Functional Genomic Approaches for Deciphering Plant-Virus Interactions

Plant-virus interactions are very complex in nature and lead to disease and symptom formation by causing various physiological, metabolic and developmental changes in the host plants. These interactions are mainly the outcomes of viral hijacking of host components to complete their infection cycles and of host defensive responses to restrict the viral infections. Viral genomes contain only a small number of genes often encoding for multifunctional proteins, and all are essential in establishing a viral infection. Thus, it is important to understand the specific roles of individual viral genes and their contribution to the viral life cycles. Among the most important viral proteins are the suppressors of RNA silencing (VSRs). These proteins function to suppress host defenses mediated by RNA silencing and can also serve in other functions, e.g. in viral movement, transactivation of host genes, virus replication and protein processing. Thus these proteins are likely to have a significant impact on host physiology and metabolism. In the present study, I have examined the plant-virus interactions and the effects of three different VSRs on host physiology and gene expression levels by microarray analysis of transgenic plants that express these VSR genes. I also studied the gene expression changes related to the expression of the whole genome of Tobacco mosaic virus (TMV) in transgenic tobacco plants. Expression of the VSR genes in the transgenic tobacco plants causes significant changes in the gene expression profiles. HC-Pro gene derived from the Potyvirus Y (PVY) causes alteration of 748 and 332 transcripts, AC2 gene derived from the African cassava mosaic virus (ACMV) causes alteration of 1118 and 251transcripts, and P25 gene derived from the Potyvirus X (PVX) causes alterations of 1355 and 64 transcripts in leaves and flowers, respectively. All three VSRs cause similar up-regulation in defense, hormonally regulated and different stress-related genes and down-regulation in the photosynthesis and starch metabolism related genes. They also induce alterations that are specific to each viral VSR. The phenotype and transcriptome alterations of the HC-Pro expressing transgenic plants are similar to those observed in some Potyvirus-infected plants. The plants show increased protein degradation, which may be due to the HC-Pro cysteine endopeptidase and thioredoxin activities. The AC2-expressing transgenic plants show a similar phenotype and gene expression pattern as HC-Pro-expressing plants, but also alter pathways related to jasmonic acid, ethylene and retrograde signaling. In the P25 expressing transgenic plants, high numbers of genes (total of 1355) were up-regulated in the leaves, compared to a very low number of down-regulated genes (total of 5). Despite of strong induction of the transcripts, only mild growth reduction and no other distinct phenotype was observed in these plants. As an example of whole virus interactions with its host, I also studied gene expression changes caused by Tobacco mosaic virus (TMV) in tobacco host in three different conditions, i.e. in transgenic plants that are first resistant to the virus, and then become susceptible to it and in wild type plants naturally infected with this virus. The microarray analysis revealed up and down-regulation of 1362 and 1422 transcripts in the TMV resistant young transgenic plants, and up and down-regulation of a total of 1150 and 1200 transcripts, respectively, in the older plants, after the resistance break. Natural TMV infections in wild type plants caused up-regulation of 550 transcripts and down-regulation of 480 transcripts. 124 up-regulated and 29 down-regulated transcripts were commonly altered between young and old TMV transgenic plants, and only 6 up-regulated and none of the down-regulated transcripts were commonly altered in all three plants. During the resistant stage, the strong down-regulation in translation-related transcripts (total of 750 genes) was observed. Additionally, transcripts related to the hormones, protein degradation and defense pathways, cell division and stress were distinctly altered. All these alterations may contribute to the TMV resistance in the young transgenic plants, and the resistance may also be related to RNA silencing, despite of the low viral abundance and lack of viral siRNAs or TMV methylation activity in the plants.

Occurrence of Akodon cursor (Rodentia, Cricetidae) with 14, 15 and 16 chromosome cytotypes in the same geographic area in Southern Brazil

The karyotype of Akodon cursor (initially identified as A. arviculoides) had been reported with chromosomal numbers 14 and 15 in the South and Southeast and 16 in Northeastern Brazil. We found the three cytotypes in a region of Southern Brazil. The G-band patterns of these specimens were the same as those from southeastern and northeastern regions. Seventeen different combinations of chromosomes due to a complex rearrangement in pair 1 and pericentric inversions in pairs 2 and 3 were identified. Seven of these combinations are new to in the literature.

