Establishment of test systems for evaluating ingestion RNA interference-induced lethality against Tetranychus urticae, a representative sucking pest

다국어 초록 (Multilingual Abstract)

Double-stranded RNA-mediated interference (RNAi) has been universally applied as a specific, potent and successful technology for gene manipulation and emerged as a potential pest control strategy when combined with the plant transgenesis. To establish the RNAi-based control strategy against the two spotted-spider mite (Tetranychus urticae Koch), a worldwide notorious sucking pests in facility cultivation, test systems for evaluating ingestion RNA interference-induced lethality were established. As a prescreening tool prior to the development of transgenic crops, protocols for the agroinfiltration in the soybean and kidney bean were established and optimized. The maximum efficiency of hairpin RNA delivery in soybean was determined to be obtained by sea sand and syringe in the soybean and kidney bean, respectively. Transient expression of target hairpin RNA reached the maximum level at 24-h post-agroinfiltration in both soybean and kidney bean. The agroinfiltrated soybean plants expressing the COPA and aquaporin 9 (AQ9) hairpin RNA induced 64.1% and 39.5% mortalities of T. urticae at 144-h post-infestation. The target gene knocksown was observed in the infested mites, confirming that the observed mortality was likely resulted from RNAi. In summary, agroinfiltration was determined as an effective pre-screening tool for evaluating the RNAi-induced lethality of plants expressing target hairpin RNA prior to the generation of transgenic plants.
To confirm the proof of concept that the constitutively expressed hairpin RNA in plant can also cause RNAi-induced lethality against T. urticae, a representative sucking pest, an Arabidopsis plant lines constitutively expressing the COPA hairpin RNA were generated via floral-dip method. When infested with T. urticae, the transgenic Arabidopsis resulted in T. urticae mortality and transcript downregulation. These findings strongly demonstrated that sap-feeding T. urticae can be actually killed by in planta expression of target lethal gene, such as COPA, and RNAi-based transgenic plants can be exploited as a novel alterative mean to control sucking pests T. urticae, as well.

목차 (Table of Contents)

• LITERATURE REVIEW 1
• 1. Tetranychus urticae 1
• 2. RNA interference 2
• 3. Agroinfiltration 3
• CHAPTER 1.Establishment of agroinfiltration system for in planta temporary expression of Tetranychus urticae hairpin RNA 5
• Abstract 6
• 1. Introduction 7
• 2. Materials and methods 10
• 2.1 Growth conditions of host plants 10
• 2.2 T. urticae strain and rearing 10
• 2.3 Construction of hairpin RNA vector 10
• 2.4 Agrobacterium tumefaciens transformation 11
• 2.5 Agroinfiltration methods 12
• 2.6 Detection of hairpin RNA in agroinfiltrated host plants 13
• 2.7 Feeding RNAi via agroinfiltrated host plants 13
• 2.8 Total RNA extraction and cDNA synthesis 14
• 2.9 Quantitative real-time PCR (qPCR) analysis 15
• 2.10 Statistical analysis 16
• 3. Results and discussion 16
• 3.1 Development of RNAi vector for in plant expression of hairpin RNA 17
• 3.2 Identification of efficient Agrobacterium delivery methods 18
• 3.3 Transient and spacial expression pattern of COPA hairpin RNA in agroinfiltrated host plants 21
• 3.4 Confirmation of COPA knockdown in T. urticae induced from host plants expressing COPA hairpin RNA 23
• CHAPTER 2. Agroinfiltration-based expression of hairpin RNA in soybean plants for RNA interference against Tetranychus urticae 25
• Abstract 26
• 1. Introduction 27
• 2. Materials and methods 29
• 2.1 Plant materials and growth conditions 29
• 2.2 T. urticiae rearing 30
• 2.3 Hairpin RNAi vector construction 30
• 2.4 A. tumefaciens transformation 31
• 2.5 Agrobacterium colony restriction analysis 31
• 2.6 Agroinfiltration 32
• 2.7 Total RNA extraction from 32
• 2.8 Quantitative real-time PCR (qPCR) 34
• 2.9 Bioassay and mortality evaluation 35
• 2.10 Data analysis 35
• 3. Results 36
• 3.1 Development of RNAi constructs for agroinfiltration 36
• 3.2 Transient expression of COPA- and AQ9-specific hairpin RNAs in agroinfiltrated soybean plants 36
• 3.3 Lethality of target hairpin RNA-expressing soybean plants against T. urticae 38
• 3.4 Downregulation of COPA and AQ9 transcripts in T. urticae fed agroinfiltrated soybean plants expressing COPA and AQ9 hairpin RNA 40
• 4. Discussion 41
• CHAPTER 3. Transgenic Arabidopsis for two spotted spider mite resistance 46
• Abstract 47
• 1. Introduction 47
• 2. Materials and methods 49
• 2.1 Plant materials and growth conditions 49
• 2.2 T. urticae strain and rearing 50
• 2.3 Hairpin RNAi vector construction 50
• 2.4 Agrobacterium tumefaciens transformation 51
• 2.5 Floral- Dip Method 51
• 2.6 Initial screening of putative transformants by using an glufosinate and PCR 52
• 2.7 Quantitative real-time PCR (qPCR) detection of dsRNA transcripts in transgenic Arabidopsis 53
• 2.8 Total RNA extraction from T. urticae 53
• 2.9 Quantitative real-time PCR (qPCR) analysis 54
• 2.10 Bioassay and mortality evaluation 55
• 3. Results 55
• 3.1 Development of gene construct for the expression of hairpin RNA in transgenic Arabidopsis for targeting COPA gene of Tetranychus urticae 55
• 3.2 Development of transgenic Arabidopsis lines 55
• 3.3 Lethality of Arabidopsis expressing COPA hairpin RNA against T. urticae 56
• 3.4 Downregulation of COPA transcripts in T. urticae fed Arabidopsis expressing COPA hairpin RNA 57
• 4. Discussion 58
• LITERATURE CITED 61
• KOREAN ABSTRACT 67