Maatalous- metsätieteellinen tiedekunta
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(Helsingin yliopisto, 2019)My doctoral thesis examines the prerequisites of replication for three positive-strand RNA viruses, Chikungunya virus (CHIKV - alphavirus), Semliki Forest virus (SFV - alphavirus) and Flock House virus (FHV - nodavirus). Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes high fever, rashes and joint pain. Semliki Forest virus (SFV) has been extensively studied as a model to comprehend the replication strategies of alphaviruses because of its low pathogenicity. A characteristic feature of alphavirus replication is the formation of membranous invaginations termed spherules, associated with the plasma membrane. Spherules act as genome factories as they are the sites of active viral replication and release nascent viral RNA strands into the cytoplasm through a bottleneck-like structure. We created a trans-replication system specific for CHIKV that would be flexible and presents no danger to the scientist. In this system, the viral replicase proteins are expressed from a DNA plasmid while the RNA template is produced from a second plasmid, in mammalian cells. This allowed for the study of viral replication without generating infectious particles. It also enabled the visualisation of spherules and labelling of all viral replicase proteins with fluorescent or small immunological tags while preserving their function. Various mutations associated with noncytotoxic phenotypes were analysed and the results showed no correlation between the level of RNA replication and cytotoxicity. Moreover, the trans-replication system was used to elucidate that the cysteine residue of CHIKV nsP2 at position 478 is responsible for its protease activity and essential for replicase polyprotein processing. Trp479 of nsP2 also plays a vital role in RNA replication. The insect nodavirus, FHV, verges upon the properties of a ‘universal virus’ as it can replicate in a wide range of hosts. Only the replicase protein A is required for its replication. An efficient FHV trans-replication system was established in mammalian cells. The outer surface of mitochondria displayed pouch-like invaginations with a ‘neck’ structure opening towards the cytoplasm. High-level synthesis of both genomic and subgenomic RNA was detected in vitro using mitochondrial pellets isolated from transfected cells. The newly synthesized RNA was found to be of positive polarity. This system was used to investigate the capping enzyme domain of protein A, both in cells and in vitro. Mutating the most conserved amino acids of the capping domain abolished or reduced viral RNA synthesis. Surprisingly, transfection of capped RNA template did not rescue the replication activity of the mutants. FHV and alphaviruses show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. The biological systems presented in this study offer valuable knowledge that could be exploited to understand the replication of other RNA viruses and also open up new avenues for the elucidation of key virus-host interactions.
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(Helsingin yliopisto, 2019)The World’s forests are facing novel stress due to climate change. Pest insects and pathogens are shifting towards new latitudes and heat stress is resulting in increased tree mortality and more frequent forest fires globally. Uncertainty in estimating the magnitude of climate change induced forest and tree decline requires new methods for unbiased estimation of tree decline. The development of remote sensing methods to detect early tree decline has been a major challenge due to the subtle nature of the early changes caused by different stressors. Multispectral lidar technology has the potential of detecting early tree decline by providing accurate three-dimensional and spectral information of tree structure simultaneously. The main objective of this thesis was to investigate the capabilities of multispectral terrestrial lidar in the detection and assessment of tree decline caused by different stressors. This was done by investigating the estimation of a remotely detectable indicator of tree decline, leaf water content (LWC). Specifically, new methods for measuring LWC using multispectral lidar intensity were developed from the leaf to the canopy scale in various environments and the relationship between LWC and tree decline induced by various stressors was investigated. Furthermore, the developed methods were tested in a forest environment to assess the applicability of multispectral lidar in the detection of bark beetle infestation in the field. Studies I-III focused on investigating the relationship between LWC and lidar intensity at multiple wavelengths. First, a hyperspectral lidar instrument was used to detect significant changes between fresh and drought-treated Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L.) trees (study I). Then, a leaf-scale study (II) with Scots pine, Norway spruce, Small-leaved lime (Tilia cordata L.), Norway maple (Acer platanoides L.) and Silver birch (Betula pendula L.) was conducted and a strong relationship (R2=0.93) between a normalized difference index (NDI) calculated from 1550 nm and 690 nm wavelengths and LWC was found. This was followed by a study (III) where LWC estimation and pathogen- and drought-induced variation in LWC was studied with Norway spruce seedlings. Blue-stain fungi (Endoconidiophora polonica) inoculated seedlings expressed a rapid decrease in LWC while drought-treated seedlings showed more stable LWC until a very severe drought. LWC of the seedlings was predicted with an R2 of 0.89 using an NDI with 1550 nm and 905 nm wavelengths. In study IV, the developed method and the relationship between LWC and tree decline was investigated in the field with European spruce bark beetle (Ips typographus L.) infested trees. It was found that of the LWC metrics studied, gravimetric water content showed significant differences in the early stages of infestation and was more sensitive to bark beetle induced tree decline than equivalent water thickness (i.e. amount of water per leaf area). Linear discriminant models that were developed between infestation severity and lidar intensity metrics from 1550 nm and 905 nm wavelengths showed that green attack stage of the infestation could classified with an overall accuracy of 90%. This dissertation contributes both to the development of an objective and automatable method for detecting and measuring tree decline in the field, and to the understanding of the relationship between LWC and tree decline with implications to remote sensing. The dissertation will be published and popularized as a music video here: http://bit.ly/idanproffa. .
