Water deficit changes the relationships between epidemiological traits of Cauliflower mosaic virus across diverse Arabidopsis thaliana accessions

Changes in plant abiotic environments may alter plant virus epidemiological traits, but how such changes actually affect their quantitative relationships is poorly understood. Here, we investigated the effects of water deficit on Cauliflower mosaic virus (CaMV) traits (virulence, accumulation, and vectored-transmission rate) in 24 natural Arabidopsis thaliana accessions grown under strictly controlled environmental conditions. CaMV virulence increased significantly in response to water deficit during vegetative growth in all A. thaliana accessions, while viral transmission by aphids and within-host accumulation were significantly altered in only a few. Under well-watered conditions, CaMV accumulation was correlated positively with CaMV transmission by aphids, while under water deficit, this relationship was reversed. Hence, under water deficit, high CaMV accumulation did not predispose to increased horizontal transmission. No other significant relationship between viral traits could be detected. Across accessions, significant relationships between climate at collection sites and viral traits were detected but require further investigation. Interactions between epidemiological traits and their alteration under abiotic stresses must be accounted for when modelling plant virus epidemiology under scenarios of climate change.

Monitoring of plant raw materials and feed for animal in 2020 for the presence of GM-ingredients

The creation and the use of genetically modified products has become a tendency in the development of agricultural and food technologies. The area of agricultural land under genetically modified plants is constantly growing. Todays the process of using GMOs and the expediency of their creation is a debatable issue. The modification of the genome of traditional agricultural cultures gives them resistance to pesticides, pests, diseases, which cause to the significant an increase of harvest and improved quality and taste characteristics. However, the effects of GMOs on the environment and the body of animals and humans have not been fully studied, and therefore the thoughts of scientists are differ on the benefits and risks of genetic engineering. Recently, the scientific literature has data on the negative effects of GMOs on animals and humans, in particular, on the morphofunctional state of organs and systems of the body, reproductive function, immune status, biochemical parameters of blood and urine. Every year the number of new genetically modified plant lines is growing, so today, the need in research of plant raw materials and feed for animal on the presence of GMOs is very important and actual. The article presents the results of research on the detection of GM ingredients in plant raw materials and in products of its processing, feed for productive and unproductive animals, etc. In 2020, 1215 samples were investigated by polymerase chain reaction with detection in real-time (PCR-RT), and it was found out that only 0.3 % from total amount were positive. From the studied samples, the most positive samples were found in samples of rapeseed, soybeans and feed for productive animals. In 27 samples of rapeseed, the number of positive samples was 7.4 %, in them were detected the target sequences of the terminator NOS (T-NOS) T plasmid Agrobacterium, and genes Pat and EPSPs. In 6 samples of soybean, the number of positive samples was 16.7 %, in them were detected the target sequences of the 35S promoter of cauliflower mosaic virus (CaMV) and the terminator NOS (T-NOS) T of the plasmid Agrobacterium. Also there was found GM ingredients in compound feeds for farm animals and poultry, in 6 samples the number of positive samples was 16.7 %, in them were detected the target sequences of the terminator NOS (T-NOS) T plasmid Agrobacterium. Conducted studies indicate that transgenic plants are in circulation in the agricultural market, so it is necessary to constantly control animal feed, plant materials and seeds for the presence of GM sources.

Plant-derived insulator-like sequences for control of transgene expression

Stable and consistent transgene expression is necessary to advance plant biotechnology. Stable expression can be achieved by incorporating enhancer-blocking insulators, which are cisregulatory elements that reduce enhancer interference in gene expression, into transgene constructs. Sufficient insulators for plant use are not available, and their discovery has remained elusive. In this work, we computationally mined the compact genome of Utricularia gibba for insulator sequences and identified short (<1 kb) sequences with potential insulator activity. Based on in vivo tests, three of these effectively mitigate the ectopic transgene expression caused by the Cauliflower Mosaic Virus 35S promoter and do so better than previously reported plant insulators. However, all sequences with apparent insulator activity also decrease the effectiveness of the CaMV 35S promoter, and thus may be more accurately classified as silencers. However, since the insulator effect is proportionately much higher than the silencing effect, these sequences are still useful for plant transformation.

