Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines.

Selectable marker genes are useful for recognizing which cells have integrated specific sequences in their genome after genetic transformation processes. They are especially important for fruit trees genetic transformation to individuate putatively genetically modified events, because most of the protocols used to genetic engineer these species are often unsuccessful or with low efficiency. Traditional selectable marker genes, mainly of bacterial origin, confer antibiotics/herbicides-resistance or metabolic advantages to transformed cells. Genes that allow the visual recognition of engineered tissues without using any selective agent, such as morphogenic regulators and reporter genes, are also used as selection tools to in vitro identify genetically modified regenerated lines. As final step, genetic engineered plants should be tested in field conditions, where selectable marker genes are no longer necessary, and strongly unpopular especially for the commercial development of the new products. Thus, different approaches, mainly based on the use of site-specific recombinases and/or editing nucleases, are being now used to recover marker-free fruit crops. This review describes and comments the most used and suitable selection tools of interest, particularly for fruit tree genetic engineering. Lastly, a spotlight highlights the biosafety aspects related to the use of selectable marker genes exploited for fruit species genetic engineering.

Strawberry fruit is highly appreciated worldwide for its organoleptic and healthy properties. However, this plant is attacked by many pathogenic fungi, which significantly affect fruit production and quality at pre- and post-harvest stages, making chemical applications the most effective but undesirable strategy to control diseases that has been found so far. Alternatively, genetic manipulation, employing plant key genes involved in defense, such as members of the NPR-like gene family, has been successful in many crops to improve resistance. The identification and use of the endogenous counterpart genes in the plant of interest (as it is the case of strawberry) is desirable as it would increase the favorable outcome and requires prior knowledge of their defense-related function. Using RNAi technology in strawberry, transient silencing of Fragaria ananassa NPR3 members in fruit significantly reduced tissue damage after Colletotrichum acutatum infection, whereas the ectopic expression of either FaNPR3.1 or FaNPR3.2 did not have an apparent effect. Furthermore, the ectopic expression of FaNPR3.2 in Arabidopsis thaliana double-mutant npr3npr4 reverted the disease resistance phenotype to Pseudomonas syringe to wild-type levels. Therefore, the results revealed that members of the strawberry FaNPR3 clade negatively regulate the defense response to pathogens, as do their Arabidopsis AtNPR3/AtNPR4 orthologs. Also, evidence was found showing that FaNPR3 members act in strawberry (F. ananassa) as positive regulators of WRKY genes, FaWRKY19 and FaWRKY24; additionally, in Arabidopsis, FaNPR3.2 negatively regulates its orthologous genes AtNPR3/AtNPR4. We report for the first time the functional characterization of FaNPR3 members in F. ananassa, which provides a relevant molecular basis for the improvement of resistance in this species through new breeding technologies.

Strawberry fruit is highly appreciated worldwide for its organoleptic and healthy properties. However, this plant is attacked by many pathogenic fungi, which significantly affect fruit production and quality at pre- and post-harvest stages, making chemical applications the most effective but undesirable strategy to control diseases, so far. Alternatively, genetic manipulation employing plant key genes involved in defense, such as members of the NPR-like gene family, has been successful in many crops to improve resistance. The identification and use of the endogenous counterpart genes in the plant of interest (as it is the case of strawberry) is desirable as it would increase the favorable outcome and requires prior knowledge of their defense related function. Using RNAi technology in strawberry, we show that transient silencing of Fragaria ananassa NPR3 members in fruit significantly reduces tissue damage after Colletotrichum acutatum infection whereas the ectopic expression of either FaNPR3.1 or FaNPR3.2 does not have an apparent effect. Furthermore, the ectopic expression of FaNPR3.2 in Arabidopsis thaliana double mutant npr3npr4 reverts the disease resistance phenotype to Pseudomonas syringe to wild type levels. Therefore, our results reveal that members of the strawberry FaNPR3 clade negatively regulate the defense response to pathogens, as do their Arabidopsis AtNPR3/AtNPR4 orthologs. Also, we provide evidence that FaNPR3 members act in strawberry (F. ananassa) as positive regulators of WRKY genes, FaWRKY19 and FaWRKY24, and that in Arabidopsis, FaNPR3.2 negatively regulates its orthologous genes AtNPR3/AtNPR4. We report for the first time the functional characterization of FaNPR3 members in F. ananassa which provides a relevant molecular basis for the improvement of resistance in this species through new breeding technologies.