Investigation of cytotoxic and mutagenic effects of Malpighia glabra L. (barbados cherry) fruit pulp and vitamin C on plant and animal test systems

Fruits are important sources of nutrients in human diet, and Barbados Cherry (Malpighia glabra L.) is of particular interest due to its high content of antioxidants. Diets rich in fruits and vegetables protect individuals against diseases and cancer, but excessive intake of vitamins may act as pro-oxidant and generate changes in DNA. To evaluate the effect of different in natura (BAN) and frozen (BAF) Barbados Cherry pulp concentrations and synthetic vitamin C in liquid form (VC) on the chromosome level and the cell cycle division, root meristeme cells of Allium cepa L. and bone marrow cells of Wistar rats Rattus norvegicus, were used as test system. In Allium cepa L., BAN, at the highest concentration (0.4 mg.mL-1) and BAF, at the lowest concentration (0.2 mg.mL-1), inhibited cell division, and there was recovery of cell division after the recovery period in water only for BAN. In the Wistar rats, all treatments with Barbados Cherry, either acute or subchronic, were not cytotoxic or mutagenic; only the highest concentration of VC increased significantly the rate of chromosomal abnormalities. The data obtained are important to reinforce the use of Barbados Cherry fruit in the diet.

Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments (similar to 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.

Management of plant communities on set-aside land and its effects on earthworm communities

Plant communities of set-aside agricultural land in a European project were managed in order to enhance plant succession towards weed-resistant, mid-successional grassland. Here, we ask if the management of a plant community affects the earthworm community. Field experiments were established in four countries, the Netherlands, Sweden, the UK, and the Czech Republic. High (15 plant species) and low diversity (four plant species) seed mixtures were sown as management practice, with natural colonization as control treatment in a randomized block design. The response of the earthworrns to the management was studied after three summers since establishment of the sites. Samples were also taken from plots with continued agricultural practices included in the experimental design and from a site with a late successional plant community representing the target plant community. The numbers and biomass of individuals were higher in the set-aside plots than in the agricultural treatment in two countries out of four. The numbers of individuals at one site (The Netherlands) was higher in the naturally colonized plots than in the sowing treatments, otherwise there were no differences between the treatments. Species diversity was lower in the agricultural plots in one country. The species composition had changed from the initial community of the agricultural field, but was still different from a late successional target community. The worm biomass was positively related to legume biomass in Sweden and to grass biomass in the UK. (C) 2005 Elsevier SAS. All rights reserved.

The effects of cattle grazing on plant-pollinator communities in a fragmented Mediterranean landscape

The main aims of this study were to assess grazing impacts on bee communities in fragmented mediterranean shrubland (phrygana) and woodland habitats that also experience frequent wildfires, and to explain the mechanisms by which these impacts occur. Fieldwork was carried out in 1999 and 2000 on Mount Carmel, in northern Israel, a known hot-spot for bee diversity. Habitats with a range of post-burn ages and varying intensities of cattle grazing were surveyed by transect recording, grazing levels, and the diversity and abundance of both flowers and bees were measured. The species richness of both bees and flowers were highest at moderate to high grazing intensities, and path-analysis indicated that the effects of both grazing and fire on bee diversity were mediated mainly through changes in flower diversity, herb flowers being more important than shrubs. The abundance of bees increased with intensified grazing pressure even at the highest levels surveyed. Surprisingly though, changes in bee abundance at high grazing levels were not caused directly by changes in flower cover. The variation in bee abundance may have been due to higher numbers of solitary bees from the family Halictidae in grazed sites, where compacted ground (nesting resource) and composites (forage resource) were abundant. The effects of grazing on plants were clearest in the intermediate-aged sites, where cattle inhibited the growth of some of the dominant shrubs, creating or maintaining more open patches where light-demanding herbs could grow, thus allowing a diverse flora to develop. Overall, bee communities benefit from a relatively high level of grazing in phrygana. Although bee and flower diversity may decrease under very heavy grazing, the present levels of grazing on Mount Carmel appear to have only beneficial effects on the bee community.

The use of bioluminescence for monitoring in planta growth dynamics of a Pseudomonas syringae plant pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

Rooting cuttings of Syringa vulgaris cv. Charles Joly and Corylus avellana cv. Aurea: the influence of stock plant pruning and shoot growth

