For the past few decades, molecular techniques and plant biotechnology have greatly aided our understanding of plant biology and have created crop plants with valuable new agronomic and nutritional traits for the benefits of farmers, consumers and the environment. Plants with stress tolerance, disease and pest resistance, herbicide resistance and superior nutritional composition have been produced through the approaches of transgene integration and RNAi (RNA interference)-gene silencing. However, transgenic plants or genetically modified organisms (GMOs) are mostly produced with a single transgene inserted into the plant genome, and the transgenes are often from heterologous organisms. It is difficult to engineer plants that are resistant to more than one pest through the single transgene integration. Engineering plants that are tolerant to a single environmental stress such as heat or drought is also a challenge, as complex reactions are elicited from plants under stress. Additionally, the integration of transgenes is random in the genome and hard to control. Technologies other than transgene integration should be explored to better engineer stress tolerance and pest resistance in plants. Increasing public concern regarding GMOs should also be addressed. Whole genome information and functional genomics have greatly enhanced our ability to engineer plant genomes in the past decade. As more information on the molecular mechanisms between plant and pest/stress interactions becomes available, it is feasible to manipulate plant genomes by disrupting the host factors that contribute to the pest and stress interactions. RNAi (RNA interference) technology has been used to produce plants that are resistant to diseases. However, RNAi normally leads to down-regulation rather than complete inhibition of target genes.The new, exciting and amazing gene editing technology termed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system that came into the spotlight about two years ago can potentially solve the problems encountered by conventional transgene and RNAi technologies. We propose to construct two CRISPR/Cas vectors for dicotyledonous and monocotyledonous plants (dicot and monocot) by targeting the negative regulators of disease resistance and stress tolerance. It is intended that these two vectors be used for any dicot or monocot plant, with at most the substitution of promoters for specific plant species. These two vectors are different from all the other CRISPR/Cas vectors used in plant gene editing: the 20-bp target sequence can be easily and efficiently substituted by other 20-bp sequences of any gene from any dicot or monocot plant with the simple Q5 site-directed mutagenesis kit (New England BioLabs) by an one-step PCR (polymerase chain reaction). Grape will be the dicot plant to be engineered for downy mildew disease resistance. Grape is an important commodity worldwide in the fruit market and wine industry. In New Jersey, the acreage of wine grapes doubled from 2002 to 2007 (Rutgers NJAES information). Currently, there are more than 190 farms statewide, in three designated American Viticulture Areas (AVAs). However, grapevine downy mildew caused by Plasmopara viticola is a serious disease affecting mainly Vitis vinifera varieties in New Jersey, as well as around the world. Cross-breeding and fungicide spray have been the main control measures for grapevine downy mildew disease for centuries. This new grape variety will require less fungicide applications, benefiting grape growers, consumers and the environment.Turfgrass will be the monocot to be engineered for dollar spot disease resistance and stress tolerance. Creeping bentgrass (Agrostis stolonifera L.) is one of the most widely used cool-season grass species on golf courses, bowling greens and tennis courts. However, there is no cultivar of creeping bentgrass that is considered to be completely resistant to dollar spot disease caused by Sclerotinia homoeocarpa F.T. Bennet. More than 70% of fungicide used on golf courses are to control dollar spot, brown patch (Rhizoctonia solani Kuhn), and anthracnose [Colletotrichum graminicola (Ces.) G.W. Wils]. Additionally, creeping bentgrass is stressed by heat and drought as other grass species during summer months. As turfgrass is a multi-billion dollar industry contributing to the national economy, disease resistant and stress tolerant turfgrass will benefit turfgrass industry and the environment.
|Effective start/end date||12/1/14 → 11/30/19|
- National Institute of Food and Agriculture (National Institute of Food and Agriculture (NIFA))