패션프루트에서 분리한 Papaya leaf curl Guandong virus의 분자적 특성 분석 = Molecular Characterization of Papaya Leaf Curl Guandong Virus Isolated from Passion Fruit

다국어 초록 (Multilingual Abstract)

Passion fruit (Passiflora edulis) is a high-value crop with increasing global consumption. However, viral diseases have recently become a serious threat due to climate change and the expansion of cultivation areas. Recently, Papaya leaf curl Guandong virus (PaLCuGdV) has become dominant in domestic passion fruit cultivation areas, making the establishment of an efficient management system and research on viral characteristics urgent. This study was conducted to establish and evaluate a rapid and precise diagnostic system for PaLCuGdV and to analyze the pathogenicity of a C4 protein mutant found in wild isolates at the molecular level. In Chapter 1, a Recombinase Polymerase Amplification combined with Lateral Flow Strip (RPA-LFS) system was developed for the on-site diagnosis of PaLCuGdV. The RPA-LFS assay developed in this study was completed within 15 minutes at 38°C and showed a sensitivity approximately 10 times higher (10⁻⁵ ng) than conventional PCR. Using this diagnostic method to investigate viral distribution by fruit tissue, the virus was detected not only in the pericarp but also in the seed coat and cotyledons. In particular, by confirming cotyledon infection in seedlings through in vitro sterile germination experiments, it was confirmed that PaLCuGdV is a seed-transmissible virus. For the viral genomic functional analysis, infectious clones constructed using Gibson Assembly induced growth stunting and leaf curling symptoms in Nicotiana benthamiana, demonstrating high biological activity along with systemic infection. Chapter 2 focused on a double mutant containing an N-terminal deletion in the C1 gene (C1-17aa) and a premature stop codon in the C4 gene (C4W59Stop), which were identified during the construction of infectious clones. In silico analysis predicted that the C1 mutant would retain function through the structural conservation of essential replication domains, whereas the C4W59Stop was predicted to undergo significant functional loss due to the deletion of 27 amino acids at the C-terminus. Frequency analysis within the wild population revealed that this mutation accounted for approximately 44% of the total. To analyze the pathogenicity of PaLCuGdV C4W59Stop, infectious clones containing the C4W59Stop mutation (pPaLCuGdV-MT) and wild-type infectious clones (pPaLCuGdV-WT) were inoculated and compared. The results showed that the symptom of pPaLCuGdV-MT was delayed until the initial infection (7 dpi), whereas viral accumulation at later stages (14-21 dpi) was similar to that of the wild-type (pPaLCuGdV-WT). The PaLCuGdV-MT exhibited mild symptoms on the upper leaves, whereas the PaLCuGdV-WT showed no significant leaf curling or stunting. Consequently, PaLCuGdV isolated from passion fruit in Korea was detected using PCR and RPA-LFS techniques, and the complete nucleotide sequence was determined. An infectious clone of PaLCuGdV was confirmed to be viable in N. benthamiana plants. The PaLCuGdV-C4-W59Stop mutant was demonstrated that the C4 protein plays a major role in viral replication and symptom in this study.

목차 (Table of Contents)

• 제 1장 패션프루트에서 분리한 PaLCuGdV의 특성 1
• 1. 서론 1
• 2. 재료 및 방법 5
• 2.1. 식물 시료 준비 5
• 2.2. Total DNA 추출 6
• 2.3. 종자 전염 검정 7
• 2.4. PCR 및 RT-PCR을 이용한 바이러스 진단 8
• 2.5. RPA를 이용한 바이러스 진단 11
• 2.6. PaLCuGdV 전체 염기서열 분석 14
• 2.7. PaLCuGdV 분리주 계통 분석 16
• 2.8. PaLCuGdV 감염성 클론 제작 17
• 2.9. Agroinfiltration을 이용한 접종 21
• 2.10. 바이러스 감염성 평가 및 유전자 분석 23
• 2.11. qPCR 정량 분석 24
• 2.12. 통계 분석 27
• 3. 결과 28
• 3.1. 패션프루트 내 바이러스병 발생 조사 28
• 3.2. RPA 진단 31
• 3.3. PaLCuGdV의 종자전염 확인 39
• 3.4. PaLCuGdV의 전체 염기서열 결정 및 계통학적 분석 45
• 3.5. PaLCuGdV-WT의 감염성 확인 50
• 3.6. PaLCuGdV-WT의 qPCR 정량분석 53
• 4. 고찰 57
• 참고문헌 61
• 제 2장 PaLCuGdV의 C4 유전자 기능 분석 71
• 1. 서론 71
• 2. 재료 및 방법 76
• 2.1. 식물 및 바이러스 확보 76
• 2.2. 단백질 구조 예측 77
• 2.3. 패션프루트에서 PaLCuGdV의 C4W59Stop 유전자 변이 빈도 분석 78
• 2.4. PaLCuGdV의 WT 및 C4W59Stop 감염성 클론 제작 79
• 2.5. Agroinfiltration을 이용한 접종 80
• 2.6. 식물의 생육조사 및 병징 관찰 81
• 2.7. 감염성클론 평가 및 C4유전자 변이분석 82
• 2.8. qPCR을 이용한 정량 분석 83
• 2.9. 통계 분석 84
• 3. 결과 85
• 3.1. PaLCuGdV의 C4W59Stop 및 C1-17aa 서열 확인 85
• 3.2. C1 및 C4 유전자가 암호화하는 단백질의 intrinsic disorder분석 87
• 3.3. C1-17aa단백질의 구조 예측 89
• 3.4. C4WT59stop단백질의 구조 예측 91
• 3.5. 패션프루트에 감염된 PaLCuGdV-WT에서 C4W59Stop빈도 분석 95
• 3.6. PaLCuGdV-C4WT59stop의 병원성 분석 97
• 3.7. C4WT59stop의 바이러스 복제 정량분석 99
• 3.8. 담배에서 PaLCuGdV-C4WT59stop변이바이러스 안전성 분석 101
• 4. 고찰 103
• 참고문헌 110