Recombination analysis of Maize dwarf mosaic virus (MDMV) in the Sugarcane mosaic virus (SCMV) subgroup of potyviruses.

Recombination among RNA viruses is a natural phenomenon that appears to have played a significant role in the species development and the evolution of many strains. It also has particular significance for the risk assessment of plants which have been genetically modified for disease resistance by incorporating viral sequences into their genomes. However, the exact recombination events taking place in viral genomes are not investigated in detail for many virus groups. In this analysis, different single-stranded positive-sense RNA potyviruses were compared using various in silico recombination detection methods and new recombination events in the Sugarcane mosaic virus (SCMV) subgroup were detected. For an extended in silico recombination analysis, two of the analyzed Maize dwarf mosaic virus full-length genomes were sequenced additionally during this work. These results strengthen the evidence that recombination is a major driving force in virus evolution, and the emergence of new virus variants in the SCMV subgroup, paired with mutations, could generate viruses with altered biological properties. The intra- and interspecific homolog recombinations seem to be a general trait in this virus group, causing little or no changes to the amino acid of the progenies. However, we found a few breakpoints between the members of SCMV subgroup and the weed-infecting distant relatives, but only a few methods of the RDP3 package predicted these events with low significance level.

Oral immunogenicity of a plant virus vector based porcine circovirus antigen - short communication.

A recombinant cucumber mosaic virus based expression system has been developed for the production of an immunogenic porcine circovirus epitope. The resulting nanoparticle was shown to elicit specific immune response in mice and pigs, when administered parenterally. To evaluate the oral applicability of this vaccine candidate, two experiments were performed. In the first one, the resistance of the vector itself to mucosal environment was tested in mice. Cucumber mosaic virus particles fed to mice were able to elicit specific mucosal and serum antibody production. In the second experiment, recombinant cucumber mosaic virus fed to piglets resulted in the appearance of porcine circovirus specific serum antibodies. The vector proved to be able to survive in the gastrointestinal tract, so that an epitope expressed on its surface could induce specific immune response. These results indicate that the developed plant virus based expression system offers an effective method for mucosal vaccine production.

The epitopes in wheat proteins for defining toxic units relevant to human health.

Wheat-related disorders are well-studied health problems. Knowledge of the composition and amounts of epitopes present in a single wheat sample represents a significant gap, and the detailed wheat proteome datasets now available can provide the necessary information to carry out an estimation of allergen prediction for a single cultivar. The combined use of genome sequence and allergen databases, prediction methodology, and cereal chemistry results in better understanding of the level of toxicity present in the end-products produced from wheat flour. The workflow presented in this review provides information about the number and distribution of epitopes at single protein, or protein fraction, levels. In addition, epitopes present in the highest frequency and harmful proteins expressed in the highest amount can be identified. The "epitope toxicity" value obtained in this way is a significant research output from the analysis of large datasets that can be applied to the food industry.

Molecular aspects of the antagonistic interaction of smoke-derived butenolides on the germination process of Grand Rapids lettuce (Lactuca sativa) achenes.

Smoke-derived compounds provide a strong chemical signal to seeds in the soil seed bank, allowing them to take advantage of the germination niche created by the occurrence of fire. The germination stimulatory activity of smoke can largely be attributed to karrikinolide (KAR(1) ), while a related compound, trimethylbutenolide (TMB), has been shown to have an inhibitory effect on germination. The aim of this study was to characterize the interaction of these potent fire-generated compounds. Dose-response analysis, leaching tests and a detailed transcriptome study were performed using highly KAR(1) -sensitive lettuce (Lactuca sativa cv 'Grand Rapids') achenes. Dose-response analysis demonstrated that the compounds are not competitors and TMB modulates germination in a concentration-dependent manner. The transcriptome analysis revealed a contrasting expression pattern induced by the compounds. KAR(1) suppressed, while TMB up-regulated ABA, seed maturation and dormancy-related transcripts. The effect of TMB was reversed by leaching the compound, while the KAR(1) effect was only reversible by leaching within the first 2 h of KAR(1) treatment. Our findings suggest that the compounds may act in concert for germination-related signaling. After the occurrence of fire, sufficient rainfall would contribute to post-germination seedling recruitment by reducing the concentration of the inhibitory compound.

Effect of light on the gene expression and hormonal status of winter and spring wheat plants during cold hardening.

