"Clean" genome editing in grapevine (Vitis ssp.)

In recent years new plant breeding techniques (NPBT), and in particular genome editing via Crispr/Cas9, emerged as breakthrough tools for the genetic improvments of agricultural species, allowing to precisely modify specific genes in shorter time compared to traditional breeding and without altering the genetic heritage of cultivars. Grapevine, the most economical valuable fruit crop in the world, may receive a major benefit from NPBT since viticulture is based on a few elite varieties. However, to date the European Coummission (EC) has not yet deliberated on the legal status of the NPBT products, whether they should or should not be covered by GMO legislation (Directive 2001/18). Waiting for the EC decision, we applied the Crispr/Cas9 system in grapevine for the inactivation of the VvMLO7 gene which plays a key role in susceptibility to powdery mildew. Our "clean" strategy aims at leaving in th eplant genome the minimal trace of exogenous DNA. It used the classical Agrobacterium tumefaciens (A.t.) to introgress Cas9, the sgRNA and the selection marker gene nptIIand allow removing the T-DNA cassette from the grapevine genome once the targeted mutations have been obtained. To this purpose, the Flp recombinase gene under the control of a heat-shock inducible promoter has been integrated in the T-DNA as well as its recognition sites (FRT), placed next to the A.t. left and right borders. NptII- and Cas9- positive lines of 'Chardonnay', 'Thompson seedless' and 'Microvine' were analyzed by next generation sequencing in order to assess the induced mutations in the target sites. Subsequently, the site-specific removal of the T-DNA cassette was evaluated in the heat-treated lines by quantifying nptII copy number with Real-time PCR method. The effect of powdery mildew infection on VvMLO7 edited plants in currently under evaluation

Up the nose of the beholder? Aesthetic perception in olfaction as a decision-making process

Is the sense of smell a source of aesthetic perception? Traditional philosophical aesthetics has centered on vision and audition but eliminated smell for its subjective and inherently affective character. This article dismantles the myth that olfaction is an unsophisticated sense. It makes a case for olfactory aesthetics by integrating recent insights in neuroscience with traditional expertise about flavor and fragrance assessment in perfumery and wine tasting. My analysis concerns the importance of observational refinement in aesthetic experience. I argue that the active engagement with stimulus features in perceptual processing shapes the phenomenological content, so much so that the perceptual structure of trained smelling varies significantly from naive smelling. In a second step, I interpret the processes that determine such perceptual refinement in the context of neural decision-making processes, and I end with a positive outlook on how research in neuroscience can be used to benefit philosophical aesthetics

Evaluation of Transgenic ‘Chardonnay' ( Vitis vinifera ) Containing Magainin Genes for Resistance to Crown Gall and Powdery Mildew

Magainins, short peptides with broad-spectrum antimicrobial activity in vitro, were assayed for their ability to confer resistance to pathogens in transgenic grapevines. Embryogenic cell suspensions of ‘Chardonnay' (Vitis vinifera L.) were co-transformed by microprojectile bombardment with a plasmid carrying the npt-II gene and a second plasmid harboring either a natural magainin-2 (mag2) or a synthetic derivative (MSI99) gene. Magainin genes and the marker gene were driven by Arabidopsis ubiquitin-3 and ubiquitin-11 promoters, respectively. A total of 10 mag2 and 9 MSI99 regenerated lines were studied by Southern blot hybridization, which showed 1-6 transgene integration events into the plant genome. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed a variable range in transcription levels among mag2 and MSI99 lines. A positive correlation between number of integration events and transcription level was observed (p<0.05). Plants were acclimated and challenged in the greenhouse with either Agrobacterium vitis strains (bacterial crown gall pathogen) at 108cfu/ml or Uncinula necator (fungal powdery mildew pathogen) at 105 conidia/ml for evaluation of disease resistance. A total of 6 mag2 and 5 MSI99 lines expressing the antimicrobial genes exhibited significant reductions of crown gall symptoms as compared to non-transformed controls. However, only two mag2 lines showed measurable symptom reductions in response to U. necator, but not strong resistance. Our results suggest that the expression of magainin-type genes in grapevines may be more effective against bacteria than fungi. Additional strategies to enhance transgene expression and the spectrum of resistance to grape diseases are suggeste

Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period

<p>Abstract</p> <p>Background</p> <p>Transgenic trees currently are being produced by <it>Agrobacterium</it>-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, <it>attacin E</it>, in the apple cultivar 'Galaxy' grown in the field for 12 years.</p> <p>Results</p> <p>Using Southern and western blot analysis, we compared transgene copy number and observed stability of expression of this gene in the leaves and fruit in several transformed lines during a 12 year period. No silenced transgenic plant was detected. Also the expression of this gene resulted in an increase in resistance to fire blight throughout 12 years of orchard trial and did not affect fruit shape, size, acidity, firmness, weight or sugar level, tree morphology, leaf shape or flower morphology or color compared to the control.</p> <p>Conclusion</p> <p>Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs. This report shows that it is possible to improve a desirable trait in apple, such as the resistance to a pathogen, through genetic engineering, without adverse alteration of fruit characteristics and tree shape.</p