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(Helsingin yliopisto, 2019)Legumes are a sustainable source of plant protein, and their production could be increased in Europe. The use of faba bean (Vicia faba L.) is limited in part due to the presence of the pyrimidine glycosides vicine and convicine. Vicine and convicine, and particularly their aglycones, can cause a form of haemolytic anaemia called favism in individuals who have genetic deficiency in the glucose-6-phosphate dehydrogenase (G6PD) enzyme. Different processing methods have reduced the vicine and convicine contents to varying levels, but the formation of the aglycones have not been studied. Practical processing methods for removal of vicine, convicine and their aglycones are still required. The main aim of this study was to implement methods for the total elimination of vicine and convicine, with a focus on the aglycones. The compounds of interest were quantified using reversed phase high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detection against an internal standard uridine. The contents of vicine and convicine were determined in cultivars grown in Finland, and the contents were compared within a selected growing year and among three growing years. Vicine and convicine were hydrolysed in faba bean extracts, in vicine and convicine fractions and in faba bean suspensions by using β-glucosidase to study the formation and stability of the aglycones. Finally, the formation and stability reactions were studied under selected model conditions and in sourdoughs and breads. The performance of the HPLC method was suitable for the analysis of vicine and convicine. The levels in the studied cultivars varied from 5.2–7.6 mg/g dry matter (DM) and 2.1–3.6 mg/g DM within one growing year for vicine and convicine, respectively. Cultivar comparison showed that the cultivar ‘Kontu’, the commonly grown cultivar in Finland, contained high amounts of vicine and convicine. No extensive variation was noted among three studied growing years, even though the weather conditions varied markedly. The aglycones were detected and monitored with the HPLC method used for vicine and convicine analysis. The aglycones formed in the vicine and convicine fractions decreased in amount and finally lost their UV absorptivity after 2 h at pH 5 at 37 °C. The need for an external enzyme source was confirmed, as losses of vicine and convicine were rather small in faba bean suspensions. Selected lactic acid bacteria (LAB) strains were able to hydrolyse vicine and convicine in faba bean sourdoughs at 25 °C, 24 h; the hydrolysis depended on the fermentation temperature. Sourdoughs lost up to 82–85% of the vicine and up to 34–47% of the convicine. The amounts of vicine and convicine in wheat breads, containing 30% faba bean, were comparable to the amounts that were in sourdoughs after 24 h of fermentation. The aglycones were measured from sourdoughs fermented at 25 °C and from the corresponding doughs, but not from the breads. This study showed that vicine and convicine can be analysed simultaneously with their aglycones with an RP-HPLC-UV method, which provides the benefit of estimation of the total elimination of these compounds. The aglycones were found to disappear in all the studied matrices. Fermentation can induce losses of vicine and convicine, but the efficiency of hydrolysis depends on the selection of strains and the fermentation conditions. Furthermore, controlled acidification is necessary for maintain acceptable sensory quality.
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(Helsingin yliopisto, 2019)Vitamin D is crucial for the human body and it has a significant role in bone growth and for maintenance of the skeleton throughout life. It is metabolized in the liver to the main circulating form, 25-hydroxyvitamin D (25(OH)D). Vitamin D binding protein (DBP) transports vitamin D metabolites into target tissues. A small amount of vitamin D is also free in the circulation. These free metabolites are suggested to be more active because they can enter the cells without a receptor. Genetic variation of DBP coding gene affects the protein’s ability to bind 25(OH)D, thereby influencing bound and free 25(OH)D concentrations. Furthermore, vitamin D concentrations have been reported to be lower in obese individuals than in their normal-weight peers. This study investigated the association of DBP genetic variation on vitamin D metabolism and bone traits. Study also aimed to examine whether obese individuals differ in their vitamin D metabolism from the normal-weight individuals and do these potential differences affect bone. These studies consisted of 233 Finnish children and adolescents (7–19 years) (Study I) and of 622 Finnish women and men (37–47 years) (Studies II and III). In Study III, the adults were stratified by body mass index into normal-weight, overweight, and obese. Intake of vitamin D and calcium, medications and physical activity were collected. 25(OH)D, parathyroid hormone (PTH), albumin, and bone turnover markers as well as single nucleotide polymorphisms (SNPs) rs4588 and 7041 in the GC gene (Study I and II) were determined. In addition, free 25(OH)D concentrations were calculated and in Study II adjusted with genotype specific affinity values. Bone traits were measured with Dual energy X-ray-absorptiometry and peripheral quantitative computed tomography. In Study I, 25(OH)D and PTH were lowest in genotype GC2/2. In boys, genotype was negatively associated with hip bone mineral content (BMC) and lumbar spine bone mineral density (BMD). In both girls and boys, genotype was associated with lumbar spine BMC and bone strength. In Study II, variation were found among genotypes in DBP, total, free, and bioavailable 25(OH)D, and PTH concentrations. DBP concentration was lowest in genotype GC2/2. Serum 25(OH)D concentrations were lowest in GC diplotypes 1S/2, 1S/1F, and 2/2, but adjusted free and bioavailable 25(OH)D concentrations were highest in GC2/2. PTH was also low in GC2/2. In Study III, free and bioavailable 25(OH)D concentrations were lower in obese women than in normal-weight. In men, both bound and free 25(OH)D were lower in obese than in those of normal-weight. In addition, DBP and PTH were higher in obese women and in both genders combined than in their normal weight peers. In obese women, 25(OH)D was negatively associated with distal radius trabecular density and tibial shaft cortical strength index (CSI). Furthermore, free and bioavailable 25(OH)D were negative determinants of distal radius CSI, radial shaft cortical density, and tibial shaft CSI in obese women. In conclusion, genetic variation in DBP is associated with bound and unbound forms of 25(OH)D, DBP and PTH concentrations and genetic variation of DBP can be one factor affecting bone mass accrual in adolescence. Associations of BMI with total, free and bioavailable 25(OH)D, DBP, and PTH concentrations demonstrate a difference in vitamin D metabolism between obese and normal-weight individuals. Furthermore, altered vitamin D metabolism in the obese may influence bone.
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(Helsingin yliopisto, 2019)Human land use affects the climate through various channels. This thesis focuses on the optimal (i.e. welfare-maximizing) regulation of land use sector climate impacts using market-based instruments, such as taxes and subsidies. The thesis consists of four articles and a summary chapter. Each article focuses on a separate aspect of land use sector climate policy. The first article outlines a comprehensive tax policy for jointly regulating carbon storage in biomass, soils and products. Considerations regarding soil carbon storage are emphasized. The second article concerns the regulation of CO2 emissions from the energy use of logging residues. The harmfulness of these emissions is compared with that of fossil emissions. A way to harmonize the carbon taxation of the both energy sources is presented. The third article regards the application of the additionality principle to forest carbon subsidies. In the stand-level context it appears that the additionality principle can be implemented without distorting the optimal rotation, by reclaiming subsidies for baseline carbon storage by a site productivity tax on forests. However, at the market-level such a tax distorts the optimal rotation and the optimal land allocation. These distortions can be avoided, if the excess subsidies are eliminated by general land taxation (which also targets other land use). The fourth article presents a new concept: the Social Cost of Forcing (SCF), which is the social cost of the marginal unit of radiative forcing at a given moment. It is a fundamental price that can be used to value different forcing agents. Forcing agents’ prices that are based on the SCF are consistent with the Social Cost of Carbon, and can therefore be consistently applied in cost-benefit analysis or utilized to harmonize the regulation of non-CO2 forcing agents. Together the four articles contribute to our understanding of land use sector climate policy design.
