Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31683
標題: 影響病毒株系專一性交互保護作用及轉基因抗性崩解之木瓜輪點病毒因子
Factors of Papaya ringspot virus affecting strain-specific cross protection and transgenic-resistance breakdown
作者: 游邦照
You, Bang-Jau
關鍵字: transgenic resistance
轉基因抗性
cross protection
strain-specificity
PRSV
broad spectrum
gene silencing
HC-Pro
3''''UTR
交互保護
株系專一性
木瓜輪點病毒
廣泛抗性
基因沉寂
3''''端非轉譯區
出版社: 植物病理學系
摘要: 木瓜輪點病毒(Papaya ringspot virus, PRSV),是熱帶及亞熱帶木瓜栽培的主要限制因子。交互保護及轉基因作物已被廣泛使用來防治PRSV。交互保護的定義是,當植物系統性感染某植物病毒後,會對類源相近的病毒株系具有抗性。PRSV夏威夷輕症病毒HA5-1,是木瓜型夏威夷重症病毒P-HA的弱病毒突變株,已廣泛使用於對抗P-HA;但是,對木瓜型台灣重症病毒P-YK、泰國重症病毒P-TH、墨西哥重症病毒P-MX,以及西瓜型木瓜輪點病毒W-CI,無法提供很好的保護效果,這種株系專一性的交互保護特性限制了HA5-1在瓜類及木瓜上的應用。另外,轉殖P-YK之鞘蛋白(CP)及3''端非轉譯區(3''UTR)的轉基因木瓜,可提供廣泛的抗性對抗P-HA、P-TH及P-MX等分離株,然而,此轉基因木瓜會被台灣的另一分離株PRSV 5-19感染。由此可見,病毒株系專一性問題,限制交互保護及轉基因作物之使用。因此,本研究主要的目的在使用雜合輕症病毒以提供對抗木瓜型及西瓜型PRSV之廣泛性抗性(第二章)、探討病毒因子對交互保護之病毒株系專一性的影響(第三章)、探討病毒因子對轉基因抗性之病毒株系專一性的影響(第四章),及利用轉入雜合基因的轉基因作物感染PRSV之策略來提升抗性,用以解決病毒株系專一性抗性之問題(第五章)。 在第二章中,以木瓜型輕症病毒HA5-1為載體換入西瓜型W-CI的CP及3''UTR,此雜合輕症病毒在瓜類上對西瓜型W-CI及木瓜型P-HA均有良好的保護效果。因此,我們可以利用雜合輕症病毒的策略去改進HA5-1,使其在瓜類作物上提供對抗西瓜型及木瓜型PRSV之廣泛性抗性。在第三章中,為了進一步探討病毒因子對交互保護之病毒株系專一性抗性的影響,我們進一步以HA5-1及P-HA為載體,分別置換W-CI、P-YK及P-TH之CP及3''UTR,並在木瓜及刺角瓜上測試各雜合輕症及重症病毒間之保護效果。研究結果指出,保護與挑戰接種病毒間共有的基因體長度、病毒3''UTR的序列相似度、病毒3''端區域組成、病毒累積量,以及寄主種類,會影響病毒株系專一性交互保護。 在在第四章中的轉基因抗性之病毒株系專一性研究上,以木瓜型P-YK為載體,置換5-19的CP加3''UTR或單獨置換HC-Pro,探討5-19的CP加3''UTR或單獨的HC-pro在瓦解轉基因抗性扮演之角色。以轉殖P-YK的CP加3''UTR之轉殖木瓜及PTY轉殖木瓜進行抗性分析,TPY轉殖木瓜是轉入無法轉譯之複合核酸片段,此核酸片段含有P-YK的CP部分基因,及木瓜畸葉病毒大里分離株(PLDMV DL)的CP部分基因。研究結果顯示,接種病毒與轉基因之序列相似度會影響轉基因抗性;然而,即使轉基因與接種病毒擁有相同的核酸序列,單獨置換5-19的HC-Pro亦會瓦解轉基因抗性。同時置換5-19的HC-Pro及CP加3''UTR,對瓦解轉基因抗性並無加成作用。經由胺基酸比對,P-YK與5-19的HC-Pro之5個胺基酸差異,主要位於決定病毒基因體複製及抑制基因沉寂的區域。 在第五章中,高度及中度抗性之TPY轉基因木瓜,分別接種PRSV 5-19及HA5-1後,高抗病性的轉基因木瓜接種PRSV 5-19後仍可完全抗PLDMV;中抗的轉基因木瓜接種HA5-1後,對PLDMV及PRSV不同地理區域分離株之抗性則有顯著提升;轉基因木瓜之轉基因表現會因感染PRSV而提升,但是,由轉基因而來的siRNA量亦會被提升。此一新的策略可用以解決交互保護及轉基因抗性之病毒株系專一性問題。 總而言之,本研究中指出,PRSV 3''端區域會影響交互保護之病毒株系專一性保護作用;PRSV 5-19的HC-Pro則會影響轉基因抗性之病毒株系專一性抗性,並造成與核酸序列專一性無關連的轉基因抗性之瓦解,此結果將嚴重衝擊轉基因作物在防治病毒之使用。此外,我們亦提出一些新穎的策略來解決病毒株系專一性抗性的問題,利用木瓜型輕症雜合病毒攜帶西瓜型PRSV之3''端區域,可在瓜類作物上提供對抗西瓜型及木瓜型PRSV之廣泛性抗性。以轉入含有PRSV及PLDMV之CP的雜合病毒基因木瓜,接種PRSV輕症病毒後可提升對抗PRSV及PLDMV的抗性,用以解決病毒株系專一性抗性的問題。
Papaya ringspot virus (PRSV) is a major limiting factor for papaya (Carica papaya L.) production in tropical and subtropical areas throughout the world. Cross protection and transgenic approaches have been conducted for PRSV control. Cross protection describes the phenomenon that plants systematically infected with one strain of a virus are protected from infection by a second related strain of the same virus. PRSV HA5-1, a mild mutant of the type P Hawaii severe strain (PRSV P-HA), has been widely used for the control of P-HA in papaya, but did not provide practical protection against PRSV type W strain W-CI in cucurbits and other type P strains in papaya, including Taiwan strain P-YK, Thailand strain P-TH, and Mexico strain P-MX. Transgenic lines carrying the coat protein (CP) and 3'' untranslated region (3''UTR) of P-YK and conferring broad-spectrum resistance against different PRSV geographic strains were found to be infected by PRSV 5-19. Therefore, the strain-specific phenomenon limits the application of cross protection and transgenic resistance. In this study, chimeric mild PRSV strain from HA5-1 to carry a heterologous 3'' terminal region of a type W virus was constructed to provide broader protection against PRSV type P and W strains in cucurbits (Chapter 2). We further identified the viral factors affecting the strain-specific resistance in cross protection (Chapter 3) and transgenic resistance (Chapter 4). We also widen the resistance by using a mild PRSV to infect transgenic papaya lines containing a chimeric construct (Chapter 5). In