RNAi-based strategies for insect pest and pathogen control: A study on RNAi delivery and dsRNA production

Leilei Yang

Plant pests and pathogens are among the biggest threats to food production, security, and safety worldwide. RNA interference (RNAi), as a powerful tool for silencing genes in eukaryotic organisms, offers a promising novel technology for management of insect pests and pathogens. However, major obstacles remain in applying RNAi, such as the lack of efficient RNAi delivery methods and high costs of dsRNA production. This study aims to address these two issues, using the important insect pest Spodoptera frugiperda and fungal pathogen Fusarium oxysporum as the targets. In the first part of my study, I employed oral feeding assays to investigate the exogenous RNAi (exo-RNAi) efficiency of G-U base-paired hairpin RNA (G:U hpRNA) against S. frugiperda. Northern blot hybridization showed that in vitro transcribed G:U hpRNA painted on corn leaves remained relatively intact with or without insect feeding, indicating its relatively high stability on corn leaves. In addition, significant amounts of G:U hpRNA were detected from insect larvae that had fed on the G:U hpRNA-painted leaves, suggesting that the RNAi molecule was successfully uptaken by the insect. Importantly, two S. frugiperda target genes were effectively silenced after three days of feeding of G:U hpRNA-painted corn leaves. These results highlighted the potential utility of G:U hpRNA for insect control via RNAi-mediated oral feeding. While the feeding assay using in vitro transcribed hpRNA in S. frugiperda suggested the potential utility of G:U hpRNA in insect control, its practical application requires a cost-effective method for producing dsRNA at large scale. While bacterium and yeast-based expression systems have been widely investigated for dsRNA production in recent years, there are still limitations in these methods. The second part of my thesis therefore focused on investigating an in planta method for hpRNA production. A truncated geminivirus (Bean yellow dwarf virus) vector was engineered to express G:U hpRNA or traditional hpRNA in Nicotiana plants via Agrobacterium infiltration. The results showed that this geminivirus vector dramatically increased the expression levels of both the G:U hpRNA and the traditional hpRNA, generating 10~20 fold more hpRNA than the traditional non-viral expression vectors. In addition, expression from the geminivirus vector was not significantly affected by sense co-suppression that occurs naturally to Agrobacterium-infiltrated transgenes, which could ensure continuous production of hpRNA. This part of my research provided a potential system for large-scale production of hpRNA or dsRNA for use in exo-RNAi. Topical application of RNAi molecules for exo-RNAi is still limited by RNA stability and delivery issues. The third part of my research was designed to investigate an alternative way for delivering exo-RNAi, based on plant endophytes. To this end, I successfully engineered an endophytic bacterium, Sinorhizobium meliloti, to express high amounts of hpRNA targeting the genes of the fungal pathogen Fusarium oxysporum. Co-culturing experiments indicated that the hpRNA-expressing S. meliloti could induce trans-kingdom gene silencing in the target F. oxysporum cells. Furthermore, co-infection of Arabidopsis and Medicago plants with F. oxysporum and hpRNA-expressing S. meliloti indicated a significant reduction of Fusarium symptoms by engineered S. meliloti in both plant species. Importantly, my study revealed that hpRNA products from plant-colonizing S. meliloti had a much longer size than those from cultured S. meliloti cells which were highly degraded. This suggests the existence of infection-induced active secretion of long hpRNA along with a hpRNA protection mechanism. Overall, this study investigated the potential application of G:U hpRNA for inducing RNAi in S. frugiperda by identifying two target genes. Moreover, the geminivirus system showed the capacity for efficient and high-level production of hpRNA, opening new possibilities for the large-scale applications of exo-RNAi in crop protection. In addition, this research also showed that the endophytic bacterium S. meliloti was capable of hpRNA expression and trans-kingdom gene silencing against the pathogenic fungus F. oxysporum by the means of microbe-induced gene silencing. Besides, this study also generated potential scientific inquiries, such as the advantages of G:U hpRNA over hpRNA in insect control, the secretion and protection of hpRNA molecules from bacteria to plants, opening new avenues of further investigations of RNAi in agriculture.

RNAi-based strategies for insect pest and pathogen control: A study on RNAi delivery and dsRNA production

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