Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production

Wildfire ignition probability in Belgium

In recent decades, large wildfires have inflicted considerable damage on valuable Natura 2000 regions in Belgium. Despite these events and the general perception that global change will exacerbate wildfire prevalence, this has not been studied yet in the Belgian context. Therefore, the national government initiated the national action plan on wildfires in order to evaluate the wildfire risk, on the one hand, and the materials, procedures, and training of fire services, on the other hand. This study focuses on the spatial distribution of the ignition probability, a component of the wildfire risk framework. In a first stage, we compile a historical wildfire database using (i) newspaper articles between 1994 and 2016 and (ii) a list of wildfire interventions between 2010 and 2013, provided by the government. In a second stage, we use a straightforward method relying on Bayes' rule and a limited number of covariates to calculate the ignition probability. It appears that most wildfire-prone areas in Belgium are located in heathland where military exercises are held. The provinces that have the largest relative areas with a high or very high wildfire risk are Limburg and Antwerp. Our study also revealed that most wildfire ignitions in Belgium are caused by humans (both arson and negligence) and that natural causes such as lightning are rather scarce. Wildfire prevention can be improved by (i) excluding military activity in fire-prone areas during the fire season, (ii) improving collaboration with foreign emergency services, (iii) concentrating the dedicated resources in the areas that display the highest ignition probabilities, (iv) improving fire detection methods, and (v) raising more awareness among the publicstatus: publishe

Isolation of tobacco DNA segments with plant promoter activity.

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA

Post transcriptional silencing of reporter transgenes in tobacco correlates whit DNA methylation

Endogenous plant genes or transgenes can be silenced on introduction of homologous gene sequences. Here we document a reporter gene-silencing event in Nicotiana tabacum that has a distinctive combination of features--i.e., (i) silencing occurs by a posttranscriptional process, (ii) silencing correlates with DNA methylation, and (iii) this de novo methylation is not restricted to cytosines located in the symmetrical motifs CG and CXG

A transgenic dTph1 insertional mutagenesis system for forward genetics in mycorrhizal phosphate transport of Petunia

The active endogenous dTph1 system of the Petunia hybrida mutator line W138 has been used in several forward-genetic mutant screens that were based on visible phenotypes such as flower morphology and color. In contrast, defective symbiotic phosphate (Pi) transport in mycorrhizal roots of Petunia is a hidden molecular phenotype as the symbiosis between plant roots and fungi takes place below ground, and, while fungal colonization can be visualized histochemically, Pi transport and the activity of Pi transporter proteins cannot be assessed visually. Here, we report on a molecular approach in which expression of a mycorrhiza-inducible bi-functional reporter transgene and insertional mutagenesis in Petunia are combined. Bi-directionalization of a mycorrhizal Pi transporter promoter controlling the expression of two reporter genes encoding firefly luciferase and GUS allows visualization of mycorrhiza-specific Pi transporter expression. A population of selectable transposon insertion mutants was established by crossing the transgenic reporter line with the mutator W138, from which the Pitransporter downregulated (ptd1) mutant was identified, which exhibits strongly reduced expression of mycorrhiza-inducible Pi transporters in mycorrhizal roots