chapter 2, we presented that the chimeric HA5-1 carrying both heterologous CP coding region and 3''UTR from W-CI provided high degrees of cross protection against W-CI and P-HA in cucurbits. Therefore, the chimeric mild virus is a potential strategy to provide broader protection against both type W and P strains in cucurbits. In chapter 3, to further identify the viral factors involved in strain-specific cross protection, the protection was conducted in horn melon and papaya by using chimeric mild and severe PRSV containing hetrologous 3'' terminal region. Our data showed that the strain-specific cross protection was determined by the length of viral genomic segment shared between the mild strain and challenge strain, sequence identity of 3''UTR, the organization of the 3'' viral genomic region, virus accumulation, and host species. In chapter 4, the study of strain-specific resistance of transgenic plants, recombinants carrying the HC-Pro coding region or the CP coding region with 3''UTR from 5-19, or both, were constructed from P-YK. The post-transcriptional gene silencing (PTGS)-mediated transgenic papaya lines carrying a translatable YK CP gene with 3''UTR or TPY transgenic lines carrying an untranslatable chimeric construct containing parts of the CP coding region with 3''UTR of YK and CP coding region of Papaya leaf-distortion mosaic virus (PLDMV) were inoculated with individual recombinant viruses, YK, or 5-19. Our data showed that the sequence divergence between the transgene and the infecting virus genome is important in overcoming the transgenic resistance. However, a heterologous HC-Pro region of 5-19 alone provided the ability to break down the transgenic resistance in a transgene sequence-homology independent manner, even though the sequence of the transgene share 100% identity with the genome of the infecting virus. There were no additive effects when the heterologous 5-19 HC-Pro was coupled with the heterologous 5-19 CP + 3''UTR. Compared with YK, five over six amino acid changes of 5-19 HC-Pro were located in the region responsible for genome amplification and gene silencing suppression. In chapter 5, highly and moderately resistant TPY transgenic lines were inoculated with a severe strain PRSV 5-19 and a mild strain Hawaii HA5-1, respectively. Highly resistant transgenic papaya lines infected with PRSV 5-19 showed completely protection against PLDMV. Moderately resistant lines infected with HA5-1 showed the synergistic effect of cross protection and transgenic resistance against PLDMV and different PRSV geographic strains. The accumulation of the chimeric transcript was increased by infection with PRSV 5-19 or HA5-1. However, siRNA accumulation derived from PLDMV CP coding region was also enhanced. This novel approach may solve the problem of strain-specificity in cross protection and transgenic resistance. Taken together, this research indicated that viral 3'' terminal region affected the strain-specific cross protection. HC-Pro of PRSV 5-19 breaks down the strain-specific transgenic resistance in a sequence-homology independent manner and will have a strong impact on the application of transgenic plant for virus control. Chimeric mild type P PRSV containing heterologous 3'' terminal region form type W strain provided broader protection against PRSV type P and W strains in cucurbits. Chimeric transgenic papaya infecting with the PRSV mild strain HA5-1 provided higher degrees of resistance against PRSV and PLDMV. Our studies have established several novel approaches to solve the problem of strain-specificity in cross protection and transgenic resistance.
URI: http://hdl.handle.net/11455/31683
Appears in Collections:植物病理學系

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