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(Helsingin yliopisto, 2019)Streptococcus pneumoniae is one of the most significant human bacterial pathogens. It is a major causative agent of community-acquired pneumonia. It is also the most common cause of bacterial otitis media and among most common bacteria causing meningitis in children. The close relatives of S. pneumoniae, the Viridans group streptococci form a central part of the human oral microbiome, but they are also a leading cause of endocarditis. The fast and reliable identification of these bacteria from clinical specimens would therefore be of prime importance for a clinical microbiology laboratory, allowing for the laboratory to provide helpful information to clinicians for identifying the infection focus and targeting antibiotic treatment. The close relationship between S. pneumoniae and Viridans group streptococci, especially their subgroup Mitis group streptococci, complicates the reliable species level identification of these bacteria. The identification of streptococci by the traditional biochemical methods is both unreliable and time-consuming: the identification takes 1-2 days after bacterial growth has been detected on a plate or in a blood culture bottle. Even sequencing of the 16S ribosomal RNA gene commonly used as a reference method for bacterial identification cannot reliably differentiate between pneumococci and other Mitis group streptococci or identify other Mitis group streptococci to the species level. For the identification of pneumococci, detection of pneumococcal-specific genes is considered a “golden standard”. For the Viridans group streptococci, multi-locus sequence typing (MLST), based on the analysis of the sequences of several genes, is considered to be the most reliable method of identification. However, the MLST is too laborious and time-consuming for the identification of streptococci in a routine clinical microbiology laboratory. Within the last decade, MALDI-TOF mass spectrometry has enabled the fast and reliable identification of most bacterial species. However, exceptionally closely related bacterial groups pose a challenge even to the MALDI-TOF technology. This holds true even for pneumococci and Mitis group streptococci. In this study, the earlier database and algorithm versions of the commonly used MALDI-TOF systems were shown to be unable to reliably differentiate between pneumococci and Mitis group streptococci. Therefore, rapid molecular detection tests, such as GenomEra S. pneumoniae™ evaluated in this study, are still needed for rapid detection of pneumococci. However, it seems that the addition of more pneumococcal and Mitis group strains in the databases and new interpretation algorithms allow for reliable differentiation between pneumococci and the Mitis group streptococci. Altogether, the combination of rapid gene detection tests and MALDI-TOF technology with enhanced databases and identification algorithms enables fast, reliable and cost-effective differentiation between pneumococci and Mitis group streptococci and the reliable group level identification of other Mitis group streptococci. This is a sufficient level of identification in a clinical microbiology laboratory, enabling the clinician to evaluate the clinical significance of the finding and target further investigations and antibiotic treatment more specifically.
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(Helsingin yliopisto, 2019)The fungal species complex Heterobasidion annosum sensu lato (s.l.) is considered one of the most devastating conifer pathogens in the boreal forest region. They affect European coniferous forests with root and butt rot, causing annual economic losses of €800 million. Despite several efforts in practical forestry to control the disease, the economic loss remains considerable. Therefore, it is still necessary to introduce alternate control measures for Heterobasidion infection. Heterobasidion spp. are infected by a diverse community of mycoviruses, mostly partitiviruses. Here, these viruses were studied to find potential viruses for biocontrol purposes. We described six novel Heterobasidion partitivirus (HetPV) species phylogenetically related to Helicobasidium mompa partitivirus V70 that infect four pathogenic Heterobasidion species. Interestingly, our study revealed that HetPV13-an1 causes severe phenotypic debilitation in its native and exotic fungal host. The RNA sequencing of isogenic virus infected and cured fungal strains showed that HetPV13-an1 affected the transcription of 683 genes. The RT-qPCR analysis showed that the response toward HetPV13-an1 infection varied between H. annosum and H. parviporum. Moreover, the wood colonization efficacy of H. parviporum infected by HetPV13-an1 was restricted in living Norway spruce trees. The ratio of polymerase and coat protein genome segments/transcripts of eight partitiviruses analysed was highly variable in mycelia. All the virus species had unique ratios of the genome segments, which were stable over different temperatures and hosts. The co-infection with HetPV13-an1 and HetPV15-pa1 reduced host growth up to 95%. Regarding the transmission efficacy of mycoviruses, HetPV15-pa1 transmission to a pre-infected host was elevated from zero to 50% by the presence of HetPV13-an1, and a double infection of these viruses in the donor resulted in an overall transmission rate of 90%. Altogether, the study demonstrated that the interplay between co-infecting viruses and their host is highly complex and that partitiviruses show potential for biocontrol. Keywords: Heterobasidion spp., wood decay, mycovirus, partitivirus, phylogenetics, RNA transcripts, transmission, growth rate.
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EFFECTS OF MICROCLIMATIC VARIATION OF SNOWMELT AND TEMPERATURE ON SUBARCTICALPINE AND ARCTIC PLANTS (Helsingin yliopisto, 2019)Climate change will be most pronounced at high latitudes where it will affect autumn, winter and spring disproportionally more than the growing season. Increasing or decreasing precipitation as snow, rising temperature and more frequent freeze-thaw cycles raise uncertainties about how the timing of snowmelt and the length of the growing season will change for northern plants. The timing of plant developmental stages (phenophases) in relation to snowmelt timing is important for avoiding harsh weather conditions in spring and ensuring a long growing season. In this doctoral dissertation, I investigate the effect of snowmelt timing and temperature conditions on plant phenology and plant stress. The first objective was to determine the natural variation in snowmelt timing on a small spatial scale in subarctic-alpine Finland. Such variation in the microclimate is created by the landscape topography and show the range of conditions plant populations are currently adapted to. Snowmelt timing varied by up to seven weeks within a few metres distance, so that early-melting plots were exposed to more frequent and more severe spring freezing events. This implies that plant populations are already exposed to the kind of climatic conditions which would be predicted from climate change. Secondly, I studied how the physiology of Vaccinium vitis-idaea and the phenology of seven subarctic-alpine plant species are affected by this natural variation in microclimate. The higher numbers of freezing events in early-melting plots were related to a higher reduction in the quantum yield of photosystem II (ΦPSII) in V. vitis-idaea, but not to increases in frost damage. This species therefore does not suffer substantial damage from the natural climatic variation in this habitat, implying that in this location it is likely highly resistant to the predicted changes in climate. Differences in snowmelt timing also led to three distinct patterns of phenological timing in subarctic-alpine plant species along the snowmelt gradient. These patterns can raise ideas about the mechanisms underlying the rate of plant development and can help researchers classify past and future phenological responses. Lastly, I compare the effect of natural versus experimental gradients of snowmelt timing on the timing of autumn senescence in High Arctic plants. The results show that the two gradients lead to different conclusions regarding the effect of snowmelt timing on the timing of autumn senescence. Selective warming only in the beginning of autumn had a delaying effect on autumn senescence in Dryas octopetala, despite the short duration of the warming treatment. Previous studies have commonly employed a warming treatment over the entire growing season. The results of this dissertation highlight that we need to integrate different approaches to studying climate change effects on plants. Natural gradients, although often disregarded, can give additional insight into plants’ adaptation to climate variation and therefore complement experiments. Experimental treatments simulating climate change need to be very selective in which period of the plant life cycle they are applied as expected changes in climate strongly depend on the season. Due to the variable sensitivity of different phenophases and plant species to climate and the importance of plants in the ecosystem carbon balance, further detailed research is needed to understand the drivers and mechanisms underlying plant phenology.
