Transformative Research

Developing a Marker-Free Genome Editing methodology for clonally-propagated crop:

New plant breeding techniques (NBPTs) like genome editing have recently received the attention of the plant scientific community because it may result in major breakthroughs for crop improvement. In grapevine, Site-Specific Nuclease-mediated editing of gene is of special interest to target genes that negatively impact economically important traits like disease resistance, drought tolerance and quality traits. Grapevine genes that confer susceptibility to Powdery Mildew or genes that are associated with the synthesis of off-flavor compounds like methoxypyrazines are being considered relevant candidates for developing new grapevine varieties resistant to PM or methoxypyrazine-free. For better public acceptance, the delivery of the ribonucleoprotein complex (SSN+single guide RNA) that does not involve DNA integration of a forgein DNA is by far the most appropriate for clonally-propagated plant material like grapevine. The transient delivery of the ribonucleoprotein complex alos offers the advantage to minimize the risk of off-targets commonly observed with SSN integrated to the genome in the context of stable transformation. However, there are several constraints for marker-free genome editing in grapevine material. It is possible to deliver into “naked” grapevine protoplasts (plant cells without cell wall) (Figure 6); but plant regeneration from grapevine protoplasts is very difficult. Major efforts should be conducted with the development of a methodology for optimized permeation and cell internalization of the RNP into regenerable tissue.
The use of embryogenic cells from which individual plants can be regenerated will address the first constraint. The use of Cell-Penetrating-Peptides to facilitate the penetration and the internalization of the RNPs could address the main issue of the delivering the cargo (Ramakrishna et al., 2014, Numata et al., 2018). We have developed a research project funded by the Wine Industry for a direct delivery of a SSN to edit grapevine material for key genes associated with performance traits. As a fisrt step, we aim to prove the feasibility of the direct delivery of a RNP into regenerable embryogenic cells.

Developing A Spray Induced Gene Silencing methodology for crop protection and improved fruit composition:

RNA-mediated interference (RNAi) is a cellular mechanism to silence gene expression, where RNA-derived molecules containing essential genetic information are degraded by small RNAs produced by a cell thus interfere with gene expression and thus cellular function. Recently, small RNAs were shown to move in both directions between the host plant and the pathogen. Pathogens use the RNAi mechanism to send small RNAs (sRNAs) that repress plant resistance genes to facilitate their development. This plant regulatory pathway can be used as a tool to suppress either virulent genes of pathogens or native genes that render the plant susceptible to pathogens. Exploiting this RNAi pathway, plants were genetically engineered to produce RNA against essential genes of the pathogen(s) to confer plant resistance against insect predation or diseases. This approach is called Host-Induced Gene Silencing (HIGS). However, the weak acceptance of GMOs by the public for food crops has hampered its broad application. A new innovative strategy, Spray-Induced Gene Silencing (SIGS), could allow RNAi technology to be utilized without the need for genetic modification of plants and would be transient on plant tissue reducing the concerns about residues on fruit and wine.
dsRNA molecules sprayed on the leaf surface were shown to be taken up by plant cells, transported to non-treated areas, and processed into sRNAs. These findings indicate that these applications can have systemic activity further enhancing their utility for pest and disease control. The premise is thus to deliver dsRNAs with a formulation made with clay particles using the same spray technology used for fungicides. Once applied, the dsRNA molecules complementary to target genes will disrupt the expression of the dsRNA-targeted genes in either the plant itself or the pathogens. The efficacy of SIGS was demonstrated for pest protection in multiple crops such as wheat, cucumber, pepper, etc.; and large-scale field trials are currently evaluated in Australia. The dsRNAs are reported to be stable and afford protection against the pathogen for up to 7-10 days, after which they degrade and leave no residue. The Fungus Erysiphe necator, responsible for Grape Powdery Mildew (GPM), is a significant threat to wine production that is mainly controlled using large quantities of fungicides. However, the emergence of fungicide resistance to chemicals suggests the need for rapid development of new strategies to control GPM. There is also a concern about the environmental impact of large-scale fungicide applications. Recent research demonstrates that agricultural pests, such as insects, nematodes, and fungi can be killed by exogenously supplied RNA molecules (Figure 7) opens opportunities to develop alternative field applications to control Powdery Mildew. We developed a SIGS to control GPM, we propose to accomplish three objectives.
In the first objective, we identified the stretches of RNA sequences with the maximum interference activity to silence fungal and native genes, EnCYP51, EnDCL1,2, and VitviMLOs 6,7, 11, respectively. In the second objective, we are currently evaluating the uptake and processing of RNA molecules by the grapevine and their systemic effects on the reduction of GPM. Finally, we will determine the efficacy of clay nanoparticles to stabilize and extend the lifespan of sprayed dsRNA molecules on grapevine leaves. Overall, we hope to provide the grape community with an RNAi-based spray technique for disease control. Our study will evaluate various aspects including the efficacy of silencing on the target genes, one adjuvant to use for long-lasting effects of dsRNA applications, the level of systemic effects, and the degree of PM-resistance afforded by the system in the laboratory studies. The results will be used for the development of field trials to evaluate the impact of SIGS system in reducing the cost of grapevine GPM management.