Reduced fire blight susceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system

The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, an MdDIPM4 knock-out has been produced in two Malus x domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty-seven transgenic lines were screened to identify CRISPR/Cas9-induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss-of-function mutation were inoculated with the pathogen. Highly significant reduction of susceptibility was observed compared to control plants. Sequencing of 5 potential off-target sites revealed no mutation event. Moreover, our construct contained a heat shock-inducible FLP/FRT recombination system designed specifically to remove the T-DNA harbouring expression cassettes for CRISPR/Cas9, marker gene and FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat-treated and screened by real-time PCR to quantify the exogenous DNA elimination. The T-DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DN

Genetic transformation of apple ( Malus x domestica ) without use of a selectable marker gene

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developing strategies (site-specific recombination, homologous recombination, transposition, and cotransformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. For the commercialization of genetically transformed plants, use of a completely marker-free technology would be desirable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations for the public. With this goal in mind, a technique for apple transformation was developed without use of any selectable marker. Transformation of the apple genotype "M.26” with the constructs pPin2Att35SGUSintron and pPin2MpNPR1 was achieved. In different experiments, 22.0-25.4% of regenerants showed integration of the gene of interest. Southern analysis in some transformed lines confirmed the integration of one copy of the gene. Some of these transformed lines have been propagated and used to determine the uniformity of transformed tissues in the plantlets. The majority of the lines are uniformly transformed plants, although some lines are chimeric, as also occurs with the conventional transformation procedure using a selectable marker gene. A second genotype of apple, "Galaxy,” was also transformed with the same constructs, with a transformation efficiency of 13

Regeneration of non-chimeric plants from DNA-free edited grapevine protoplasts

The application of New Breeding Techniques (NBTs) in Vitis vinifera is highly desirable to introduce valuable traits while preserving the genotype of the elite cultivars. However, a broad application of NBTs through standard DNA-based transformation is poorly accepted by public opinion and law regulations in Europe and other countries due to the stable integration of exogenous DNA, which leads to transgenic plants possibly affected by chimerism. A single-cell based approach, coupled with a DNA-free transfection of the CRISPR/Cas editing machinery, constitutes a powerful tool to overcome these problems and maintain the original genetic make-up in the whole organism. We here describe a successful single-cell based, DNA-free methodology to obtain edited grapevine plants, regenerated from protoplasts isolated from embryogenic callus of two table grapevine varieties (V. vinifera cv. Crimson seedless and Sugraone). The regenerated, non-chimeric plants were edited on the downy- and powdery-mildew susceptibility genes, VviDMR6 and VviMlo6 respectively, either as single or double mutant

VvEPFL9-1 Knock-Out via CRISPR/Cas9 reduces stomatal density in grapevine

10openInternationalInternational coauthor/editorEpidermal Patterning Factor Like 9 (EPFL9), also known as STOMAGEN, is a cysteine-rich peptide that induces stomata formation in vascular plants, acting antagonistically to other epidermal patterning factors (EPF1, EPF2). In grapevine there are two EPFL9 genes, EPFL9-1 and EPFL9-2 sharing 82% identity at protein level in the mature functional C-terminal domain. In this study, CRISPR/Cas9 system was applied to functionally characterize VvEPFL9-1 in ‘Sugraone’, a highly transformable genotype. A set of plants, regenerated after gene transfer in embryogenic calli via Agrobacterium tumefaciens, were selected for evaluation. For many lines, the editing profile in the target site displayed a range of mutations mainly causing frameshift in the coding sequence or affecting the second cysteine residue. The analysis of stomata density revealed that in edited plants the number of stomata was significantly reduced compared to control, demonstrating for the first time the role of EPFL9 in a perennial fruit crop. Three edited lines were then assessed for growth, photosynthesis, stomatal conductance, and water use efficiency in experiments carried out at different environmental conditions. Intrinsic water-use efficiency was improved in edited lines compared to control, indicating possible advantages in reducing stomatal density under future environmental drier scenarios. Our results show the potential of manipulating stomatal density for optimizing grapevine adaptation under changing climate conditions.openClemens, Molly; Faralli, Michele; Lagreze, Jorge; Bontempo, Luana; Piazza, Stefano; Varotto, Claudio; Malnoy, Mickael; Oechel, Walter; Rizzoli, Annapaola; Dalla Costa, LorenzaClemens, M.; Faralli, M.; Lagreze, J.; Bontempo, L.; Piazza, S.; Varotto, C.; Malnoy, M.; Oechel, W.; Rizzoli, A.; Dalla Costa, L

Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine

10openInternationalItalian coauthor/editorErysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasionopenPessina, S.; Lenzi, L.; Perazzolli, M.; Campa, M.; Dalla Costa, L.; Urso, S.; Vale, G.; Salamini, F.; Velasco, R.; Malnoy, M.Pessina, S.; Lenzi, L.; Perazzolli, M.; Campa, M.; Dalla Costa, L.; Urso, S.; Vale, G.; Salamini, F.; Velasco, R.; Malnoy, M.A