Heterologous expression of Arabidopsis SOS3 increases salinity tolerance in Petunia.

BACKGROUND: Salinity stress is one of the most important rising problems worldwide. It significantly reduces plant growth and development, mainly by provoking excessive uptake of ions such as Na+. The Salt Overly Sensitive (SOS) machinery is a well-known signaling pathway that help plants to maintain ion homeostasis by reducing Na+ accumulation in plant cells. Overexpression of key components of this pathway has been reported to increase salinity stress tolerance in some plant species. METHODS AND RESULTS: In this study, SOS3 cDNA isolated from Arabidopsis seedlings was transferred into the Petunia genome by two common plant transformation methods, Agrobacterium and biolistic gun. Transgene integration and expression in putative lines were evaluated by PCR and RT-PCR techniques. In vitro and greenhouse evaluation of transgenic plants for salt tolerance showed that, compared to the wild type, transgenic plants overexpressing AtSOS3 gene exhibit enhanced salt tolerance in response to high NaCl concentrations. CONCLUSIONS: These results not only demonstrate the potential of SOS pathway components to improve salt tolerance in Petunia, but also provide more evidence for functional conservation of the SOS salt tolerance signaling pathway among different plant families.

Alfalfa mosaic virus nanoparticles-based in situ vaccination induces antitumor immune responses in breast cancer model.

Background: Preclinical and clinical studies show that local and systemic antitumor efficacy is achievable by in situ vaccination (ISV) using plant virus nanoparticles in which immunostimulatory reagents are directly administered into the tumor rather than systemically. Aim: To investigate a minimally studied plant virus nanoparticle, alfalfa mosaic virus (AMV), for ISV treatment of 4T1, the very aggressive and metastatic murine triple-negative breast cancer model. Materials & methods: AMV nanoparticles were propagated and characterized. Their treatment impact on in vivo tumors were analyzed using determination of inherent immunogenicity, cytokine analysis, western blotting analysis and immunohistochemistry methodologies. Results: AMV used as an ISV significantly slowed down tumor progression and prolonged survival through immune mechanisms (p < 0.001). Conclusion: Mechanistic studies show that ISV with AMV increases costimulatory molecules, inflammatory cytokines and immune effector cell infiltration and downregulates immune-suppressive molecules.

Plant viral nanoparticles for packaging and in vivo delivery of bioactive cargos.

Nanoparticles have unique capabilities and considerable promise for many different biological uses. One capability is delivering bioactive cargos to specific cells, tissues, or organisms. Depending on the task, there are multiple variables to consider including nanoparticle selection, targeting strategies, and incorporating cargo so it can be delivered in a biologically active form. One nanoparticle option, genetically controlled plant viral nanoparticles (PVNPs), is highly uniform within a given virus but quite variable between viruses with a broad range of useful properties. PVNPs are flexible and versatile tools for incorporating and delivering a wide range of small or large molecule cargos. Furthermore, PVNPs can be modified to create nanostructures that can solve problems in medical, environmental, and basic research. This review discusses the currently available techniques for delivering bioactive cargos with PVNPs and potential cargos that can be delivered with these strategies. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Isolation and identification of Pseudomonas azotoformans for induced calcite precipitation.

Biomineralization is a process by which living organisms produce minerals. The extracellular production of these biominerals by microbes has potential for various bioengineering applications. For example, crack remediation and improvement of durability of concrete is an important goal for engineers and biomineral-producing microbes could be a useful tool in achieving this goal. Here we report the isolation, biochemical characterization and molecular identification of Pseudomonas azotoformans, a microbe that produces calcite and which potentially be used to repair cracks in concrete structures. Initially, 38 bacterial isolates were isolated from soil and cements. As a first test, the isolates were screened using a urease assay followed by biochemical tests for the rate of urea hydrolysis, calcite production and the insolubility of calcite. Molecular amplification and sequencing of a 16S rRNA fragment of selected isolates permitted us to identify P. azotoformans as a good candidate for preparation of biotechnological concrete. This species was isolated from soil and the results show that among the tested isolates it had the highest rate of urea hydrolysis, produced the highest amount of calcite, which, furthermore was the most adhesive and insoluble. This species is thus of interest as an agent with the potential ability to repair cracks in concrete.

Genetic analysis of Iranian population of Potato leafroll virus based on ORF0.

Potato leafroll virus (PLRV) is a destructive virus of potatoes and responsible for high yield losses wherever potatoes are grown. In this study, DNA fragments containing ORF0 from each of nine PLRV isolates was sequenced. Sequence analysis data using 36 isolates from 12 different countries including 14 Iranian isolates showed that the identities of ORF0 at both nucleotide and amino acid levels between the Iranian isolates were 96-100 % and these isolates were more similar to the European PLRV isolates than to the other isolates. Furthermore, phylogenetic and population genetic analysis were carried out on the basis of full-length ORF0 and overlapping and non-overlapping regions of ORF0 and ORF1 (ORF0/1) which revealed that PLRV isolates were not geographically resolved. Also, we identified negative selection with different ratios for each of the mentioned genomic regions suggesting effects of F-box motif and -1 frameshift on ORF0 non-overlapping region and ORF0/1 in the selection pressure, respectively. Five recombination events were detected in the Iranian, Australian, and European isolates suggesting an important role for this phenomenon in influencing genetic diversity within this virus population.

