SummaryDELLA proteins are plant nuclear factors that restrain growth and proliferation in response to hormonal signals. The effects of the manipulation of the DELLA pathway in the making of a berry-like fruit were investigated. The expression of the Arabidopsis thaliana gain-of-function DELLA allele Atgai del in tomato (Solanum lycopersicum L.) produced partially sterile dwarf plants and compacted influorescences, as expected for a constitutively activated growth repressor. In contrast, antisense silencing of the single endogenous tomato DELLA gene homologue (SlDELLA) produced slender-like plants with elongated flower trusses. Interestingly, the depletion of SlDELLA in tomato was sufficient to overcome the growth arrest normally imposed on the ovary at anthesis, resulting in parthenocarpic fruits in the absence of pollination. Antisense SlDELLA-engineered fruits were smaller in size and elongated in shape compared with wild type. Cell number estimations showed that fruit set, resulting from reduced SlDELLA expression, arose from activated cell elongation at the longitudinal and lateral axes of the fruit pericarp, bypassing phase-II (post-pollination) cell divisions. Parthenocarpy caused by SlDELLA depletion is facultative, as hand pollination restored wild-type fruit phenotype. This indicates that fertilization-associated SlDELLA-independent signals are operational in ovary-fruit transitions. SlDELLA was also found to restrain growth in other reproductive structures, affecting style elongation, stylar hair primordial growth and stigma development.
Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.
Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC-DAD and the volatile products by GC/MS. β-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9',10') bonds and generating β-ionone, although β-cyclocitral resulting from a 7,8 (7',8') cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates.
Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.
Animals are continuously threatened by pathogens entering the body through natural openings. Here we show that in chicken ( Gallus gallus ), secretory IgA (sIgA) protects the epithelia lining these natural cavities. A gene encoding a chicken polymeric Ig receptor ( GG-pIgR ), a key component of sIgA, was identified, and shown to be expressed in the liver, intestine and bursa of Fabricius. All motifs involved in pIgR function are present, with a highly conserved Ig-binding motif in the first Ig-like domain. Physical association of GG-pIgR with pIgA in bile and intestine demonstrates that this protein is a functional receptor. Thus, as shown for mammals, this receptor interacts with J-chain-containing polymeric IgA (pIgA) at the basolateral epithelial cell surface resulting in transcytosis and subsequent cleavage of the pIgR, releasing sIgA in the mucosal lumen. Interestingly, the extracellular portion of GG-pIgR protein comprises only four Ig-like domains, in contrast with the five domain structure found in mammalian pIgR genes. The second Ig-like domain of mammalian pIgR does not have an orthologous domain in the chicken gene. The presence of pIgR in chicken suggests that this gene has evolved before the divergence of birds and reptiles, indicating that secretory Igs may have a prominent role in first line defence in various non-mammalian species.
Petal senescence is an example of a highly reproducible cell death programme. In this programme, DNA is fragmented internucleosomally and cells with condensed nuclei containing an increased number of 5' ends can be detected with the TUNEL technique. The pea homologue of defender against apoptotic death (dad), a gene described to suppress endogenous programmed cell death in Caenorhabditis elegans and mammals was isolated. Expression studies show that dad declines dramatically upon flower anthesis disappearing in senescent petals, and is down-regulated by the plant hormone ethylene.
Crocetin, one of the few colored apocarotenoids known in nature, is present in flowers and fruits and has long been used medicinally and as a colorant. Saffron is the main source of crocetin, although a few other plants produce lower amounts of this apocarotenoid. Notably, Buddleja davidii accumulates crocetin in its flowers. Recently, a carotenoid dioxygenase cleavage enzyme, CCD2, has been characterized as responsible for crocetin production in Crocus species. We searched for CCD2 homologues in B. davidii and identified several CCD enzymes from the CCD1 and CCD4 subfamilies. Unexpectedly, two out of the three CCD4 enzymes, namely BdCCD4.1 and BdCCD4.3, showed 7,8;7',8' activity in vitro and in vivo over zeaxanthin. In silico analyses of these enzymes and CCD2 allowed the determination of key residues for this activity. Both BdCCD4 genes are highly expressed during flower development and transcripts levels parallel the accumulation of crocins in the petals. Phylogenetic analysis showed that BdCCD4.2 grouped with almost all the characterized CCD4 enzymes, while BdCCD4.1 and BdCCD4.3 form a new sub-cluster together with CCD4 enzymes from certain Lamiales species. The present study indicates that convergent evolution led to the acquisition of 7,8;7',8' apocarotenoid cleavage activity in two separate CCD enzyme families.
The current CoVid-19 crisis is revealing the strengths and the weaknesses of the world’s capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. In this work, we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. For the design of the antibodies, we took advantage, among other data sources, of the DNA sequence information made rapidly available by other groups in preprint publications. mAbs were engineered as single-chain fragments fused to a human gamma Fc and transiently expressed using a viral vector. In parallel, we also produced the recombinant SARS-CoV-2 N protein and the receptor binding domain (RBD) of the Spike protein in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies, we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Our results indicate that gram amounts of anti-SARS-CoV-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using N. benthamiana as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses.
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