Kiểm soát đồng thời virus dưa bằng vaccine bivalent nhẹ - Luận án tiến sĩ

BỊ 3 PRK 1ñ 1/0315 18 St A Department of Plant Pathology National Chung Hsing University EERE Doctoral Dissertation wig- F-H-ReEER TR PP ich EP? ñnijá2 TR a Concurrent control of two different virus species in cucurbits by two-in-one or bivalent mild vaccine eB: RHRGL SPA AI: PRISE Advisor: Dr. Shyi-Dong Yeh Co-advisor: Dr. Yuh-Kun Chen Student: Thi-Ngoc-Bich Tran PEA R- pots e#- 3 July, 2023 B] in Pl A 81/025 EE SA Tỷ + A AL a % M8 :š015—@—3 -ifE/EMEIRI)6M/S#+† mae 7 a i Š wa 4: RR EB # FH: _ 8107035003 & U 3Ä lễ 38 FF UL šế OA oh C48 FAIS: Air PRK #1 12 75 F AeA Ain PRK 1ñ 1 1ã 1E eS RAGE = wm R B+ HAY l l2 MARA: AHR # & R # ER 8|53% B ĐÁ, mk © HH + Ể BỊ >.82kÉ 4Š AEE HAE & ARE AR ‡ 8 wf + Bl ie #33 AMA AE PALE “at aS 2 TL #:iM|4- 4H14 + eit 6 34 mY Ro # + 4 BỊ ừ †? KE EAE FE BAS to 1 ÚÉ H+ Z3 Bib RAB Mee ARE BR e A 3S 8 t+ 6 § ae 19+ 14 re Đế. BỊ] từ PEK 181/075 18 St RAGE 4 Bh AEN IEE A aE > rễ ⁄ 8 wt oo (.

bp # R AB # --=~ w A +A HäH Acknowledgments I would like to sincerely thank Dr. Shyi-Dong Yeh and Dr. Yuh-Kun Chen for their guidance, encouragement, and support throughout my research. I would also like to thank Dr.

Fuh-Jyh Jan, Dr. Hui-Liang Wang, Dr. Shih-Shun Lin, Dr. Jun-Yi Yang, Dr.

Tsung-Chi Chen, and Dr. Chin-Chih Chen for participating in my dissertation committee. Their guidance has been invaluable throughout this entire process. Likewise, I would like to thank Dr.

Hao-Wen Cheng, Ms. Xing-Yun Xie, and Dr. Chung-Hao Huang for their teaching and assistance in my understanding of techniques during the experiment period. This work would not have been possible without their expertise.

I would like to thank all my lab members, Dr. Wen-Chi Hu, Ms. Ya-Ling Huang, Dr. Duy-Hung Do, Dr.

Thi-Thu-Yen Tran, Dr. Ya-Chi Lin, Ms. Reun-Ping Goh, Ms. Sine-Lan Wang, Mr.

Sang-You Lin, Ms. Fang-Yu Chang, Ms. Thi-Thuy-Linh Le, and Mr. Xuan-Tung Ngo for their abundant help in each of their own special ways from the first day that I came to the lab, and for making such a friendly environment in which to work.

Special thanks go to Dr. Nguyen Ngoc Thuy, and Dr. Shaw-Yhi Hwang for encouraging me to apply to the Ministry Of Education (MOE) Taiwan Scholarship. I would like to thank the Department of Plant Pathology, National Chung Hsing University (Taiwan), and the Faculty of Agronomy at Nong Lam University - Ho Chi Minh City (Viet Nam) for supporting me during my Ph.

Finally, I special like to thank my husband, Nguyen Van Hong, for caring for my daughter Nguyen Ngoc Kim Ngan from 18 months old in Vietnam. Thank him and my family for their unconditional support, encouragement, and everything they have done for me during this time in NCHU. Also, I would like to thank all my teachers in Vietnam, who taught me knowledge and made me believe that biology is very interesting. Abstract Plants of the family Cucurbitaceae, such as cucumber, pumpkin, muskmelon, watermelon, and squash, are economically important cucurbit crops.