The effect of light on gene expression and hormonal status during the development of freezing tolerance was studied in winter wheat (Triticum aestivum var. Mv Emese) and in the spring wheat variety Nadro. Ten-day-old plants (3-leaf stage) were cold hardened at 5°C for 12 days under either normal (250 µmol m(-2) s(-1) ) or low (20 µmol m(-2) s(-1) ) light conditions. Comprehensive analysis was carried out to explore the background of frost tolerance and the differences between these wheat varieties. Global genome analysis was performed, enquiring about the details of the cold signaling pathways. The expression level of a large number of genes is affected by light, and this effect may differ in different wheat genotypes. Photosynthesis-related processes probably play a key role in the enhancement of freezing tolerance; however, there are several other genes whose induction is light-dependent, so either there is cross-talk between signaling of chloroplast originating and other protective mechanisms or there are other light sensors that transduce signals to the components responsible for stress tolerance. Changes in the level of both plant hormones (indole-3-acetic acid, cytokinins, nitric oxide and ethylene precursor 1-aminocyclopropane-1-carboxylic acid) and other stress-related protective substances (proline, phenolics) were investigated during the phases of the hardening period. Hormonal levels were also affected by light and their dynamics indicate that wheat plants try to keep growing during the cold-hardening period. The data from this experiment may provide a new insight into the cross talk between cold and light signaling in wheat.

The unique carnation stunt-associated pararetroviroid.

The discovery and description of a pararetroviroid associated with carnation stunt was one of the most outstanding achievements gained by the research team directed by Ricardo Flores, as it opened up a whole new world in plant virology and quickly led to the identification of further viroid diseases. The carnation stunt-associated viroid-like RNA has been proved to exist in both RNA and DNA form, a discovery that pointed to new ways of studying the co-evolution of plants and viroid-like RNAs. This paper aims to summarise the scientific work of Ricardo Flores, a source of inspiration to both present and future generations of scientists.

Identification a coat protein region of cucumber mosaic virus (CMV) essential for long-distance movement in cucumber.

To characterise the long-distance movement determinant of cucumoviral coat proteins (CPs), five mutants were engineered into the CMV CP bearing the corresponding tomato aspermy virus (TAV) loops exposed on the surface of the virion. Both viruses can move long-distance in Nicotiana clevelandii, but only CMV can move long-distance in cucumber. Investigation of the CMV chimeras identified three amino acids of the ßB-ßC loop that were essential for the CMV long-distance movement in cucumber. Introducing these mutations into the TAV CP was not sufficient for long-distance movement, indicating that this is not the sole region causing long-distance movement deficiency.

Bioeconomy: Biomass and biomass-based energy supply and demand.

This paper addresses the challenges of the transition from a fossil fuel-dependent to a bio-based economy and implications related to the production of food, feed, bioenergy and other bio-based materials. The objective is to provide a comprehensive review of global biomass and biomass-based energy supplies and demand, with particular attention to the EU. Furthermore, factors related to setting priorities in the use of non-food biomass are discussed, as food security will remain the top priority. Finally, the changes in the bioenergy balance indicators in the Member States of the EU and new plant breeding technologies are analyzed. Overall, this study describes the complexity of the bio-based value chains in making decisions on how best to use biomass. The article presents a comprehensive review on global biomass and biomass based energy supplies and demand, discusses the European chemical industry perspective, analyzes the changes in the biomass based energy balance indicators in the Member States of the EU, and considers the challenges of the new plant breeding technologies.

Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR1.

BACKGROUND: Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR1), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR1 action. RESULTS: In this paper we demonstrate that although smoke-water and KAR1 treatment of maize kernels result in a similar physiological response, the gene expression and the protein ubiquitination patterns are quite different. Treatment with smoke-water enhanced the ubiquitination of proteins and activated protein-degradation-related genes. This effect was completely absent from KAR1-treated kernels, in which a specific aquaporin gene was distinctly upregulated. CONCLUSIONS: Our findings indicate that the array of bioactive compounds present in smoke-water form an environmental signal that may act together in germination stimulation. It is highly possible that the smoke/KAR1 'signal' is perceived by a receptor that is shared with the signal transduction system implied in perceiving environmental cues (especially stresses and light), or some kind of specialized receptor exists in fire-prone plant species which diverged from a more general one present in a common ancestor, and also found in non fire-prone plants allowing for a somewhat weaker but still significant response. Besides their obvious use in agricultural practices, smoke and KAR1 can be used in studies to gain further insight into the transcriptional changes during germination.

Natural insertions within the N-terminal region of the coat protein of Maize dwarf mosaic potyvirus (MDMV) have an effect on the RNA stability.

A 13 amino acid residue insertion was found in the N-terminal region of the coat protein of several Maize dwarf mosaic virus isolates (MDMV). These insertions seem to be the result of a direct duplication event, but differ in some positions. In order to evaluate the influence of the insertion on the RNA secondary structure and stability, the RNA secondary structures and minimum free energies (MFE) of all existing MDMV coat protein sequences were estimated using three different softwares, the Vienna RNA Package, NUPACK, and UNAFold, and compared to the secondary structure and MFE of various random sequence collections preserving the nucleotide distribution of MDMV. The bioinformatic analysis showed that the insertion stabilizes the RNA structure of the coat protein gene.