The relationship between shoot growth and rooting was examined in two, 'difficult-to root' amenity trees, Syringa vulgaris L. cv. Charles Joly and Corylus avellana L. cv. Aurea. A range of treatments reflecting severity of pruning was imposed on field-grown stock prior to bud break. To minimise variation due to the numbers of buds that developed under different treatments, bud number was restricted to 30 per plant. Leafy cuttings were harvested at different stages of the active growth phase of each species. With Syringa, rooting decreased with later harvests, but loss of rooting potential was delayed in cuttings collected from the most severe pruning treatment. Rooting potential was associated with the extent of post-excision shoot growth on the cutting but regression analyses indicated that this relationship could not entirely explain the loss of rooting with time, nor the effects due to pruning. Similarly, in Corylus rooting was promoted by severe pruning, but the relationship between apical growth on the cutting and rooting was weaker than in Syringa, and only at the last harvest did growth play a critical role in determining rooting. Another unusual factor of the last harvest of Corylus was a bimodal distribution of roots per cutting, with very few rooted cuttings having less than five roots. This implies that, for this harvest at least, the potential of an individual cutting to root is probably not limited by the number of potential rooting sites.

Quantitative plant proteomics

Quantitation is an inherent requirement in comparative proteomics and there is no exception to this for plant proteomics. Quantitative proteomics has high demands on the experimental workflow, requiring a thorough design and often a complex multi-step structure. It has to include sufficient numbers of biological and technical replicates and methods that are able to facilitate a quantitative signal read-out. Quantitative plant proteomics in particular poses many additional challenges but because of the nature of plants it also offers some potential advantages. In general, analysis of plants has been less prominent in proteomics. Low protein concentration, difficulties in protein extraction, genome multiploidy, high Rubisco abundance in green tissue, and an absence of well-annotated and completed genome sequences are some of the main challenges in plant proteomics. However, the latter is now changing with several genomes emerging for model plants and crops such as potato, tomato, soybean, rice, maize and barley. This review discusses the current status in quantitative plant proteomics (MS-based and non-MS-based) and its challenges and potentials. Both relative and absolute quantitation methods in plant proteomics from DIGE to MS-based analysis after isotope labeling and label-free quantitation are described and illustrated by published studies. In particular, we describe plant-specific quantitative methods such as metabolic labeling methods that can take full advantage of plant metabolism and culture practices, and discuss other potential advantages and challenges that may arise from the unique properties of plants.

Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.661025 and 1.161026, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

A method for under-sampled ecological network data analysis: plant-pollination as case study

In this paper, we develop a method, termed the Interaction Distribution (ID) method, for analysis of quantitative ecological network data. In many cases, quantitative network data sets are under-sampled, i.e. many interactions are poorly sampled or remain unobserved. Hence, the output of statistical analyses may fail to differentiate between patterns that are statistical artefacts and those which are real characteristics of ecological networks. The ID method can support assessment and inference of under-sampled ecological network data. In the current paper, we illustrate and discuss the ID method based on the properties of plant-animal pollination data sets of flower visitation frequencies. However, the ID method may be applied to other types of ecological networks. The method can supplement existing network analyses based on two definitions of the underlying probabilities for each combination of pollinator and plant species: (1), pi,j: the probability for a visit made by the i’th pollinator species to take place on the j’th plant species; (2), qi,j: the probability for a visit received by the j’th plant species to be made by the i’th pollinator. The method applies the Dirichlet distribution to estimate these two probabilities, based on a given empirical data set. The estimated mean values for pi,j and qi,j reflect the relative differences between recorded numbers of visits for different pollinator and plant species, and the estimated uncertainty of pi,j and qi,j decreases with higher numbers of recorded visits.

Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum

The genome structure of Colletotrichum lindemuthianum in a set of diverse isolates was investigated using a combination of physical and molecular approaches. Flow cytometric measurement of genome size revealed significant variation between strains, with the smallest genome representing 59% of the largest. Southern-blot profiles of a cloned fungal telomere revealed a total chromosome number varying from 9 to 12. Chromosome separations using pulsed-field gel electrophoresis (PFGE) showed that these chromosomes belong to two distinct size classes: a variable number of small (< 2.5 Mb) polymorphic chromosomes and a set of unresolved chromosomes larger than 7 Mb. Two dispersed repeat elements were shown to cluster on distinct polymorphic minichromosomes. Single-copy flanking sequences from these repeat-containing clones specifically marked distinct small chromosomes. These markers were absent in some strains, indicating that part of the observed variability in genome organization may be explained by the presence or absence, in a given strain, of dispensable genomic regions and/or chromosomes.