A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

Background The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging. Results The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua. Conclusions A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

QUALITY OF HELIANTHUS ANNUUS HONEY OBTAINED IN THE CONDITIONS OF RADIOACTIVE CONTAMINATION

Natural honey is a source of vital amino acids, easily digestible carbohydrates, macro, microelements, biologically active substances that determine nutritional, antibacterial and antioxidant properties. In the conditions of man-caused pollution of Polissya of Ukraine due to the accident at the Chernobyl nuclear power plant, systematic control of the quality and safety of beekeeping products is important. To conduct such research, we created a group of twelve bee families - analogs of the Ukrainian breed, medium strength. Families were kept in unified multifunctional hives. At the beginning of the honey harvest, the bee families were transported to the sunflower fields, where they stayed during the blossoming of the plants. The density of radioactive contamination of 137Cs soils where sunflower was grown was 47.0 kBq / m2. We used organoleptic, physicochemical, microscopic, microbiological, and radiological methods in the study. According to standard methods, we studied the species composition of pollen grains, physicochemical parameters of centrifugal, honeycomb, and «zabrus» sunflower honey.(zabrus honey was obtained from wax caps, which we cut with an apiary knife from honeycombs filled with nectar and sealed by bees). The content of lead (Pb) in honey from sunflower obtained in the conditions of Polissya is 1.8 - 2.1 times higher than the State sanitary norms. The largest amount of it is in the centrifugal honey. In acceptable amounts, the heavy metals cadmium (Cd), arsenic (As), and 137Cs were present in honey. Pesticides, dichlorodiphenyltrichloromethylmethane, and hexachlorane were not detected in the samples. We investigated the bactericidal action against bacterial growth of typical cultures of Proteus vulgaris, Escherichia coli, Klebsiella pneumonia, Salmonella Typhimurium, and Staphylococcus aureus. Zubrus sunflower honey showed the highest antimicrobial and antioxidant properties. We found that the value of antioxidant activity (AOA) of sunflower honey depends on the method of its production, duration of storage, and solutions of extracts (alcohol, aqueous) used in research. Laboratory control of transgenic organisms in flowers and sunflower pollen did not reveal the target sequences of the cauliflower mosaic virus (CaMV) 35S promoter and the NOS terminator (nopaline synthase) of the plasmid Agrobacterium tumefaciens.

Novel Plantibodies Show Promise to Protect Citrus from Greening Disease

Candidatus Liberibacter asiaticus (CLas), the bacteria responsible for citrus greening disease [huanglongbing (HLB)], has become a worldwide threat to citrus (Citrus sp.) production. HLB has proven difficult to study and treat because of the complex interactions between CLas, the citrus host, and insect vectors. We have selected for single chain fragment variable (scFv) antibodies from a specialized bacteriophage library for binding activity against CLas proteins InvA and TolC. Portions of each protein were chosen as antigens based on predicted binding availability and theorized necessary functions in pathogenicity. Binding affinity for individual scFv-expressing clones was confirmed by phage enzyme-linked immunosorbent assay (ELISA). The scFv sequences were stably transformed under the control of a tandem Cauliflower mosaic virus 35S (CaMV 2x35S) promoter by Agrobacterium tumefacien–mediated transformation into ‘Carrizo’ citrange (Citrus sinensis × Poncirus trifoliate), a citrus rootstock cultivar. Replicated plants of single transformations were inoculated by infestation with CLas positive asian citrus psyllid (Diaphorina citri), a CLas vector. Inoculation and disease progression was monitored through quantitative real-time polymerase chain reaction. Inoculated transgenic plants showed significantly reduced CLas titer compared with wild types. A subpopulation of transgenic plants displayed no measurable surviving bacteria after 12 months. Interestingly, individual replicated plants from the same transgenic events strongly segregated into two populations by resistance phenotype: a minority that were indistinguishable from wild-type plants and a majority that were highly resistant. Our results are the first step in developing a novel protection strategy for HLB.

