Analysis of viral (zucchini yellow mosaic virus) genetic diversity during systemic movement through a Cucurbita pepo vine.
ABSTRACT: Determining the extent and structure of intra-host genetic diversity and the magnitude and impact of population bottlenecks is central to understanding the mechanisms of viral evolution. To determine the nature of viral evolution following systemic movement through a plant, we performed deep sequencing of 23 leaves that grew sequentially along a single Cucurbita pepo vine that was infected with zucchini yellow mosaic virus (ZYMV), and on a leaf that grew in on a side branch. Strikingly, of 112 genetic (i.e. sub-consensus) variants observed in the data set as a whole, only 22 were found in multiple leaves. Similarly, only three of the 13 variants present in the inoculating population were found in the subsequent leaves on the vine. Hence, it appears that systemic movement is characterized by sequential population bottlenecks, although not sufficient to reduce the population to a single virion as multiple variants were consistently transmitted between leaves. In addition, the number of variants within a leaf increases as a function of distance from the inoculated (source) leaf, suggesting that the circulating sap may serve as a continual source of virus. Notably, multiple mutational variants were observed in the cylindrical inclusion (CI) protein (known to be involved in both cell-to-cell and systemic movement of the virus) that were present in multiple (19/24) leaf samples. These mutations resulted in a conformational change, suggesting that they might confer a selective advantage in systemic movement within the vine. Overall, these data reveal that bottlenecks occur during systemic movement, that variants circulate in the phloem sap throughout the infection process, and that important conformational changes in CI protein may arise during individual infections.
Project description:For any organism, population size, and fluctuations thereof, are of primary importance in determining the forces driving its evolution. This is particularly true for viruses--rapidly evolving entities that form populations with transient and explosive expansions alternating with phases of migration, resulting in strong population bottlenecks and associated founder effects that increase genetic drift. A typical illustration of this pattern is the progression of viral disease within a eukaryotic host, where such demographic fluctuations are a key factor in the emergence of new variants with altered virulence. Viruses initiate replication in one or only a few infection foci, then move through the vasculature to seed secondary infection sites and so invade distant organs and tissues. Founder effects during this within-host colonization might depend on the concentration of infectious units accumulating and circulating in the vasculature, as this represents the infection dose reaching new organs or "territories". Surprisingly, whether or not the easily measurable circulating (plasma) virus load directly drives the size of population bottlenecks during host colonization has not been documented in animal viruses, while in plants the virus load within the sap has never been estimated. Here, we address this important question by monitoring both the virus concentration flowing in host plant sap, and the number of viral genomes founding the population in each successive new leaf. Our results clearly indicate that the concentration of circulating viruses directly determines the size of bottlenecks, which hence controls founder effects and effective population size during disease progression within a host.
Project description:The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Despite their high mutation rate and rapid evolution, this parameter is poorly documented experimentally in viruses, particularly plant viruses. All available studies, however, have demonstrated the existence of huge within-host demographic fluctuations, drastically reducing Ne upon systemic invasion of different organs and tissues. Notably, extreme bottlenecks have been detected at the stage of systemic leaf colonization in all plant viral species investigated so far, sustaining the general idea that some unknown obstacle(s) imposes a barrier on the development of all plant viruses. This idea has important implications, as it appoints genetic drift as a constant major force in plant virus evolution. By co-inoculating several genetic variants of Cauliflower mosaic virus into a large number of replicate host plants, and by monitoring their relative frequency within the viral population over the course of the host systemic infection, only minute stochastic variations were detected. This allowed the estimation of the CaMV Ne during colonization of successive leaves at several hundreds of viral genomes, a value about 100-fold higher than that reported for any other plant virus investigated so far, and indicated the very limited role played by genetic drift during plant systemic infection by this virus. These results suggest that the barriers that generate bottlenecks in some plant virus species might well not exist, or can be surmounted by other viruses, implying that severe bottlenecks during host colonization do not necessarily apply to all plant-infecting viruses.
Project description:DIR1 is a lipid transfer protein (LTP) postulated to complex with and/or chaperone a signal(s) to distant leaves during Systemic Acquired Resistance (SAR) in Arabidopsis. DIR1 was detected in phloem sap-enriched petiole exudates collected from wild-type leaves induced for SAR, suggesting that DIR1 gains access to the phloem for movement from the induced leaf. Occasionally the defective in induced resistance1 (dir1-1) mutant displayed a partially SAR-competent phenotype and a DIR1-sized band in protein gel blots was detected in dir1-1 exudates suggesting that a highly similar protein, DIR1-like (At5g48490), may contribute to SAR. Recombinant protein studies demonstrated that DIR1 polyclonal antibodies recognize DIR1 and DIR1-like. Homology modeling of DIR1-like using the DIR1-phospholipid crystal structure as template, provides clues as to why the dir1-1 mutant is rarely SAR-competent. The contribution of DIR1 and DIR1-like during SAR was examined using an Agrobacterium-mediated transient expression-SAR assay and an estrogen-inducible DIR1-EGFP/dir1-1 line. We provide evidence that upon SAR induction, DIR1 moves down the leaf petiole to distant leaves. Our data also suggests that DIR1-like displays a reduced capacity to move to distant leaves during SAR and this may explain why dir1-1 is occasionally SAR-competent.
