The fungi Botrytis cinerea and Erysiphe necator are responsible for gray mold and powdery mildew diseases, respectively, which are among the most devastating diseases of grapes. Two endochitinase (ech42 and ech33) genes and one N-acetyl-β-D-hexosaminidase (nag70) gene from biocontrol agents related to Trichoderma spp. were used to develop a set of 103 genetically modified (GM) 'Thompson Seedless' lines (568 plants) that were established in open field in 2004 and evaluated for fungal tolerance starting in 2006. Statistical analyses were carried out considering transgene, explant origin, and plant response to both fungi in the field and in detached leaf assays. The results allowed for the selection of the 19 consistently most tolerant lines through two consecutive years (2007-2008 and 2008-2009 seasons). Plants from these lines were grafted onto the rootstock Harmony and established in the field in 2009 for further characterization. Transgene status was shown in most of these lines by Southern blot, real-time PCR, ELISA, and immunostrips; the most tolerant candidates expressed the ech42-nag70 double gene construct and the ech33 gene from a local Hypocrea virens isolate. B. cinerea growth assays in Petri dishes supplemented with berry juices extracted from the most tolerant individuals of the selected population was inhibited. These results demonstrate that improved fungal tolerance can be attributed to transgene expression and support the iterative molecular and physiological phenotyping in order to define selected individuals from a population of GM grapevines.

The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of ‘Thompson Seedless’ somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

Successfully gene editing (GE) in Prunus spp. has been delayed due to its woody nature presenting additional difficulties in both, proper regeneration protocols and designing efficient gene transfer techniques. The availability of adequate, single cell culture techniques for GE such as protoplast regeneration, is a limiting step for the genus and for this reason, the improvement of regular regeneration protocols and finding more efficient techniques for the delivery of the “editing reagents” seem to be a reasonable strategy to incorporate GE in the genus. During the last 10 years, we have focused our efforts optimizing some previous regeneration and gene transfer procedures for Japanese plum (P. salicina), sweet cherry (P. avium) and peach (P. persica) to incorporate them into a GE technology on these species. In parallel, delivery techniques for the CRISPR/Cas9 editing components, i.e., guide RNA (gRNA) and Cas9, have been developed with the aim of improving gene targeting efficiencies. In that line, using DNA virus-based replicons provides a significant improvement, as their replicational release from their carriers enables their enhanced expression. Here, we make a brief overview of the tissue culture and regeneration protocols we have developed for P. salicina, P. avium and P. persica, and then we proceed to describe the use of Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the editing reagents in vivo and to evaluate their editing capability on individuals derived from Agrobacterium-mediated gene transfer experiments of these species. We show part of our characterization assays using new BeYDV-derived vectors harboring multiple gRNAs, the Cas9 gene, and the green fluorescent protein reporter gene. We also describe a dedicated genome analysis tool, by which gRNA pairs can be designed to address gene deletions of the target genes and to predict off-target sequences. Finally, as an example, we show the general results describing GE of the peach TERMINAL FLOWER 1 gene and some preliminary characterizations of these materials.