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Characterization of Watermelon Silver Mottle Virus by Genomic Sequencing, Serological Properties and Transgenic Resistance
|關鍵字:||watermelon silver mottle virus|
|摘要:||番茄斑萎病毒屬（the genus Tospovirus）之西瓜銀斑病毒（watermelon silver mottle virus, WSMV）為目前臺灣西瓜、甜瓜、洋香瓜、冬瓜等葫蘆科植物，生產之重大限制因子。Tospovirus 屬病毒為 Bunyaviridae 科中唯一可感染植物的屬，其特徵為具有套膜的球型病毒，核心並有大（L），中（M），小（S）三條單股基因體RNA。在分類上，西瓜銀斑病毒之核鞘蛋白與血清型 I（serogroup I）的番茄斑萎病毒（tomato spotted wilt virus, TSWV）、血清型II的花生輪斑病毒（groundnut ringspot virus, GRSV）、番茄黃斑病毒（tomato chlorotic spot virus, TCSV）及血清型 III 的鳳仙花疽斑病毒（impatiens necrotic spot virus, INSV），並無血清學的關係，故被列為血清型 IV。 而與花生頂芽壞疽病毒（peanut bud necrosis virus, PBNV）屬於同血清型之不同種病毒。因此，為了進一步研究 WSMV 在分子層次上的特性，本研究乃對其 M 及 L RNA 進行解序及分析，並且進一步製備核鞘蛋白的多元及單元抗體供血清檢定，另外並構築核鞘蛋白轉基因菸草，評估其抗病性狀，以做為防治西瓜銀斑病毒之模式。
在本論文第二章節中分析 M RNA 之核甘酸序列，全長共有4,880個核甘酸，其 RNA 具有雙極性轉譯蛋白策略，病毒股對映產生35 kDa 的非結構性蛋白（NSm），而互補股則對映產生127.6 kDa的醣蛋白（G1/G2）。其 NSm 及 G1/G2 蛋白，與 TSWV 及 INSV 比對，僅有30.5-40.9﹪的相同度且無核酸雜合反應關係，顯示WSMV與血清型 I 及 III 之關聯性非常低，而與 PBNV 則分別有83.4-88.7﹪的相同度。為探討 WSMV M RNA 在植物體中的基因表現策略，本研究從感染 WSMV 的菸草中萃取出植物體的全部 RNA，以對應 NSm 或G1/G2 之正負股共四個核酸探針（riboprobes），分別進行北方雜合反應，除了證實罹病的植物體中同時具有病毒股及互補股的全長度 RNA 外，並可測到1.0 及 3.4 kb 片段，顯示 NSm 及 G1/G2 確實是分別由病毒股及互補股對應的 mRNA 所產生。同樣的，對由核鞘蛋白純化出的病毒 RNA 進行北方雜合反應，結果顯示，除了病毒股及病毒的互補股皆可被核鞘蛋白包被外，其 mRNA 亦可能被核鞘蛋白所包被。
本論文第三章節則完成了 L RNA 的解序，WSMV的 L RNA 全長有8,917個核甘酸，其由病毒的互補股對應產生331.8 kDa 的蛋白，其蛋白與 TSWV 及 INSV 僅有22.3-46.5﹪的相同度，而與 PBNV 則有91.3﹪的相同度。WSMV 產生的各個蛋白與其他的病毒相較，此蛋白最為相似，其與 Bunyaviridae 科中的其他病毒相較，它具有複製脢之功能要素（motif），而從複製脢的演化關係樹狀圖，可見 Tospovirus 屬與 Bunyavirudae 科中Bunyavirus 屬的關係較與Phlebovirus 屬及 Hantavirus 屬的關係要接近。因此，在完成了 L RNA 基因體序列之解讀後，我們獲得了 WSMV 之所有遺傳分子的基本資料。
本論文第四章節，為血清學性狀之探討。由於純化 WSMV 完整粒子極為困難，故以表面活性劑 Triton-X 處理將套膜打破、配合離心及電泳等過程將 WSMV 的核鞘蛋白純化出來，並進行多元及單元抗體的製備，以間接酵素聯結血清免疫吸附法 (indirect ELISA) 法檢測，所得之多元抗體血清力價可達16,000倍，在培養液之單元抗體具400倍，而腹水抗體則可高達106倍，腹水及培養液中的單元抗體皆可與 WSMV 病毒感染的植物汁液及純化的鞘蛋白強烈反應，在田間檢測方面，單元抗體之背景值較多元抗體降低至少二倍，顯示所得之單元抗體可更精確檢測田間的罹病植物。而在分類方面，以 TSWV、GRSV、INSV 及 WSMV 之核鞘蛋白多元或WSMV 之核鞘蛋白單元抗體以上述二次抗體酵素聯結血清法與西方轉漬法檢測，再度確認 WSMV確實與 TSWV、GRSV 及 INSV 無血清學關係。而 TSWV 及 GRSV 之核鞘蛋白多元抗體會有強烈的交互反應，顯示此二病毒應重新歸類為同一血清型。
第五章節的部份是將 WSMV N 基因轉殖菸草，經康黴素 (kanamycin) 篩選後，獲得27個品系，以聚合脢連鎖反應確認及接種試驗後，獲得三個抗病品系，此三品系以自交所獲得之子代之抗病性均為3:1之分離結果，顯示其轉基因均為單一顯性遺傳。其中之 12-12 品系經由三次自交得到同合子（homozygote），其分別作為父母本再與非轉型之菸草雜交，得到半合子（hemizygote）。分析其抗病性狀，同合子於 WSMV 接種後，無病徵出現，然而，半合子則較健康之對照組延緩二到三天發病，且接種葉上之病斑數較少。至於對其他 TSMV、INSV、GRSV 及同血清型之 HT-1 病毒則無抗病性，顯示其專一性強。基於 WSMV 之N 基因轉基因菸草之構築提供之成功模式，未來應可用於葫蘆科及番茄之經濟作物上達抗 WSMV 之功效。|