Among aphid- bore viruses, papaya ringspot virus (PRSV), zucchini yellow mosaic virus (ZYMV) and cucumber mosaic virus (CMV) are three common and prevalent viruses, causing severe losses in a wide range of cucurbit crops globally. In this study, co-infection of horn melon plants with two mild strains, HAS-1 and ZAC, previously developed mild mutants of PRSV P and ZYMV, respectively, confers concurrent protection against PRSV P type and ZYMV. In addition, mild mutants of PRSV W, denoted WAC, were created by site -directed mutagenesis through modifications of the pathogenicity motifs FRNK and PD in helper component-protease (HC-Pro). Plant of horn melon, muskmelon and watermelon pre-infected with WAC and ZAC showed no synergistic effects on severity and provided near complete protection against both PRSV W and ZYMV.

Subsequently, WAC was used as a backbone for the construction of recombinants carrying movement protein (MP) or different lengths of coat protein (CP) coding sequence of cucumber mosaic virus (CMV). The bivalent recombinant WAC-CP provided a high degree of cross protection for concurrent control of PRSV W and CMV in cucurbits. This dissertation is divided into three chapters as follows. Chapter 1 “Literature review and research objectives” This chapter describes all relevant references related to this study.

The objectives and experimental approaches of this study are also described. Chapter 2 “Concurrent control of two aphid-borne potyviruses in cucurbits by two-in-one vaccine”. In this chapter, we first disclosed that co-infection of horn melon plants with two mild strains, HA5-1 and ZAC, the previously developed mild mutants of PRSV P and ZYMY\V, respectively, confers concurrent protection against PRSV P- type and ZYMV. Consequently, mild mutants of PRSV W, denoted WAC, were generated by site-directed mutagenesis through modifications of the pathogenicity motifs FRNK and PD in helper component-protease (HC-Pro).

Horn melon (Cucumis metuliferus) plants pre-inoculated with WAC and ZAC showed no apparent interference on viral accumulation with no synergistic effects on severity and provided complete protection against both PRSV W and ZYMV. Similar results were reproduced in muskmelon and watermelon plants, indicating the feasibility of a two-in-one vaccine for concurrent control of PRSV W and ZYMV in cucurbits. Chapter 3 “Development of mild bivalent vaccines for the concurrent control of papaya ringspot virus and cucumber mosaic virus in cucurbits”. A stable mild strain WAC was used as a backbone for generating bivalent vaccines for concurrent control of PRSV and CMV in cucurbits.

WAC recombinants carrying the CMV movement protein (MP) and different lengths of CMV-CP were constructed. The infectivity assay of individual recombinant constructs in horn melon plants indicated that the recombinants WAC-MP, WAC-CP, WAC-CPn, and WAC-CPc cause symptomless infection similar to WAC. In Chenopodium quinoa plants, all recombinants caused infection without local lesion. WAC-CP, WAC-CPn, and WAC- CPc were stable after six-generation transfers.

All recombinants retain a zigzag accumulation pattern and biological characteristics of WAC, inducing recovered mild symptoms on infected horn melon plants. For cross-protection evaluation, WAC-CP provided complete protection against the individual PRSV W-CI or CMV challenge in horn melon plants. Furthermore, WAC-CP provided 93% and 77% protection against mixed inoculation of PRSV W-CI and CMV in horn melon and muskmelon plants, respectively. Although other recombinants, including WAC-MP, WAC-CPn, and WAC-CPc were ineffective against PRSV W-CI or CMV, alone or together, they still delayed the symptom development for at least 10-15 days, indicating partial protection effectiveness.

Our results showed that WAC-derived bivalent vaccines have great potential for managing aphid-borne cucurbit viruses. Table of Contents Acknowledgment T7. 1 dÀIDNETH Laipss6t0gR43ELDEGGESEGEDEIBEUSEEICEHGBESEGEEEUENHARDERISSEGESEHSIAVEEEIEIEGH-DDEEHSREEIERESRGSNSiiS@moasassi il TEAS CLE CONE IRG sitactescres sates dbs tri es scalable diel da lcs iv ListOf table su nang ngu0 tá Ga 4Ä8138351SSGIARIEGBIGIGSi30000800ã0-006033ãẸ8 SSES.GIRSRSHERRGSHìAG4ã0500034N8A4ãSĐOG8SG3X3SBggsaSE Vili LAS Of T1SUPES. ccrecereaneseeeresenunaennencennnrereannmencmcreninmcmner arcane ix Chapter 1: Literature review and research obj€CfÏV€S.- ch nghệ, 1 Literature TEC OW ten .nesapcimsnan set sarmeneadlnm ame Gere eas eee aR 2 Aphid-borne viruses threatening the production of cucurbits worÏdwide.- --- --‹--s>-+ 2 Papaya ringspot virus (PRSV).