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(Finnish Meteorological Institute, 2019)This thesis applies cost-benefit analysis (CBA) to certain environmental questions and through its results contributes to both the theoretical literature on CBA in environmental economics and practical issues in the application of CBA to environmental problems. The work comprises an introduction and four articles, which address three common thematic areas: 1) distributional issues, 2) climate change adaptation, and 3) urban ecosystem services. Article I contributes to the literature through analyses that i) provide a connection between the income effect and distributional issues; ii) compare weighting schemes both theoretically and empirically; iii) show how weights could be incorporated into a CBA in practice; and iv) demonstrate that results are sensitive not only to whether weights are applied, but also to the choice of the inequality parameter and spatial resolution. Article II analyzes whether over-investment in disaster risk reduction and climate change adaptation is a legitimate economic concern and examines how the public reacts to major infrastructure investments. The results constitute a contribution to both the theoretical and empirical literature on the economics of climate change adaptation. Article III evaluates how potential innovations in weather services can reduce weather sensitivity and, consequently, decrease the negative effects of climate change on transport, particularly in the road transport sector. The article illustrates how innovations in the provision and use of weather and climate information can be beneficial for adapting to the changing climate and contributes to the empirical literature on the economics of climate change adaptation. Article IV presents a CBA of a relatively novel feature in the urban green portfolio: green roofs. The specific objectives of the research are i) to facilitate benefit-transfer of ecosystem services from one urban area to another by providing detailed information on valuation methods and the role of different assumptions and parameter values and ii) to include scenic values as a benefit item based on a formal and trackable analysis rather than on a guess. The article contributes to the empirical literature related to both the cost-benefit analysis of urban ecosystem services and the economics of climate change adaptation.
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(2019)Emissions of biogenic volatile organic compounds (BVOCs) cool down the global climate via their impacts on aerosol and cloud formation. Climate change will likely have a major impact on BVOC fluxes from the biosphere, including soils, due to temperature-driven plant biosynthesis of volatile organic compounds (VOCs), compound volatility and microbial activity. Soils are a poorly quantified source of VOCs, where the diversity of driving factors creates high spatial and temporal variability in soil VOC fluxes. The aim of this study was to analyse the magnitude and variability of forest floor VOC fluxes, to determine the role of the boreal forest floor in the forest stand BVOC exchange and to estimate plant ecophysiological and microbiological processes, which drive forest floor VOC exchange. Forest floor VOC exchange was determined using a steady-state flow-through chamber technique coupled with mass spectrometry in the boreal and hemiboreal climates. We revealed that the boreal forest floor contributes significantly to forest stand fluxes, but its importance varies between seasons. The forest floor accounted only a few per cent of the total forest stand fluxes of monoterpenes in summer, while in spring and autumn it could be up to 90%. The forest floor VOC exchange was stable between years, while fluxes had clear seasonal dynamic. Monoterpenes and oxygenated VOCs originated from fresh litter, microbial activity, and ground vegetation VOC biosynthesis. Air inside soil layers was found to contain diverse compounds. Forest floor VOC fluxes varied strongly depending on climate and tree species. Atmospheric chemistry may be strongly affected by soils during periods when plant-related BVOC biosynthesis and fluxes are low. In the future, we need continuous and simultaneous VOC exchange measurements from forest floors and forest stands in various ecosystems and climate zones. The global budget for soil VOC emissions should also be defined based on existing studies.
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(Helsingin yliopisto, 2019)The fungus Heterobasidion parviporum is considered as the most destructive disease agent of conifers. The main host of H. parviporum is Norway spruce (Picea abies), and the primary infection is mediated by aerial sexual spores (basidiospores) landing on fresh stump surfaces or tree injuries. The secondary infection is mediated by vegetative spread of the fungus via root contacts formed between neighboring trees. H. parviporum features a dual and flexible lifestyle (saprotrophy and necrotrophy). Due to the unavailability of a reference genome, studies of the molecular pathology have heavily relied on parallel studies on its sibling species H. irregulare. The rapid development of next-generation sequencing techniques has revolutionized the scalability, reliability, and resolution of sequencing in life sciences. The first aim of this study was to provide a reference genome for H. parviporum by application of whole-genome sequencing. We selected a virulent isolate (isolate 96026) as the reference, which presented us a genome assembly of 37.76 Mb, hosting 10,502 protein-coding genes. To identify genomic variations potentially accountable for the virulence of the reference isolate, 14 less virulent isolates were also sequenced. Comparative genomic analysis uncovered not only the remarkable intraspecific level of polymorphism, but also two genomic regions exclusive to the reference isolate. The proteins encoded by genes situated in these two regions included a cytochrome P450, a major facilitator superfamily transporter, and secreted proteins, suggestive of their potential implication in the virulence of H. parviporum reference isolate. To propose candidate virulence factors for functional characterizations, secreted protein-coding genes under genome-wide selection pressure or possessing featured variants were explored, and examples were listed. We then targeted small-secreted proteins (SSPs) in H. parviporum reference isolate to provide promising SSP candidate(s) with functional evidence in terms of promoting disease development. A particular SSP (HpSSP35.8) was found capable of causing rapid cell death on the leaves of Nicotiana benthamiana and of triggering the expression of some defense-related genes in N. benthamiana. In addition, HpSSP35.8 was also highly expressed during the pre-symptomatic phase of H. parviporum infection of Norway spruce seedlings, in which some defense-related genes were also induced. Collectively, all evidence suggests this SSP as a potentially important virulence candidate, presumably in the early stage of pathogenic interaction with the host. Lastly, the transcriptome and DNA methylome profiles of the reference isolate growing as a necrotroph and as a saprotroph were obtained. A group of carbohydrate-active enzymes was found essential for its both necrotrophic and saprotrophic growth, and a small set of signaling- and transcription factors might be associated with the transition of saprotrophic to necrotrophic growth. DNA methylation was demonstrated to mostly target and repress the activity of transposable elements in the genome. A small group of genes with significantly varied methylation and expression levels were identified, implying the potential of DNA methylation as one of the transcriptional regulatory mechanisms in this fungus. Our study provided the reference genome and first methylome of H. parviporum. Follow-up research into the molecular mechanisms of Heterobasidion pathogenesis could be greatly facilitated by the availability of the genome, transcriptome, methylome and secretome data.
