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.

Pathogen derived resistance to Potato virus Y: mechanisms and risks

Selostus: Patogeenivälitteinen, siirtogeeninen kestävyys perunan Y-virusta vastaan: mekanismit ja riskit

Real-time Imaging and Mosaicking of Planar Surfaces

Laajojen pintojen kuvaaminen rajoitetussa työskentelytilassa riittävällä kuvatarkkuudella voi olla vaikeaa. Kuvaaminen on suoritettava osissa ja osat koottava saumattomaksi kokonaisnäkymäksi eli mosaiikkikuvaksi. Kuvauslaitetta käsin siirtelevän käyttäjän on saatava välitöntä palautetta, jotta mosaiikkiin ei jäisi aukkoja ja työ olisi nopeaa. Työn tarkoituksena oli rakentaa pieni, kannettava ja tarkka kuvauslaite paperi- ja painoteollisuuden tarpeisiin sekä kehittää palautteen antamiseen menetelmä, joka koostaaja esittää karkeaa mosaiikkikuvaa tosiajassa. Työssä rakennettiin kaksi kuvauslaitetta: ensimmäinen kuluttajille ja toinen teollisuuteen tarkoitetuista osista. Kuvamateriaali käsiteltiin tavallisella pöytätietokoneella. Videokuvien välinen liike laskettiin yksinkertaisella seurantamenetelmällä ja mosaiikkikuvaa koottiin kameroiden kuvanopeudella. Laskennallista valaistuksenkorjausta tutkittiin ja kehitetty menetelmä otettiin käyttöön. Ensimmäisessä kuvauslaitteessa on ongelmia valaistuksen ja linssivääristymien kanssa tuottaen huonolaatuisia mosaiikkikuvia. Toisessa kuvauslaitteessa nämä ongelmat on korjattu. Seurantamenetelmä toimii hyvin ottaen huomioon sen yksinkertaisuuden ja siihen ehdotetaan monia parannuksia. Työn tulokset osoittavat, että tosiaikainen mosaiikkikuvan koostaminen megapikselin kuvamateriaalista on mahdollista kuluttajille tarkoitetulla tietokonelaitteistolla.

Spatiotemporal Features of Coastal Waters in Southwest Finland

In coastal waters, physico-chemical and biological properties and constituents vary at different time scales. In the study area of this thesis, within the Archipelago Sea in the northern Baltic Sea, seasonal cycles of light and temperature set preconditions for intra-annual variations, but developments at other temporal scales occur as well. Weather-induced runoffs and currents may alter water properties over the short term, and the consequences over time of eutrophication and global changes are to a degree unpredictable. The dynamic characteristics of northern Baltic Sea waters are further diversified at the archipelago coasts. Water properties may differ in adjacent basins, which are separated by island and underwater thresholds limiting water exchange, making the area not only a mosaic of islands but also one of water masses. Long-term monitoring and in situ observations provide an essential data reserve for coastal management and research. Since the seasonal amplitudes of water properties are so high, inter-annual comparisons of water-quality variables have to be based on observations sampled at the same time each year. In this thesis I compare areas by their temporal characteristics, using both inter-annual and seasonal data. After comparing spatial differences in seasonal cycles, I conclude that spatial comparisons and temporal generalizations have to be made with caution. In classifying areas by the state of their waters, the results may be biased even if the sampling is annually simultaneous, since the dynamics of water properties may vary according to the area. The most comprehensive view of the spatiotemporal dynamics of water properties would be achieved by means of comparisons with data consisting of multiple annual samples. For practical reasons, this cannot be achieved with conventional in situ sampling. A holistic understanding of the spatiotemporal features of the water properties of the Archipelago Sea will have to be based on the application of multiple methods, complementing each other’s spatial and temporal coverage. The integration of multi-source observational data and time-series analysis may be methodologically challenging, but it will yield new information as to the spatiotemporal regime of the Archipelago Sea.

Grass-endophyte coevolution and ploidy levels in fescues

ABSTRACT Fescues consist of wild and cultivated grasses that have adapted to a wide range of environmental conditions. They are an excellent model species for evolutionary ecology studies that investigate symbiosis and polyploidization and their effects on plant performance. First, they are frequently infected with symbiotic endophytic fungi known to affect a plant’s ability to cope with biotic and abiotic environmental factors. Second, fescue species have been reported to have substantial intraspecific variation in their ploidy level and morphology. In my thesis, I examined large-scale generalizations for frequency of polyploidy and endophyte infections and their effects on plant morphology. As a model species, I selected red (Festuca rubra) and viviparous sheep’s (F. vivipara) fescues. They are closely related, but they differ in terms of distribution and endophyte infection frequency. I investigated the biogeographic pattern and population biology of 29 red and 12 viviparous sheep’s fescue populations across ≈300 latitudes in Europe (400-690 N). To examine plant ploidy levels, I implemented time- and cost-efficient plate-based high throughput flow cytometric analysis. This efficient procedure enabled me to analyze over 1000 red fescue individuals. I found three ploidy levels among them: overall 84 %, 9 % and 7 % of the red fescue plants were hexaploid, tetraploid and octoploid, respectively. However, all viviparous sheep’s fescue plants were tetraploid. Ploidy level of red fescue appeared to some extent follow gradients in latitude and primary production as suggested by previous studies, but these results could be explained better by taking the sampling design and local adaptation into account. Three Spanish populations were mostly tetraploids and one high elevation population in northernmost Finland (Halti) was octoploid, while most other populations (25 sites) were dominated by hexaploids. Endophyte infection frequencies of wild fescue populations varied from 0 to 81 % in red fescue populations and from 0 to 30 % in viviparous sheep’s fescue populations. No gradients with latitude or primary production of the sites were detected. As taxonomy of red fescues is somewhat unclear, I also studied morphology, ploidy variation and endophyte status of proposed subspecies of European red fescues. Contrary to previous literature, different ploidy levels occurred in the same subspecies. In addition to wild fescues, I also used two agronomically important cultivars of meadow and tall fescue (Schedonorus phoenix and S. pratensis). As grass-legume mixtures have an agronomic advantage over monocultures in meadows, I carried out a mixture/competition experiment with fescues and red clover to find that species composition, nutrient availability and endophyte status together determined the total biomass yield that was higher in mixtures compared to monocultures. The results of this thesis demonstrate the importance of local biotic and abiotic factors such as grazing gradients and habitat types, rather than suggested general global geographical or environmental factors on grass polyploidization or its association with symbiotic endophytic fungi. I conclude that variation in endophyte infection frequencies and ploidy levels of wild fescues support the geographic mosaic theory of coevolution. Historical incidents, e.g., glaciation and present local factors, rather than ploidy or endophyte status, determine fescue morphology.