Arboreal thorn-dwelling ants use domatia morphology to partition nest sites on the savanna ant-plant, Vachellia erioloba

Nest site selection in arboreal, domatia-dwelling ants, particularly those coexisting on a single host plant, is little understood. To examine this phenomenon we studied the African savannah tree Vachellia erioloba, which hosts ants in swollen-thorn domatia. We found four ant species from different genera (Cataulacus intrudens, Tapinoma subtile, Tetraponera ambigua and an unidentified Crematogaster species). In contrast to other African ant plants, many V. erioloba trees (41 % in our survey) were simultaneously co-occupied by more than one ant species. Our study provides quantitative field data describing: (1) aspects of tree and domatia morphology relevant to supporting a community of mutualist ants, (2) how ant species occupancy varies with domatia morphology and (3) how ant colony size varies with domatia size and species. We found that Crematogaster sp. occupy the largest thorns, followed by C. intrudens, with T. subtile in the smallest thorns. Thorn age, as well as nest entrance hole size correlated closely with ant species occupant. These differing occupancy patterns may help to explain the unusual coexistence of three ant species on individual myrmecophytic trees. In all three common ant species, colony size, as measured by total number of ants, increased with domatia size. Additionally, domatia volume and species identity interact to predict ant numbers, suggesting differing responses between species to increased availability of nesting space. The proportion of total ants in nests that were immatures varied with thorn volume and species, highlighting the importance of domatia morphology in influencing colony structure.

Impacts of plant-based foods in ancestral hominin diets on the metabolism and function of gut microbiota in vitro

Ancestral human populations had diets containing more indigestible plant material than present-day diets in industrialized countries. One hypothesis for the rise in prevalence of obesity is that physiological mechanisms for controlling appetite evolved to match a diet with plant fiber content higher than that of present-day diets. We investigated how diet affects gut microbiota and colon cells by comparing human microbial communities with those from a primate that has an extreme plant-based diet, namely, the gelada baboon, which is a grazer. The effects of potato (high starch) versus grass (high lignin and cellulose) diets on human-derived versus gelada-derived fecal communities were compared in vitro. We especially focused on the production of short-chain fatty acids, which are hypothesized to be key metabolites influencing appetite regulation pathways. The results confirmed that diet has a major effect on bacterial numbers, short-chain fatty acid production, and the release of hormones involved in appetite suppression. The potato diet yielded greater production of short-chain fatty acids and hormone release than the grass diet, even in the gelada cultures, which we had expected should be better adapted to the grass diet. The strong effects of diet on hormone release could not be explained, however, solely by short-chain fatty acid concentrations. Nuclear magnetic resonance spectroscopy found changes in additional metabolites, including betaine and isoleucine, that might play key roles in inhibiting and stimulating appetite suppression pathways. Our study results indicate that a broader array of metabolites might be involved in triggering gut hormone release in humans than previously thought. IMPORTANCE: One theory for rising levels of obesity in western populations is that the body's mechanisms for controlling appetite evolved to match ancestral diets with more low-energy plant foods. We investigated this idea by comparing the effects of diet on appetite suppression pathways via the use of gut bacterial communities from humans and gelada baboons, which are modern-day primates with an extreme diet of low-energy plant food, namely, grass. We found that diet does play a major role in affecting gut bacteria and the production of a hormone that suppresses appetite but not in the direction predicted by the ancestral diet hypothesis. Also, bacterial products were correlated with hormone release that were different from those normally thought to play this role. By comparing microbiota and diets outside the natural range for modern humans, we found a relationship between diet and appetite pathways that was more complex than previously hypothesized on the basis of more-controlled studies of the effects of single compounds.

Parasitoids select plants more heavily infested with their caterpillar hosts: a new approach to aid interpretation of plant headspace volatiles

Plants produce volatile organic compounds (VOCs) in response to herbivore attack, and these VOCs can be used by parasitoids of the herbivore as host location cues. We investigated the behavioural responses of the parasitoid Cotesia vestalis to VOCs from a plant–herbivore complex consisting of cabbage plants (Brassica oleracea) and the parasitoids host caterpillar, Plutella xylostella. A Y-tube olfactometer was used to compare the parasitoids' responses to VOCs produced as a result of different levels of attack by the caterpillar and equivalent levels of mechanical damage. Headspace VOC production by these plant treatments was examined using gas chromatography–mass spectrometry. Cotesia vestalis were able to exploit quantitative and qualitative differences in volatile emissions, from the plant–herbivore complex, produced as a result of different numbers of herbivores feeding. Cotesia vestalis showed a preference for plants with more herbivores and herbivore damage, but did not distinguish between different levels of mechanical damage. Volatile profiles of plants with different levels of herbivores/herbivore damage could also be separated by canonical discriminant analyses. Analyses revealed a number of compounds whose emission increased significantly with herbivore load, and these VOCs may be particularly good indicators of herbivore number, as the parasitoid processes cues from its external environment