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(Helsingin yliopisto, 2019)Information needs associated with forest monitoring have become increasingly complex. Data to support these information needs are required to be systematically generated, spatially exhaustive, spatially explicit, and to capture changes at a spatial and temporal resolution that is commensurate with both natural and anthropogenic impacts. Moreover, reporting obligations impose additional expectations of transparency, repeatability, and data provenance. The overall objective of this dissertation was to address these needs and improve capacity for large-area monitoring of forest disturbance and subsequent recovery. Landsat time series (LTS) enhance opportunities for forest monitoring, particularly for post-disturbance recovery assessments, while best-available pixel (BAP) compositing approaches allow LTS approaches to be applied over large forest extents. In substudies I and IV, forest monitoring information needs were identified and linked to image compositing criteria and data availability in Canada and Finland. In substudy II, methods were developed and demonstrated for generating large-area, gap-filled Landsat BAP image composites that preserve detected changes, generate continuous change metrics, and provide foundational, annual data to support forest monitoring. In substudy III a national monitoring framework was prototyped at scale over the 650 Mha of Canada’s forest ecosystems, providing a detailed analysis of areas disturbed by wildfire and harvest for a 25-year period (1985–2010), as well as characterizing short- and long-term recovery. New insights on spectral recovery metrics were provided by substudies V and VI. In substudies V, the utility of spectral measures of recovery were evaluated and confirmed against benchmarks of forest cover and height derived from airborne laser scanning data. In substudy VI the influence of field-measured structure and composition on spectral recovery were examined and quantified. By focusing on four key aspects of forest monitoring systems: information needs, data availability, methods development, and information outcomes, the component studies demonstrated that combining BAP compositing and LTS analysis approaches provides data with the requisite characteristics to support large-area forest monitoring, while also enabling a more comprehensive assessment of forest disturbance and recovery.
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(Helsingin yliopisto, 2019)The objective of the research described in this thesis was to evaluate the potential and sustainability of protein feed use of different non-defatted microalgae species in the nutrition of lactating dairy cows. Four physiological experiments (Publications I-III) were conducted with dairy cows to compare dry matter (DM) intake, milk production, energy metabolism, nitrogen (N) utilisation and amino acid (AA) metabolism using diets containing microalgae (Spirulina platensis, Chlorella vulgaris and Nannochloropsis gaditana) and conventional protein feeds (rapeseed meal, soya bean meal and faba beans). The microalgae diets constituted of S. platensis (Exp. 2, 3 and 4; I-III), C. vulgaris (Exp. 3; II), mixture of S. platensis and C. vulgaris (Exp. 1; I), and mixture of C. vulgaris and N. gaditana (Exp. 3; II). These microalgae substituted protein in rapeseed meal partially (Exp. 1, 2 and 4; I, III) or completely (Exp. 1 and 2; I), soybean meal completely (Exp. 3; II), and faba beans partially (Exp. 4; III). In addition, the effect of protein supplementation with rapeseed meal and microalgae in comparison to unsupplemented diet (Exp. 2; I) was studied. The crude protein (CP; Kjeldahl-N × 6.25) concentration of microalgae was on average 690 g/kg DM for S. platensis, 597 g/kg DM for C. vulgaris and 385 g/kg DM for N. gaditana. These microalgae were relatively low in crude fat, with highest concentration of 192 g/kg DM measured for N. gaditana. Compared to conventional protein feeds, all microalgae were lower in histidine, but higher in methionine. The DM intake (DMI) of microalgae containing feeds was lower than that of conventional protein feeds in three out of four experiments. When forage and concentrates were fed separately, cows compensated the decreased intake of microalgae containing concentrates by increasing silage intake (Exp. 2 and 3; I-II). Thus, total DMI remained unchanged (Exp. 2 and 3; I, II). This decreased the proportion of concentrate in the diet as much as 11.4 %-units. When all feed components were offered as total mixed ration (TMR), total DMI was decreased (on average 0.65 kg/d) as cows no longer were able to avoid microalgae (Exp. 4; III). The poor palatability of microalgae was not related to amount of microalgae in the diet, dietary crude fat concentration or stage of lactation. Milk yield was unaffected by microalgae inclusion in the diet in three out of four experiments. The partial substitution of rapeseed meal or faba beans with S. platensis decreased milk yield on rapeseed, but increased on faba bean supplemented diets (Exp. 4; III). The same was observed as a tendency for protein yield. Also in Experiment 2 (I), S. platensis tended to result in lower milk protein yield than rapeseed meal. Microalgae resulted in milk, energy corrected milk (ECM) and protein yields similar to soya bean meal (Exp. 3; II). S. platensis inclusion in the diet increased milk fat concentration on soya (Exp. 3; II) and rapeseed meal (Exp. 4; III) supplemented diets, which might have been caused by more acetate intensive ruminal fermentation, increased body mobilisation or higher methionine intake. Protein supplementation increased N excretion in urine and faeces, and decreased the conversion efficiency of dietary N into milk protein (milk N:N intake; NUE) (Exp. 2; I). The responses of microalgae inclusion in the diet depended on the source of protein in the basal diet. In Exp. 2 (I), the substitution of rapeseed meal with S. platensis decreased NUE and human-edible protein efficiency. The same was observed in Exp. 4 (III) on rapeseed meal supplemented diets, whereas on faba bean supplemented diets NUE was increased and human-edible protein efficiency was unaffected by S. platensis inclusion in the diet. In addition, the partial substitution of both rapeseed meal and faba beans resulted in increased urinary and total excretion of N (Exp. 4; III). The complete substitution of soya bean meal with different microalgae did not affect NUE, but decreased urinary N excretion and tended to increase human-edible protein efficiency. The substitution of all conventional protein feeds with microalgae decreased the intake of histidine and increased that of methionine. In two out of four experiments, these changes did not convert to major changes in arterial concentrations of these AA and their metabolites. The changes observed with S. platensis or mixture of S. platensis and C. vulgaris were decreased (Exp. 1; I) or tendency for decreased (Exp. 2; I) arterial histidine concentration, decreased arterial carnosine concentration in Exp. 1 and 2 (I), and decreased mammary uptake of histidine in Exp. 2 (I) and methionine in Exp. 4 (III). In conclusion, the results demonstrated no biological or physiological constraints for protein feed use of different microalgae. Moreover, microalgae seemed to be suitable substitute for soya bean meal and faba beans in dairy cow nutrition. However, microalgae are slightly inferior to rapeseed meal as indicated by results of milk production and N utilisation. The greatest challenge limiting the feed use of microalgae for lactating dairy cows is their poorer palatability relative to conventional feeds.