Project description:Not only does leaf shape vary between Passiflora species, but between sequential nodes of the vine. The profound changes in leaf shape within Passiflora vines reflect the temporal development of the shoot apical meristem from which leaves are derived and patterned, a phenomenon known as heteroblasty. We perform a morphometric analysis of more than 3,300 leaves from 40 different Passiflora species using two different methods: homologous landmarks and Elliptical Fourier Descriptors (EFDs). Changes in leaf shape across the vine are first quantified in allometric terms; that is, changes in the relative area of leaf subregions expressed in terms of overall leaf area. Shape is constrained to strict linear relationships as a function of size that vary between species. Statistical analysis of leaf shape, using landmarks and EFDs, reveals that species effects (regardless of node) are the strongest, followed by interaction effects between species and heteroblasty (i.e., species-specific patterns in leaf shape across nodes) and that heteroblasty effects across nodes (regardless of species) are negligible. The ability of different nodes to predictively discriminate species and the variability of landmark and EFD traits at each node is then analyzed. Heteroblastic trajectories, the changes in leaf shape between the first and last measured leaves in a vine, are then compared between species in a multivariate space. Leaf shape diversity among Passiflora species is expressed in a heteroblastic-dependent manner, unique to each species. Leaf shape is constrained by linear, allometric relationships related to leaf size that vary between species. There is a strong species × heteroblasty interaction effect for leaf shape, suggesting that different leaf shapes between species arise through changes in shape across nodes specific to each species. The first leaves in the series are not only more like each other, but are also less variable across species. From this similar, shared leaf shape, subsequent leaves in the heteroblastic series follow divergent morphological trajectories. The disparate leaf shapes characteristic of Passiflora species arise from a shared, juvenile morphology.
Project description:Genetic bottlenecks facilitate the fixation and extinction of variants in populations, and viral populations are no exception to this theory. To examine the existence of genetic bottlenecks in cell-to-cell movement of plant RNA viruses, we prepared constructs for Soil-borne wheat mosaic virus RNA2 vectors carrying two different fluorescent proteins, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Coinoculation of host plant leaves with the two RNA2 vectors and the wild-type RNA1 showed separation of the two vector RNA2s, mostly within seven to nine cell-to-cell movements from individual initially coinfected cells. Our statistical analysis showed that the number of viral RNA genomes establishing infection in adjacent cells after the first cell-to-cell movement from an initially infected cell was 5.97 +/- 0.22 on average and 5.02 +/- 0.29 after the second cell-to-cell movement. These results indicate that plant RNA viruses may generally face narrow genetic bottlenecks in every cell-to-cell movement. Furthermore, our model suggests that, rather than suffering from fitness losses caused by the bottlenecks, the plant RNA viruses are utilizing the repeated genetic bottlenecks as an essential element of rapid selection of their adaptive variants in trans-acting genes or elements to respond to host shifting and changes in the growth conditions of the hosts.
Project description:BACKGROUND:Leaves of the medicinal plant Ampelopsis grossedentata, which is commonly known as vine tea, are used widely in the traditional Chinese beverage in southwest China. The leaves contain a large amount of dihydromyricetin, a compound with various biological activities. However, the transcript profiles involved in its biosynthetic pathway in this plant are unknown. RESULTS:We conducted a transcriptome analysis of both young and old leaves of the vine tea plant using Illumina sequencing. Of the transcriptome datasets, a total of 52.47 million and 47.25 million clean reads were obtained from young and old leaves, respectively. Among 471,658 transcripts and 177,422 genes generated, 7768 differentially expressed genes were identified in leaves at these two stages of development. The phenylpropanoid biosynthetic pathway of vine tea was investigated according to the transcriptome profiling analysis. Most of the genes encoding phenylpropanoid biosynthesis enzymes were identified and found to be differentially expressed in different tissues and leaf stages of vine tea and also greatly contributed to the biosynthesis of dihydromyricetin in vine tea. CONCLUSIONS:To the best of our knowledge, this is the first formal study to explore the transcriptome of A. grossedentata. The study provides an insight into the expression patterns and differential distribution of genes related to dihydromyricetin biosynthesis in vine tea. The information may pave the way to metabolically engineering plants with higher flavonoid content.
