Engineering novel types of wheat plants through RNA interference and genome editing

Crop plants suffer severe yield losses due to significant damages caused by aphids. The level of nitrogen fertilization has a major impact on the population of aphids (nymphs and adults). A high-level nitrogen fertilizer application produces the greatest aphid population. The objectives of this thesis are to develop effective and environmentally friendly pest management strategies for aphid control and to increase nitrogen use efficiency (NUE) for the development of sustainable agriculture. The main results are as following:
RNA interference (RNAi) technology is a versatile and environmentally friendly method for pest management in crop protection. Transgenic plants expressing siRNA/dsRNA and non-transformative methods, such as microinjection, feeding, and nanocarrier-delivered mediated RNAi approach, have been successfully applied in agricultural practice for insect pest control. Grain aphid (Sitobion miscanthi) is one of the most dominant and devastating insect pests in wheat, which causes substantial losses to wheat production each year. Engineering transgenic plants expressing double strand RNA (dsRNA) targeting an insect-specific gene has been demonstrated to provide an alternative environmentally friendly strategy for aphid management through plant-mediated RNA interference (RNAi). Here we identified and characterized a novel potential RNAi target gene (SmDSR33) which was a gene encoding a putative salivary protein. Stable transgenic wheat lines expressing dsRNA for targeted silencing of SmDSR33 in grain aphids through plant-mediated RNAi were generated. After feeding on transgenic wheat, the attenuated expression levels of SmDSR33 in aphids were observed when compared to aphids feeding on wild-type plants. The decreased SmDSR33 expression levels reduced significantly aphid fecundity and survival. We also observed altered aphid feeding behaviors such as longer duration of intercellular stylet pathway and shorter duration of passive ingestion in electropenetrography assays. Furthermore, both surviving aphids and their offspring exhibited decreased survival rates and fecundity, indicating that the silencing effect could be persistent and transgenerational in grain aphids. SmDSR33 can be selected as an effective RNAi target for wheat aphid control. Silencing of an essential salivary protein gene involved in ingestion through plant-mediated RNAi was a second target and could be exploited as an effective strategy for aphid control in wheat.
The fast-developing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been extensively applied for functional genomics studies and crop improvement. Nitrogen (N) is applied in relatively large amounts in agricultural practice to promote plant growth and development. To maximize yield, semi-dwarf crop varieties of the Green Revolution (GRV) require a high nitrogen fertilizer input. With the world's population growing and environmental pollution increasing, enhanced crop productivity with limited N supplies is a critical barrier. Improved nitrogen use efficiency (NUE) will be necessary for agricultural sustainability in the future. Wheat (Triticum aestivum L.) is a staple food crop consumed by more than 30% of world population. Nitrogen (N) fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production. However, undue N fertilizer application and the low N use efficiency (NUE) of modern wheat varieties are aggravating environmental pollution and ecological deterioration. Under nitrogen-limiting conditions, the rice (Oryza sativa L.) abnormal cytokinin response1 repressor1 (are1) mutant exhibits increased NUE, delayed senescence and consequently, increased grain yield. However, the function of ARE1 ortholog in wheat remains unknown. Here, we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698. We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of mutant lines either with partial or triple-null taare1 alleles. All mutant lines showed enhanced tolerance to N starvation, delayed senescence and increased grain yield in field conditions. In particular, the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control. Together, our results underscored the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE.
In conclusion, we engineered novel aphid-resistant and nitrogen-use efficient wheat germplasms through plant-mediated RNAi technology and CRISPR/Cas9-mediated genome editing technology.