Genetic diversity of Hungarian Maize dwarf mosaic virus isolates.

The genetic diversity of the coat-protein (CP) region and the untranslated C-terminal region (3'UTR) of Maize dwarf mosaic virus (MDMV) was analyzed to evaluate the variability between isolates (inter-isolate sequence diversity). The results of inter-isolate sequence diversity analysis showed that the diversity of the MDMV CP gene is fairly high (p-distance: up to 0.136). During sequence analysis, a 13 amino-acid residue insertion and an 8 amino-acid residue deletion were found within the N-terminal region of the CP gene. The phylogenetic analysis showed that-unlike other potyvirus species in this subgroup-the MDMV isolates could not be distinguished on the basis of their host plants or geographic origins.

Stress-related genes define essential steps in the response of maize seedlings to smoke-water.

Smoke from burning vegetation is widely recognised as a germination cue for seed germination and recent reports suggest that smoke treatments can improve seedling vigour also. We investigated the effect of smoke-water on seedling vigour and changes of the global transcriptome in the early post-germination phase in maize. Application of smoke-water improved the germination characteristics and seedling vigour. The transcriptional response of embryos and emerging radicles 24 and 48 h after the onset of smoke treatment was investigated. The microarray study revealed a number of smoke-responsive genes amongst which stress- and abscisic acid (ABA)-related genes were over-represented. The global promoter analysis of the smoke-responsive genes revealed a tight correlation with the results obtained from Gene Ontology annotations. This concerted over-expression shows that smoke treatment induces stress and ABA-related responses in the early post-germination phase which leads to better adaptation to environmental stress factors occurring during germination, eventually resulting in greater seedling vigour.

Characterization of a natural Plum pox virus isolate bearing a truncated coat protein.

Plum pox virus (PPV) isolates were collected in Hungary from plum varieties. PCR targeting the 3' genomic region resulted in a shorter PCR product in the case of the B1298 isolate bearing a 135-nucleotide deletion in frame in the N-terminal part of the coat protein (CP). The isolate was aphid-transmissible and the virion diameter was reduced compared to PPV-SK68. Detectability of this isolate by Western blot varied according to the antibody used. Integration of the deleted CP gene into an infectious PPV clone had no effect on infectivity and symptomatology. In competition experiments, B1298 had a considerable advantage in virus accumulation.

Molecular Modeling for Better Understanding of Cucumovirus Pathology.

Cucumber mosaic virus (CMV) is a small RNA virus capable of infecting a wide variety of plant species. The high economic losses due to the CMV infection made this virus a relevant subject of scientific studies, which were further facilitated by the small size of the viral genome. Hence, CMV also became a model organism to investigate the molecular mechanism of pathogenesis. All viral functions are dependent on intra- and intermolecular interactions between nucleic acids and proteins of the virus and the host. This review summarizes the recent data on molecular determinants of such interactions. A particular emphasis is given to the results obtained by utilizing molecular-based planning and modeling techniques.

A single point mutation on the cucumber mosaic virus surface induces an unexpected and strong interaction with the F1 complex of the ATP synthase in Nicotiana clevelandii plants.

A previous study showed that a single amino acid difference in the cucumber mosaic virus (CMV) capsid protein (CP) elicits unusual symptoms. The wild-type strain (CMV-R) induces green mosaic symptoms and malformation while the mutant strain (CMV-R3E79R) causes chlorotic lesions on inoculated leaves and strong stunting with necrosis on systemic leaves. Virion preparations of CMV-R and CMV-R3E79R were partially purified from Nicotiana clevelandii A. Gray and analysed by two-dimensional gel electrophoresis. Their separated protein patterns showed remarkable differences at the 50-75 kDa range, both in numbers and intensity of spots, with more protein spots for the mutant CMV. Mass spectrometry analysis demonstrated that the virion preparations contained host proteins identified as ATP synthase alpha and beta subunits as well as small and large Rubisco subunits, respectively. Virus overlay protein binding assay (VOPBA), immunogold electron microscopy and modified ELISA experiments were used to prove the direct interaction between the virus particle and the N. clevelandii ATP synthase F1 motor complex. Protein-protein docking study revealed that the electrostatic change in the mutant CMV can introduce stronger interactions with ATP synthase F1 complex. Based on our findings we suggest that the mutation present in the CP can have a direct effect on the long-distance movement and systemic symptoms. In molecular view the mutant CMV virion can lethally block the rotation of the ATP synthase F1 motor complex which may lead to cell apoptosis, and finally to plant death.

The role of the coat protein region in symptom formation on Physalis floridana varies between PVY strains.