Project description:PREMISE:The size and shape (physiognomy) of woody, dicotyledonous angiosperm leaves are correlated with climate. These relationships are the basis for multiple paleoclimate proxies. Here we test whether Vitis exhibits phenotypic plasticity and whether physiognomy varies along the vine. METHODS:We used Digital Leaf Physiognomy (DiLP) to measure leaf characters of four Vitis species from the USDA Germplasm Repository (Geneva, New York) from the 2012-2013 and 2014-2015 leaf-growing seasons, which had different environmental conditions. RESULTS:Leaf shape changed allometrically through developmental stages; early stages were more linear than later stages. There were significant differences in physiognomy in the same developmental stage between the growing seasons, and species had significant differences in mean physiognomy between growing seasons. Phenotypic plasticity was defined as changes between growing seasons after controlling for developmental stage or after averaging all developmental stages. Vitis amurensis and V. riparia had the greatest phenotypic plasticity. North American species exhibited significant differences in tooth area:blade area. Intermediate developmental stages were most likely to exhibit phenotypic plasticity, and only V. amurensis exhibited phenotypic plasticity in later developmental stages. CONCLUSIONS:Leaves have variable phenotypic plasticity along the vine. Environmental signal was strongest in intermediate developmental stages. This is significant for leaf physiognomic-paleoclimate proxies because these leaves are likely the most common in leaf litter and reflect leaves primarily included in paleoclimate reconstructions. Early season and early developmental stages have the potential to be confounding factors but are unlikely to exert significant influence because of differential preservation potential.
Project description:Background:Leaf shape among Passiflora species is spectacularly diverse. Underlying this diversity in leaf shape are profound changes in the patterning of the primary vasculature and laminar outgrowth. Each of these aspects of leaf morphology-vasculature and blade-provides different insights into leaf patterning. Results:Here, we morphometrically analyze >3300 leaves from 40 different Passiflora species collected sequentially across the vine. Each leaf is measured in two different ways: using 1) 15 homologous Procrustes-adjusted landmarks of the vasculature, sinuses, and lobes; and 2) Elliptical Fourier Descriptors (EFDs), which quantify the outline of the leaf. The ability of landmarks, EFDs, and both datasets together are compared to determine their relative ability to predict species and node position within the vine. Pairwise correlation of x and y landmark coordinates and EFD harmonic coefficients reveals close associations between traits and insights into the relationship between vasculature and blade patterning. Conclusions:Landmarks, more reflective of the vasculature, and EFDs, more reflective of the blade contour, describe both similar and distinct features of leaf morphology. Landmarks and EFDs vary in ability to predict species identity and node position in the vine and exhibit a correlational structure (both within landmark or EFD traits and between the two data types) revealing constraints between vascular and blade patterning underlying natural variation in leaf morphology among Passiflora species.
Project description:The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Populations having high Ne adapt faster, as selection acts more intensely, than populations having low Ne, where random effects of genetic drift dominate. Estimating Ne for various steps of plant virus life cycle has been the focus of several studies in the last decade, but no estimates are available for the vertical transmission of plant viruses, although virus seed transmission is economically significant in at least 18% of plant viruses in at least one plant species. Here we study the co-dynamics of two variants of Pea seedborne mosaic virus (PSbMV) colonizing leaves of pea plants (Pisum sativum L.) during the whole flowering period, and their subsequent transmission to plant progeny through seeds. Whereas classical estimators of Ne could be used for leaf infection at the systemic level, as virus variants were equally competitive, dedicated stochastic models were needed to estimate Ne during vertical transmission. Very little genetic drift was observed during the infection of apical leaves, with Ne values ranging from 59 to 216. In contrast, a very drastic genetic drift was observed during vertical transmission, with an average number of infectious virus particles contributing to the infection of a seedling from an infected mother plant close to one. A simple model of vertical transmission, assuming a cumulative action of virus infectious particles and a virus density threshold required for vertical transmission to occur fitted the experimental data very satisfactorily. This study reveals that vertically-transmitted viruses endure bottlenecks as narrow as those imposed by horizontal transmission. These bottlenecks are likely to slow down virus adaptation and could decrease virus fitness and virulence.
Project description:To better understand the long-term impact of Ophiostoma novo-ulmi Brasier on leaf physiology in 'Dodoens', a Dutch elm disease-tolerant hybrid, measurements of leaf area, leaf dry mass, petiole anatomy, petiole hydraulic conductivity, leaf and branch water potential, and branch sap flow were performed 3 years following an initial artificial inoculation. Although fungal hyphae were detected in fully expanded leaves, neither anatomical nor morphological traits were affected, indicating that there was no impact from the fungal hyphae on the leaves during leaf expansion. In contrast, however, infected trees showed both a lower transpiration rate of branches and a lower sap flow density. The long-term persistence of fungal hyphae inside vessels decreased the xylem hydraulic conductivity, but stomatal regulation of transpiration appeared to be unaffected as the leaf water potential in both infected and non-infected trees was similarly driven by the transpirational demands. Regardless of the fungal infection, leaves with a higher leaf mass per area ratio tended to have a higher leaf area-specific conductivity. Smaller leaves had an increased number of conduits with smaller diameters and thicker cell walls. Such a pattern could increase tolerance towards hydraulic dysfunction. Measurements of water potential and theoretical xylem conductivity revealed that petiole anatomy could predict the maximal transpiration rate. Three years following fungal inoculation, phenotypic expressions for the majority of the examined traits revealed a constitutive nature for their possible role in Dutch elm disease tolerance of 'Dodoens' trees.