MSI4/FVE interacts with CUL4-DDB1 and a PRC2-like complex to control epigenetic regulation of flowering time in Arabidopsis.

Flowering at the right time is crucial to ensure successful plant reproduction and seed yield and is dependent on both environmental and endogenous parameters. Among the different pathways that impinge on flowering, the autonomous pathway promotes floral transition independently of day length through the repression of the central flowering repressor flowering locus C (FLC). FLC blocks floral transition by repressing flowering time integrators such as flowering locus T (FT). MSI4/FVE is a key regulator of the autonomous pathway that reduces FLC expression. Here we report that the MSI4 protein is a DDB1 and CUL4-associated factor that represses FLC expression through its association with a CLF-Polycomb Repressive Complex 2 (PRC2) in Arabidopsis. Thus, the lack of MSI4 or decreased CUL4 activity reduces H3K27 trimethylation on FLC, but also on its downstream target FT, resulting in increased expression of both genes. Moreover, CUL4 interacts with FLC chromatin in an MSI4-dependant manner, and the interaction between MSI4 and CUL4-DDB1 is necessary for the epigenetic repression of FLC. Overall our work provides evidence for a unique functional interaction between the cullin-RING ubiquitin ligase (CUL4-DDB1(MSI4)) and a CLF-PRC2 complex in the regulation of flowering timing in Arabidopsis.

The Arabidopsis CUL4-DDB1 complex interacts with MSI1 and is required to maintain MEDEA parental imprinting.

Protein ubiquitylation regulates a broad variety of biological processes in all eukaryotes. Recent work identified a novel class of cullin-containing ubiquitin ligases (E3s) composed of CUL4, DDB1, and one WD40 protein, believed to act as a substrate receptor. Strikingly, CUL4-based E3 ligases (CRL4s) have important functions at the chromatin level, including responses to DNA damage in metazoans and plants and, in fission yeast, in heterochromatin silencing. Among putative CRL4 receptors we identified MULTICOPY SUPPRESSOR OF IRA1 (MSI1), which belongs to an evolutionary conserved protein family. MSI1-like proteins contribute to different protein complexes, including the epigenetic regulatory Polycomb repressive complex 2 (PRC2). Here, we provide evidence that Arabidopsis MSI1 physically interacts with DDB1A and is part of a multimeric protein complex including CUL4. CUL4 and DDB1 loss-of-function lead to embryo lethality. Interestingly, as in fis class mutants, cul4 mutants exhibit autonomous endosperm initiation and loss of parental imprinting of MEDEA, a target gene of the Arabidopsis PRC2 complex. In addition, after pollination both MEDEA transcript and protein accumulate in a cul4 mutant background. Overall, our work provides the first evidence of a physical and functional link between a CRL4 E3 ligase and a PRC2 complex, thus indicating a novel role of ubiquitylation in the repression of gene expression.

Viral suppression of RNA silencing by destabilisation of ARGONAUTE 1.

RNA silencing is a manifestation of a ubiquitous phenomenon that acts, at least in plants and some insects, as a natural defense mechanism against viruses. As a counter-strategy, viruses have evolved to encode silencing suppressor proteins (SSPs) that can block the defense response and evade the host immunity. Although numerous SSP have been identified, little information is available on the molecular basis of their mode of action. Among SSPs, the polerovirus protein P0 functions as an F-box protein that targets an essential actor of the silencing pathway. Our work demonstrates that one of the main targets is ARGONAUTE 1 (AGO1), a key component of the RISC effector complex. By a physical interaction with AGO1, P0 mediates AGO1 protein degradation in planta. This is the first report of a plant virus that exploits components of the host ubiquitination machinery to overcome RNA silencing. This unusual mode of action may provide some clues concerning the mechanism governing phloem tropism of poleroviruses.

The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0's silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0's expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.

F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function.

Plants employ small RNA-mediated posttranscriptional gene silencing as a virus defense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode of action of most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 of two Arabidopsis-infecting poleroviruses interacts by means of a conserved minimal F-box motif with Arabidopsis thaliana orthologs of S-phase kinase-related protein 1 (SKP1), a component of the SCF family of ubiquitin E3 ligases. Point mutations in the F-box-like motif abolished the P0-SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity of P0. Knockdown of expression of a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus infection. Together, the results support a model in which P0 acts as an F-box protein that targets an essential component of the host posttranscriptional gene silencing machinery.