náo ho da 22 0 da Hà ngà 2 Zucchini yellow mosaic’ virus (ZY M VQ Mạ. 4 Cucumber mosaic virus (CMV). 5 The role of helper component-protease (HC-Pro) and coat protein genes (CP) of OfyVHTUSSGS. ee on ee oe ee ee ý The role of the movement protein (MP) and the coat protein (CP) of CMV.

- 9 Strategies for controlling aphid-borne viruses infecting cucurb1S. Agricultural practi ces csssesscsssesensnacenaeniamnennmeanmnnee terme 10 2. 10 Se PCACIIS LOT TESISIANCE puo tong vesensemsntnrvenasriaearianwumrer eens 11 4. PLANS PENIS TESISLANCE wesvicscasncannmsannenerunsannsuartas ceents oxen zenteneassiaaies rset asninanat eneesetedcovamanes 11 5.

Cross protection = vaccination with a mild VirUs. cece eeccecceeteeeceeneeeeeeeneeeees 12 MMechanilsffi öf6†055 Pr Ol6 Ct OD line sònndsbbotibtoaGITG1ABEOSSA181i54599G1G408338810011ÐĐTSEISSSSEESENHBIS4E22300054 12 Methods to create effective mild strains for cross DTOf€CfIOH.- ¿52 22+ S2 *+£+svesrerrrsres l5 A proper mild strain is a prerequisite for cross DTOf€CfIOH1.-- 56 2c SE S+vsreeerrsreree 14 The advantages and disadvantages of cross-protectiOn .:cceccecceceeceesceseeseeseeseeeeceneeerensenes 15 Control the mixed infection by cross protection 1n CUCULDItS.eceeceeeesceeteeeeceneceseeeseeeeees 16 Objectives of the Study 121. 18 FRGICTON CGS sua gsxscottisg65200210554054G1133gã0605308800856038/25855.ã680ữ/35 gi48308S23538ò302S3E5801G3086i08S0388g880đ/0:G001830Ó80590/588::3385888 19 Chapter 2: Concurrent control of two aphid-borne potyviruses in cucurbits by fwo-in-one yaccine. Ee sun Biaariane CS Men neeneneesvneesonesvenesnornerenenneesnaesensneeseerssenns 33 AD StHA CL as csscssssussaszasscsanass ous svanaausnascsiss LÔ, CME AEN ẤP.gE+ 34 TNtOdUCHON: cessceseeaxee cease emcee Ea womweneons means 00001161500 Đ801901554GE0910000151830880298 35 Materials and methods 7.

38 Plant néatertals and wirUSfSouff6S. 38 Co-infection of mild mutant PRSV HA5-1 and ZYMV-ZAC against two different severe potyviruses of PRSV P-HA and ZYMV-GFP in horn melon.-- --- 38 Modification of HC-Pro for the generation of mild mutant PRSV-WAC. 39 Inoculation of horn melon plants with PRSV-WAC, ZYMV-ZAC or both. 40 Examination of titer of PRSV and ZYMV.:ccccccsscssecsscssscesssencesneeseseecesscestecseenatonnee 41 Analysis of the RNA silencing suppression ability of mutated HC-Pros of PRSV and ZAC by aproInflÏlralOTiussescedsesssoiesoaaidsnpAG154082103841300135Y0ESGSĐNNSSEIRNEYSRERMESSASEESEISSS3088 41 Cross protection effectiveness of PRSV-WAC and ZYMV-ZAC alone or together, against severe strains of PRSV W-CI and ZYMV-GFP under greenhouse CONCILL OTIS gọn no bn no ng A0 BEBE118014G118BASBEESILERGRISHISIRSESHASBEEAASEESGIGTOSKEISRESHHSEXEIGEIESERICHSSREEEDBSS4 42 SH ÍB cececemeunenin cone eeceieiidie ices Gaerne errr ee eerie 43 Co-infection of mild viruses PRSV P HAS-1 (HAS-1) and ZYMV-ZAC (ZAC) provides protection against severe isolates of cognate PRSV P-HA and ZYMV- GPP nho rire lon: DAHIESisssssssseseboseiineiibidBibLEihbsgSdlndlss2ddo39tE4B880X031AL2380G000813001010480888452880g186 43 Generation of attenuated mutant from PRSV W-CI by modification of the pathopenicity factOt Cs PO -erecccsessvesessmmsnersesinnay earner eecorasrersereenn este 91003100042070501301190930003888 43 Symptoms and accumulation dynamics of WAC and ZAC in co-infected horn melon pÏanfs.

