selection on observed recombination patterns. Moreover, a highly virulent recombinant genotype dominating virus populations within one plant has, apparently, the potential to be maintained in the natural population according to its infectivity, within-host accumulation, and transmission efficiency - all of which were similar or intermediate to those of the parent genotypes. Our results anticipate the outcomes of natural encounters between TYLCV and ToLCKMV

The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology

Is it possible to be "better" than wild type TYLCV ?

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Randomness and selection in the emergence of begomovirus recombinants

National audienc

Frequency and nature of recombinant genomes isolated in plants co-infected with two begomoviruses

National audienceBegomoviruses (Family Geminiviridae) are circular ssDNA viruses transmitted by the whitefly Bemisia tabaci. They replicate in the nucleus of infected cells by recombination prone mechanisms, rolling circle replication and recombination dependant replication. Recombinant begomoviruses were frequently reported, some of them being implicated in emerging diseases. Consistently, Garcia Andres et al. (2007) showed that recombinant viral genomes occurred frequently in tomato plants 120 days after their inoculation with two tomato yellow leaf curl (TYLC) associated begomoviruses. About 50% of the isolated viral genomes were recombinants and nearly 100% after vector transmission (whitely Bemisia tabaci) to a new tomato plant. This study brought up several questions regarding the delay until recombinants are detected, the distribution of recombination breakpoint and the influence of the vector which is expected to stimulate recombination after transmission in host plant. We addressed these questions by studying the frequency and nature of recombinant genomes in tomato plants coinfected with two TYLC associated begomoviruses, either by artificial inoculation with Agrobacterium tumefaciens, or naturally with B. tabaci. The coinfected viruses were Tomato yellow leaf curl virus (TYLCV) and Tomato leaf curl Mayotte virus (ToLCYTV) which were previously used for generating artificial recombinants using DNA-shuffling (see F. Vuillaume presentation). These two begomoviruses exhibit 18% nucleotide differences spread throughout the genome. Viral genomes isolated from co-infected plants using the commercial TempliPhi kit were cloned and analysed with RFLP assay and sequencing. Several of our results contrast or complete those of Garcia Andres

Stochasticité et sélection dans l'émergence de génomes recombinants chez les begomovirus

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Frequency of TYX, TOX and recombinant (REC) genomes in co-infected tomato plants.

<p>Following co-infection of plants with <i>Tomato yellow leaf curl virus</i> (TYX) and <i>Tomato leaf curl Comoros virus</i> (TOX), the frequency of parental and recombinant genomes was monitored within plants at different days post-inoculation (dpi). <b>A</b>) Frequencies in four tomato plants co-infected by agro-inoculation and sampled at 30, 60 and 150 days post inoculation (dpi). The 330 dpi results were from four plants of a separate experiment as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058375#pone-0058375-g001" target="_blank">Figure 1</a>. <b>B</b>) Frequencies in five tomato plants co-infected by vector-inoculation and sampled at 60 and 150 days after the agro-inoculation of the source plants used for vector transmission as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058375#pone-0058375-g001" target="_blank">Figure 1</a>. The numbers at the top of the graphs indicate the total number of genomes analyzed by restriction analysis. The error bars are 95% confidence intervals of the mean frequency.</p

Origin of samples collected on tomato plants co-infected with TYX and TOX.

<p>Samples were collected from four independent experiments as indicated. Tomato plants were co-infected with <i>Tomato yellow leaf curl virus</i> (TYX) and <i>Tomato leaf curl Comoros viru</i>s;(TOX) either by agro-inoculation (blue lines) or by vector-inoculation (red lines). The vertical dotted red lines represent the vector transmission in which plant 1 and 4 were used as source plants. In Experiments 1–3, the percentage of recombinant genomes and the number of genomes analyzed (between brackets) were indicated at each sampling time. When the same recombinant genome was isolated from at least two distinct plants it is indicated by a symbol (▪, ▴, □) or by its name, (R10). (-) indicates no data obtained. In Experiment 4, the frequency of viral DNA of TYX and recombinant genomes (REC) in the viral population were estimated at 18 and 30 days post inoculation (dpi) by real time PCR. The frequency was estimated from the five plants which were detected positive for recombinants at 18 dpi and from the 22 plants detected positive at 30 dpi.</p

Relative infectivities of the recombinant R4 and the parental clones TYX and TOX in tomato.

<p>The infectivities were determined 30 days after co-inoculation of tomato plants with <i>Tomato yellow leaf curl virus</i> (TYX) and <i>Tomato leaf curl Comoros virus</i> (TOX) (A) or with TYX, TOX and recombinant R4 (B). Infectivity (number of infected plants/number of inoculated plants) was determined from four independent tests (1–4) comprising 30 to 45 plants each in (A) and 60 to 90 plants each in (B). In both graphs, the infectivity of TYX is arbitrarily set to 1 (blue line).</p

Effects of the co-inoculation method and the time after inoculation on the frequency of <i>Tomato yellow leaf curl virus</i> (TYX), <i>Tomato leaf curl Comoros vir</i>us (TOX) and recombinant genomes (REC) in co-infected tomato plants.

<p>Generalized linear mixed model, df = 1.</p>1<p>Significant effects (*) <i>P</i><0.05. (**) <i>P</i><0.001 and (***) <i>P</i><0.0001.</p