Cauliflower mosaic virus P6 Dysfunctions Histone Deacetylase HD2C to Promote Virus Infection

Histone deacetylases (HDACs) are vital epigenetic modifiers not only in regulating plant development but also in abiotic- and biotic-stress responses. Though to date, the functions of HD2C—an HD2-type HDAC—In plant development and abiotic stress have been intensively explored, its function in biotic stress remains unknown. In this study, we have identified HD2C as an interaction partner of the Cauliflower mosaic virus (CaMV) P6 protein. It functions as a positive regulator in defending against CaMV infection. The hd2c mutants show enhanced susceptibility to CaMV infection. In support, the accumulation of viral DNA, viral transcripts, and the deposition of histone acetylation on the viral minichromosomes are increased in hd2c mutants. P6 interferes with the interaction between HD2C and HDA6, and P6 overexpression lines have similar phenotypes with hd2c mutants. In further investigations, P6 overexpression lines, together with CaMV infection plants, are more sensitive to ABA and NaCl with a concomitant increasing expression of ABA/NaCl-regulated genes. Moreover, the global levels of histone acetylation are increased in P6 overexpression lines and CaMV infection plants. Collectively, our results suggest that P6 dysfunctions histone deacetylase HD2C by physical interaction to promote CaMV infection.

A Core35S Promoter of Cauliflower Mosaic Virus Drives More Efficient Replication of Turnip Crinkle Virus

The 35S promoter with a duplicated enhancer (frequently referred to as 2X35S) is a strong dicotyledonous plant-specific promoter commonly used in generating transgenic plants to enable high-level expression of genes of interest. It is also used to drive the initiation of RNA virus replication from viral cDNA, with the consensus understanding that high levels of viral RNA production powered by 2X35S permit a more efficient initiation of virus replication. Here, we showed that the exact opposite is true. We found that, compared to the Core35S promoter, the 2X35S promoter-driven initiation of turnip crinkle virus (TCV) infection was delayed by at least 24 h. We first compared three versions of 35S promoter, namely 2X35S, 1X35S, and Core35S, for their ability to power the expression of a non-replicating green fluorescent protein (GFP) gene, and confirmed that 2X35S and Core35S correlated with the highest and lowest GFP expression, respectively. However, when inserted upstream of TCV cDNA, 2X35S-driven replication was not detected until 72 h post-inoculation (72 hpi) in inoculated leaves. By contrast, Core35S-driven replication was detected earlier at 48 hpi. A similar delay was also observed in systemically infected leaves (six versus four days post-inoculation). Combining our results, we hypothesized that the stronger 2X35S promoter might enable a higher accumulation of a TCV protein that became a repressor of TCV replication at higher cellular concentration. Extending from these results, we propose that the Core35S (or mini35S) promoter is likely a better choice for generating infectious cDNA clones of TCV.

Complete Genome Sequence of Isolate Bari 1, a Mild Strain of Cauliflower Mosaic Virus

We present here the complete genome sequence of isolate Bari 1, a mild strain of Cauliflower mosaic virus (CaMV). The isolate was collected from Diplotaxis tenuifolia (perennial wall-rocket) in Bari, Italy. The genome was 8,020 nucleotides long and shared ≤85.4% nucleotide identity to other CaMV isolates.

Cauliflower Mosaic Virus Utilizes Processing Bodies to Escape Translational Repression in Arabidopsis

Viral infections impose extraordinary RNA stress on a cell, triggering cellular RNA surveillance pathways like RNA decapping, nonsense-mediated decay and RNA silencing. Viruses need to maneuver between these pathways to establish infection and succeed in producing high amounts of viral proteins. Processing bodies (PBs) are integral to RNA triage in eukaryotic cells with several distinct RNA quality control pathways converging for selective RNA regulation. In this study, we investigate the role of Arabidopsis thaliana PBs during Cauliflower Mosaic Virus (CaMV) infection. We find that several PB components are co-opted into viral replication factories and support virus multiplication. This pro-viral role was not associated with RNA decay pathways but instead, we could establish PB components as essential helpers in viral RNA translation. While CaMV is normally resilient to RNA silencing, PB dysfunctions expose the virus to this pathway, similar to previous observations on transgenes. Transgenes, however, undergo RNA Quality Control dependent RNA degradation, whereas CaMV RNA remains stable but becomes translationally repressed through decreased ribosome association, revealing a unique dependence between PBs, RNA silencing and translational repression. Together, our study shows that PB components are co-opted by the virus to maintain efficient translation, a mechanism not associated with canonical PB functions.