ge BR aches ebay bi, BUD. Mee vaseasccteseonsinseeseavacanertecnesesenevnenstaves 44 Mutated HC-Pros of both PRSV and ZYMV have lower RNA silencing suppression abilities but without additive effect. cece eseeeeeeeeeeeeeeeeeeeeeteneeeeens 45 Co-infection of WAC and ZAC in horn melon, muskmelon, and watermelon plants provides complete or high degree of cross protection against both PRSV W-Cl and ZYMV-GED secsnsnoooorBEoonooooe ERUEEoosisoooEoooooikđ Ân nho nga gg sen 46 Discussion. 48 Acknowled ements s5 cá neo tu UIẤ couse 01641411013511E11NG1911835634815 138815) l srst02110001511661848338138% 32 References sine NATIONAL CHUNG HSIMG.

n1 vê, Đa Chapter 3: Development of bivalent vaccines for the concurrent control of papaya ringspot virus and cucumber mosaic virus in cucurbÏfS. - ---- 555cc ss+ecsxeecee 73 PS DSEL TỔ oi soucugiigtgix6055640534854054100638136i05g03036090335883ã28E5383039E0:80103809401a9880038G30084303546.G66 180116 SSO BaG SDRAM EE 74 Tntto duction ssssscsc2266560111100213401101401038100400340068081850S01SLREEGGEEEESSESAGEESEEIESUEAGEESEEIEEELEAXSEEEESZ9EEE4EE 75 Materials and methods. 78 ‘Vitus SOUICES and PLOPACALION boáa 6c iti6146L68%5036i240L9 38063 U5ug án GỆ4%SUNS 383 šg0g881438.5938205g BạxSgRukSi 78 Construction of PRSV W type mild strain WAC carrying CMV MP reading frame. 78 vi Construction of PRSV W mild strain WAC carrying different lengths of CMV CP T68 IHB (ATG eco cncteeniereennernnluniusies reentrant meuniere tere eES Evaluation for the pathogenicity of recombinant Viruses.

5 +5 +55 <++<xs++ Time-course accumulation levels of recombinants in horn melon planfs. Accumulation of the severe virus PRSV W-CI (W-C]) and CMV, alone or topether 1n HOt MELON Plats ie scessssrssaevrssnsmaveansaenscaenss 1853E814331G00.IAASIGSL-44GESESGSEEESSB The stability of individual recombinant viruses in C. Cross-protection effectiveness of individual recombinants against two unrelated Infectivity and cross-protection effectiveness of recombinant virus WAC-MP in hos Melon Plants ccccccceveserescmensee Me AO Mls enerecseenmcemmemmermreeme nme Infectivity of mild strain PRSV WAC carrying different lengths of the CP gene of Genetic stability of PRSV WAC carrying different lengths of CMV CP. Time-course accumulation levels of the PRSV WAC carrying different lengths of CMV-CP-vfusesNATIONAI.CHUHNG HSING UNIVERSITY.

scsssmmemsrnetatt eaves The mild recombinant WAC-CP provides high degrees of cross protection against both PRSV W-CI and CMV, alone or together, in horn melon and muskmelon vii List of tables Chapter 1 Table 1. The function of protein encoded by CMV RNAS .eecceeseeseeseceeeeteenseeeeeeeees 9 Chapter 2 Table 1. Cross protection effectiveness (in percentages) of PRSV-WAC and ZYMV-ZAC alone or in combination, against two different severe potyvirus strains of PRSV W-CI and ZYMYV-GFPP. c1 2 2211222111 2511 15211151511 eree 59 Supplementary Table S1.

Details of primers used in this study .