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(Helsingin yliopisto, 2019)This dissertation focuses on analysing the feelings of ownership that the owners and other users of forests have developed towards privately-owned forest resources. These resources play a major part in providing forest-based benefits to society, as a large proportion of the forests in Europe and the US are privately owned by so called non-industrial private forest owners (NIPF). Therefore, the decisions the NIPF owners make regarding their forest resources have a direct impact on the availability of forest-based ecosystem services. Due to the importance of the private forest resources at global, national and local levels, it is not surprising that a vast number of regulations and land use practices have been developed, that set the regulatory framework for the use of forests. Thus also, users other than the owners feel that they have the right to speak about the use of forests. Due to these demands and the expectations from the wider society, the forest owners do not have sole control over their forest areas. In the best case, nevertheless, the objectives of both private forest owners and various society’s objectives for the forests could be met at the same time. This necessitates a profound understanding of the forest owners’ own objectives, values and motivations regarding their forests. This dissertation contributes to the research on forest ownership by introducing a novel concept, psychological ownership, as a potential approach to understanding the possessive feelings towards privately-owned forests, and via that, a better understanding of the role of these feelings in the behaviour of forest owners and other forest users (in this case nature-based tourism entrepreneurs). Psychological ownership is based on the idea that ownership should not be understood only as a legal construct, but also to include certain psychological elements i.e. to the feeling “it is mine”. Originally, psychological ownership was introduced in the field of organizational research, but it has since been applied increasingly in other sectors. In this study, it is used as the theoretical background to understand the ownership feelings about private forest resources. Psychological ownership can also bring a new approach to study the co-operation relationships related to the use of forests by multiple stakeholders, for example, when introducing new potential uses of forest resources (in this case nature-based entrepreneurship). The study is qualitative in nature and the data consist of thematic interviews with private forest owners and nature tourism entrepreneurs. The results summarise the findings from three published journal articles. They show that both the legal owners and the nature-based entrepreneurs utilizing private forest areas seem to have developed psychological ownership feelings towards these forests. However, these feelings are not necessarily dependent on the legal ownership of the resource. Furthermore, the psychological ownership experienced seems to influence the behaviour of the persons expressing these feelings, for example, related to the private forest owners’ forest management decisions. The results also illustrate that recognizing psychological ownership can help in understanding successful co-operation relationships and potential conflict situations relating to the multiple use of forest resources. In practice, it could help to foresee or even manage the potential conflicts.
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(Helsingin yliopisto, 2019)In the wake of the modernization of agriculture and agricultural markets, the role of the farmer has increasingly moved towards that of an owner and investor in agricultural producer cooperatives. Competitive pressures, internationalization, and the growth of cooperatives call for an examination of new avenues for acquiring investment capital, as the traditional cooperative structure may fail to provide sufficient incentives to urge members to contribute to the long-term success of the cooperative. Despite an emerging multitude of new cooperative structures, the investment preferences of cooperative members are not sufficiently understood. Moreover, the preferences of potential non-member investors beyond cooperative boundaries remain practically unexplored. This dissertation consists of four essays around the theme of investment behavior in agricultural producer cooperatives. The analyses are based on questionnaire data from Finnish dairy farmers and financial market professionals. The farmer survey examined member preferences concerning the use of cooperative surplus for investments as well as their views on new cooperative investment instruments. The investor survey studied the willingness of non-members to invest in agricultural production and the behavioral motivations affecting their investment decisions. The study methods applied here are novel to the context of investment in cooperatives. The results offer insights into new possibilities to develop capital sourcing strategies for use by growth-seeking agricultural producer cooperatives. An understanding of investor preferences will facilitate the design of new financing mechanisms for cooperatives.
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(Helsingin yliopisto, 2019)My research is about the sense of home comprehended as the entirety of meanings. My approach was to look at the sense of home from the phenomenological stance and from the experiences of the consumer. Utilizing the theory of practice I examined the elements of the sense of home and their significance by means of a theme interview. The theme interviews were organized in Finland and in the Portuguese Algarve. The analysis of the interview transcripts was done as a text analysis using Atlas.Ti7. The analysis is primarily based on themes-analysis. Resulting from the analysis, there emerged several different entireties of the sense of home. Each sense of home entirety is framed by a concrete observed space, an abstract imagined space, and a living space. The practical space means easy everyday life and the functionality of home-based practices. The space is relatively clean and tidy. The homely lair manifested itself as snuggling down and emphasizing all kinds of comfort. The homely lair is a private space. The objects at home remind the occupant of his or her personal history and the loved ones. For many, a homely lair was made to the second home. In the space, the loved ones — even the late ones — are present. The feeling of a homely lair was also brought into the second home in the Algarve. Specifically, the second home in the Algarve is clearly a place for the peace of mind, which is best described by the desire for freedom, freedom from duties, external timetables and pressures. The sense of home can be merely an inner state of mind, which is not necessarily tied to a particular geographical place. The sense of home can be present here and now or in an imagination. The stimulating ambience is a comprehensive feeling conveyed by multiple senses. Experiences rise in the home space, of the home space, of the environment and of the imagination, and of the virtual world that complements the visible reality. The creator of the ambience actualizes herself and wants to involve other people as part of the ambience both as evaluators and as audience. One significant result relates to material. Home in Finland is furnished with objects and things, intangible objects and virtual material. In the second home abroad, however, the mobile consumers want to efface the solid personal meaning of things. Housing, furnishing and moving around are preferentially organized through services. The study complements and updates the definition of comfort. Earlier layers of domestic comfort include privacy, leisure, ease, comfortable furnishings, heat, light, ventilation, household appliances, efficiency, running water, indoor toilet and bathroom. New layers of comfort are abstract requirements of time management, to synchronize one's everyday life with the rhythms of the environment, and the desire to take control of one's own life.