The Potato virus Y (PVY) cDNA full-length clone created by Jakab et al. [Jakab, G., Droz, E., Brigneti, G., Baulcombe, D., Malnoë, P., 1997. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y. J. Gen. Virol. 78, 3141-3145] was stabilized by inserting three introns into putatively toxic genes. Using this clone, hybrid viruses were constructed by in vitro recombination. The PVY-N/NTN and PVY-N/O chimeras carried the 3' end of NIb, the whole CP and 3'UTR region of PVY(NTN) and PVY(O), respectively, in a PVY(N) genetic background. The clones proved to be stable after several passages by re-sequencing the exchanged region. Both hybrid viruses showed reduced infectivity in particle bombardment experiments, but they were suitable for further mechanical plant inoculation. In five of the six host plant species, inoculated with the two chimeras and three parental strains, the chimeras produced similar symptoms to those of PVY(N). By contrast, Physalis floridana reacted with different pattern of symptoms. In this species, the symptoms caused by the N/O hybrid were similar to those of the 3'NIb-CP-donating PVY(O) strain, and not to those of the background (PVY(N)). The results suggest that symptom determinants may be different even between strains of the same virus species in a particular host.

Comprehensive Analysis of DWARF14-LIKE2 (DLK2) Reveals Its Functional Divergence from Strigolactone-Related Paralogs.

Strigolactones (SLs) and related butenolides, originally identified as active seed germination stimulants of parasitic weeds, play important roles in many aspects of plant development. Two members of the D14 α/ß hydrolase protein family, DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are essential for SL/butenolide signaling. The third member of the family in Arabidopsis, DWARF 14-LIKE2 (DLK2) is structurally very similar to D14 and KAI2, but its function is unknown. We demonstrated that DLK2 does not bind nor hydrolyze natural (+)5-deoxystrigol [(+)5DS], and weakly hydrolyzes non-natural strigolactone (-)5DS. A detailed genetic analysis revealed that DLK2 does not affect SL responses and can regulate seedling photomorphogenesis. DLK2 is upregulated in the dark dependent upon KAI2 and PHYTOCHROME INTERACTING FACTORS (PIFs), indicating that DLK2 might function in light signaling pathways. In addition, unlike its paralog proteins, DLK2 is not subject to rac-GR24-induced degradation, suggesting that DLK2 acts independently of MORE AXILLARY GROWTH2 (MAX2); however, regulation of DLK2 transcription is mostly accomplished through MAX2. In conclusion, these data suggest that DLK2 represents a divergent member of the DWARF14 family.

Homology modelling and protein structure based functional analysis of five cucumovirus coat proteins.

Coat proteins (CP) of five cucumovirus isolates, Cucumber mosaic virus (CMV) strains R, M and Trk7, Tomato aspermy virus (TAV) strain P and Peanut stunt virus (PSV) strain Er, were constructed by homology modelling. The X-ray structure of the Fny-CMV CP subunit B was used as a template. Models of cucumovirus CPs were built by the MODELLER program. Model refinements were carried out using the Kollman molecular mechanical force field. Models were analyzed by the PROCHECK programs. Electrostatic potential calculations were applied to all models and functional site search was performed with the PROSITE software, a web based tool for searching biologically significant sites. Symptom determinants published up to the present were compared with the PROSITE hits in the light of 3D models and electrostatic information. In all cases, we analyzed the effect of mutations on the structure, electrostatic potential patterns and function of CPs, respectively. We found that high flexibility of the betaE-alphaEF loop starting with the residue 129 is required, but it is not sufficient for the symptom appearance. Furthermore, phosphorylation of the CP is prospective to be important in the host response mechanism. All analyzed mutations were related to the modifications of the predicted phosphorylation sites. Based on our conclusions we predicted the infectivity of the examined viruses.

Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment.

Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid.

The necrotic pathotype of the cucumber mosaic virus (CMV) ns strain is solely determined by amino acid 461 of the 1a protein.

The unique Ns isolate of Cucumber mosaic virus (CMV) induces necrotic lesions on several Nicotiana spp. in contrast to other strains that cause systemic mosaic on these plants. By using biologically active RNA transcripts from cDNAs of Ns-CMV and a reference subgroup I strain Rs-CMV, we confined the genetic determinant solely responsible for necrosis induction to amino acid 461 of the la protein translated from genomic RNA1. An Arg to Cys change at this position (R461C) rendered Rs-CMV necrotic, whereas the reciprocal C461R mutation reverted the necrotic phenotype of Ns-CMV. Necrotic (Ns-CMV, R461C) and non-necrotic (Rs-CMV and C461R) viruses accumulated to similar levels in Nicotiana clevelandii protoplasts. Deletion of the residue at position 461 abolished replicase activity of the Ns-CMV 1a protein. The R461C mutation also was introduced into the 1a protein of Trk7-CMV, a subgroup II isolate. Symptoms induced by the Trk7/R461C mutant were identical to those caused by wild-type Trk7-CMV, even when the mutant Trk7 RNA1 was co-inoculated with RNA2 and 3 of the necrotic Ns strain.