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(Helsingin yliopisto, 2019)Global food production will face several challenges in the future. Population continues growing, and expansion of agricultural area is becoming more difficult. A common view is that yields need to double by 2050. The goal is not without challenges: expansion will contribute to deforestation, habitat loss and carbon emissions, while productivity increases could contribute to nutrient run-offs, increased greenhouse gas emissions and land degradation. The scientific community recommends sustainable intensification as the best way to reckon with the challenges. However, adequate policy measures need to be in place for that end to be achievable. This dissertation provides new findings to support better informed policy decisions. The essays in this dissertation examine the interrelationship between agricultural policies and land use from various angles. The approach is empiric, and the dissertation aims to further define how policy measures affect farmers' land use decisions. The empirical analysis aims to improve predictions on the effects of policy measures. Special focus is on how policies steer land use in the direction of sustainable intensification. Essay I examines the modeling of intensive margin adjustment of agricultural land use in computable general equilibrium (CGE) models, which are widely used to analyze land use change. The analysis is based on empirical estimation of agricultural production functions with Finnish farm-level data. The results indicate that the CRESH functional form should be favored over its alternatives. The subsequent CGE model simulations showed that both the elasticity estimates and the choice of functional form affect the model results significantly. Consequently, the widely-applied CES functional forms likely underestimate the elasticity of the intensive margin adjustment, and thus the adaptation possibilities in the agricultural sector as a whole. Essay II examines how policies and markets interact in the structural change of agricultural sector. The model applied is based on Fisher's principle, which explains variables' evolution as regards underlying fitness factors. This essay examines both land input and output markets separately with respect to both market and subsidy income. The empirical analysis employs quantile regression techniques that can shed light on the effects of the whole range of distribution values in addition to the mean effects. The results show that decoupled subsidies have increased the market-orientation of the sector concurrently with increasingly inefficient allocation of land. However, the latter effect was found to dominate, which indicates a negative net effect. Additionally, the distinction between coupled and decoupled subsidies matters very little in comparison to the effects that the subsidies have when paid in one form or another. Subsidies and market incomes attenuate each other, and thus they direct structural change in different directions. Decoupling has increased land market rigidities and thus inefficient allocation of land. Essay III examines how farmers adjust their land use to coupled, crop-specific subsidy payments. It exploits an idiosyncratic policy change in the Agenda 2000 reform as a quasi-experimental setting. The causality between the subsidy payments and production decisions could be reliably established with the standard differences-in-differences model. A range of empirical strategies suitable for the task are explored, while the results of OLS fixed effects and fractional response models are reported as they were considered the most reliable. The results vary between the models, which nevertheless does not affect the main conclusions. The results show that the reform affected farmers' land use decisions as predicted by economic theory: a crop's area increases (decreases) if its subsidy payment increases (decreases). Furthermore, allocation decisions are very elastic to coupled subsidies, especially for feed crops.
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(Helsingin yliopisto, 2019)Sahateollisuuden liiketoimintastrategiat perustuvat perinteiseen (tuotelähtöiseen) liiketoimintalogiikkaan. Perinteiset kilpailukyvun lähteet, kuten alhaisemmat hinnat tai tuotannon tehokkuus, eivät ole enää riittäviä turvaamaan sahateollisuuden kannattavuutta. Siirtyminen valmistusyrityksestä palveluliiketoimintaan sisältää merkittävää potentiaalia asiakkaan kokeman arvon kasvattamiseen ja siten sahateollisuuden kilpailukyvyn parantamiseen. Palveluliiketoiminnan perusajatuksena on arvonluonti yhteistyössä palvelun tuottajan, asiakkaan ja muiden osapuolten kanssa. Näin ollen asiakas on liiketoiminnan keskiössä, haastaen perinteistä tuotelähtöistä ajattelutapaa. Työn tavoitteena on selvittää sahateollisuuden liiketoimintamallin muutosta kohti asiakasläjtöisyyttä ja palveluliiketoimintaa. Koska arvoketjun toimijat ovat kytköksissä toisiinsa, tutkimuksessa tarkastellaan koko puutuoteteollisuutta. Muutosta kuvataan palvelulogiikan avulla (service logic), joka korostaa yrityksen ja asiakkaan välistä vuorovaikutusta. Työn uutuusarvo on asiakaslähtöisyyden ja palvelulogiikan soveltaminen sahateollisuuden kontekstissa. Lisäksi siinä tarkastellaan innovaation ja digitalisaation merkitystä alan kilpailukyvyn lähteenä. Tulokset perustuvat sekä tapaus- että haastattelututkimuksiin (n = 36). Tulosten perusteella saha- ja puutuoteteollisuudella on paljon hyödyntämätöntä potentiaalia kilpailukykyvyn kasvattamiseksi, jonka se voi savuttaa siirtymällä kohti asiakaslähtöisyyttä ja palveluiliikeotimintaa. Siirtyminen vaatii merkittäviä strategisia muutoksia, kyseenalaistaen nykyiset toimintatavta ja periaatteet. Muutos alkaa ymmärryksellä siitä, mitä asiakaslähtöisyys merkitsee. Tarvitaan lisää ymmärrystä asiakkaiden tarpeista ja prosesseista, joiden perusteella yritykset voivat arvioida ja kehittää omaa tarjoomaansa. Keskeistä palveluliiketoiminnan onnistumisessa on asenteiden muutos kohti asiakaslähtöisyyttä – ei pelkästään omassa organisaatiossa, vaan läpi arvoketjujen.
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(Helsingin yliopisto, 2019)Previous findings suggest that bone mineral density and bone loss are highly genetically determined but the further details of bone genetics remain partly unknown. In thesis, it was studied for the first time whether the single nucleotide polymorphisms of lactase and apolipoprotein E (APOE) genes are associated with the peripheral quantitative computed tomography (pQCT) bone traits of radius and tibia. In addition to the gene polymorphisms, the associations of physical activity in childhood and adulthood with various bone traits as outcomes were examined. This is also a current topic since despite the fact that bone-loading physical activities are essential in building healthy bones; it seems that modern communities do not encourage most of us to move and exercise enough. Study subjects of the present thesis are part of the Cardiovascular Risk in Young Finns Study which was initiated in 1980 with 3596 persons aged 3, 6, 9, 12, 15 and 18. The findings presented are additionally based on the follow-ups conducted in 1986, 2001 and 2007. The pQCT and quantitative ultrasound bone measurements and fracture data were collected in 2008 when the participants were 31-46 years old. Lactase C/T-13910 genotyping (rs4988235) was performed using a 5’ nuclease assay and the APOE genotypes (rs429358 and rs7412) and the APOE promoter polymorphisms -219 and +113 (rs405509 and rs440446) using TaqMan SNP genotyping assays. Dietary intakes were collected through 48-hour dietary recall interviews and later in 2007 with a 131-item food frequency questionnaire. Physical activity in childhood was assessed using a questionnaire including items on leisure-time physical activity, organised exercise, participation in competitions, and intensity level. In adulthood, pedometer-determined steps were used to quantify the amount of physical activity undertaken in 2007. In Study I, small differences in trabecular densities at the distal sites of radius and tibia were found in men between the lactase genotypes. Men with the T/T genotype had ~3% higher values than the carriers of T/C and C/C genotypes. They also had higher calcium intake than men carrying the C allele. In Study II, the carriers of the APOE ԑ4 allele had higher total-cholesterol and LDL-cholesterol serum concentrations than the non-carriers. In addition, the ԑ4 allele was associated with some lower cortical density but greater bone mineral content (BMC) at the tibial diaphysis. Women with the APOE promoter -219T/T allele had on average 7-8% lower cortical and compressive strengths at the distal sites of radius and tibia than the female G/G carriers. At the tibial shaft, the mean values of cortical strength were 2-2.5% lower in subjects with the -219T/T allele compared to the G/G carriers. Men with the -219T/T allele also had 1-2% lower cortical density and cortical strength at the radial shaft but 4-5% greater total areas at the radius than men with the G/G allele. In addition, women with the -219T/T allele whose longitudinal saturated fat intake was the highest (≥35.5 g/day) had the lowest total area and torsional strength at the radial shaft. Almost identical results were found in women and men with the +113G/C genotypes (G/G, G/C and C/C) as reported for the -219G/T genotypes (G/G, G/T and T/T). In Study III, there were no statistically significant differences in the risk of low-energy fractures between the different physical activity groups of 3-18-year-old children and adolescents. In addition, no differences were found in adult tibial traits between the physical activity groups of 3-6-year-olds. In females, the highest level of physical activity at the age of 9-18 years was significantly associated with a lower likelihood of below median values of compressive strength at the distal tibia, and total and cortical areas, and BMC and strength indices at the tibial shaft compared to the lowest activity level in adolescence (ORs 0.33-0.53). Similarly in males, total area at the distal site and cortical area and strength at the tibial shaft were less likely to be below the median values in the highest physical activity group compared to the lowest activity group of adolescents (ORs 0.48-0.53). In Study IV, women within the highest tertile of daily steps had 3.8% and 0.5% greater broadband ultrasound attenuation and speed of sound at the calcaneus compared to women in the lowest tertile. In tibia, women in the highest tertile (> 8765 daily steps) had on average 1-5.4% greater bone cross-sectional area, BMC, trabecular density and compressive strength at the distal site and 1.6-2.7% greater total and cortical bone areas, BMC and torsional strength at the shaft compared to their study peers. Similarly, BMC and BSI at the distal radius and bone areas, BMC and torsional strength at radial shaft were 1.7-3.4% greater in women within the highest tertile of daily steps compared to the women with a lower number of daily steps. In men, the differences in studied bone traits were mainly non-significant across the tertiles of daily steps. Statistically significant results presented have p-values of ≤0.05. In conclusion, the genetic heterogeneity in lactase gene or the APOE ԑ2/ԑ3/ԑ4 genetic variation seemed to have little effect on the studied bone traits at the radius and tibia. Instead, the APOE promoter -219T/T and +113C/C alleles were associated with lower cortical bone phenotypes in both genders compared to the G/G carriers. In women, these supposed risk alleles of APOE promoter polymorphisms were also associated with lower total area and torsional strength at the radial shaft when the intake of saturated fat was high. In addition, a high level of physical activity at the age of 9-18 years, but not in younger children, was associated with wider and stronger weight-bearing tibia in both females and males. In adulthood, a higher amount of physical activity measured as daily steps was associated with greater bone cross-sectional area, mineral mass and strength at the calcaneus, tibia and radius in women.
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(Helsingin yliopisto, 2019)Potyviral helper component proteinase (HCPro) is a quintessential example of a multifunctional viral protein. Its name comes from two of its earliest identified functions- ‘Helper Component’ involved in aphid-mediated plant-plant transmission of the virus, and cysteine proteinase responsible for its self-cleavage from the rest of the viral polyprotein. HCPro’s best-studied function is its ability to suppress RNA silencing. One of the factors underlying the multifunctionality of HCPro is its ability to interact with a wide range of host factors causing perturbations in several cellular pathways. In this study, interaction of HCPro with the host proteins S-adenosyl-L-methionine synthase (SAMS), S-adenosyl-L homocysteine hydrolase (SAHH), ARGONAUTE 1 (AGO1) and VARICOSE (VCS) was addressed and implications of these interactions in potato virus A (PVA; genus Potyvirus) infection, and potato virus X (PVX)-PVA mixed infection were studied. In this study, HCPro was found to interact with the host methionine cycle enzymes SAMS and SAHH and to inhibit SAMS activity. Disruption of the methionine cycle promoted PVA infection. Methionine cycle plays a crucial role in the smooth running of RNA silencing by providing S-adenosyl methionine (SAM) for methyltransferase hua enhancer 1 (HEN1). Small RNA (sRNA) duplexes, which are methylated by HEN1, are stable and capable to act in RNA silencing, whereas, the unmethylated sRNAs are polyuridylated and targeted to degradation. A blockage in sRNA methylation via HCPro-mediated methionine cycle disruption was proposed to act as a circuit breaker of RNA silencing pathway for the benefit of PVA infection. SAHH was also found to be involved in PVX-PVA synergism. Blockage of the methionine cycle at SAHH, coupled with synergism-specific downregulation of closely associated glutathione (GSH) biosynthesis pathway enhanced PVX genomic RNA accumulation and subgenomic RNA expression. Moreover, depletion of cellular antioxidant GSH was suggested to be the reason behind induction of severe oxidative stress during potex–potyvirus mixed infection. In another line of this study, formation of HCPro-associated high molecular weight (HMW) complexes and their functions were studied. Interaction between HCPro and a WD40 domain containing scaffolding protein VCS was shown to be crucial for formation and stability of HCPro-associated HMW complexes. Importance of HCPro-VCS interaction in governing the assembly of PVA-induced granules (PGs) was demonstrated. This study reinforced the correlation between the PGs and RNA silencing suppression. Interestingly, HCPro, AGO1, VPg and CI were detected in the ribosome-associated HMW-complexes. Association of AGO1 with ribosomes may indicate occurrence of RISC-mediated translational repression as an additional defense mechanism against PVA infection. While, presence of HCPro, VPg and CI therein suggested a putative mechanism by which HCPro derived ribosome-associated HMW complexes might participate in relieving PVA translational repression. Accordingly, co-operation between HCPro, VCS and VPg was shown to act in favor of active PVA translation. Intriguingly, importance of HCPro-VCS interaction was also found to be important in PVA encapsidation. In conclusion this study provides evidence for interaction between HCPro and host proteins SAMS, SAHH, VCS and AGO1 in planta. Furthermore, importance of these interactions are demonstrated to play crucial role in governing various viral processes during PVA infection.