Watermelon silver mottle virus (WSMV), a member of the genus Tospovirus, is the major limiting factor for growing watermelon and other cucurbits in Taiwan. The genus Tospovirus is the only genus in the family Bunyaviridae that infects plants. The genus is characterized by quasi-spherical enveloped particles containing three ssRNA segments, denoted S RNA, M RNA and L RNA. The nucleocapsid protein (NP) of WSMV is serologically unrelated to tospoviruses of serogroup I (tomato spotted wilt virus, TSWV), serogroup II (groundnut ringspot virus, GRSV; tomato chlorotic spot virus, TCSV), and serogroup III (impatiens necrotic spot virus, INSV). However, WSMV and peanut bud necrosis virus (PBNV) are two related and yet distinct tospovirus species of serogroup IV. To further analyze the molecular characteristics of WSMV, the complete nucleotide sequences of the genomic M RNA and L RNA were determined. Also, polyclonal and monoclonal antibodies against the purified NP were prepared for serological studies. Moreover, transgenic resistance to WSMV in the N-gene transgenic tobacco was evaluated under greenhouse conditions. In the chapter 2, the nucleotide sequence of the M RNA segment of WSMV was determined. The M RNA is 4880 nucleotides in length with two open reading frames (ORFs) in an ambisense organization. The NSm ORF located on the viral strand encodes a protein of 35 kDa, and the G1/G2 ORF located on the viral complementary strand encodes a protein of 127.6 kDa. The RNA probe corresponding to NSm or G1/G2 ORF of WSMV failed to hybridize with M dsRNAs of TSWV and INSV. The low amino acid identities of the NSm and G1/G2 proteins (30.5-40.9%) with those of TSWV and INSV indicate that WSMV belongs to the genus Tospovirus but is phylogenetically distinct from viruses in serogroups I and III. WSMV and PBNV share 83.4% and 88.7% amino acid identities for their NSm and G1/G2 proteins, respectively. It is concluded that they are two related but distinct species of serogroup IV. In addition to the viral or viral complementary full length M RNA, two putative RNA messages for the NSm gene and the G1/G2 gene, 1.0 kb and 3.4 kb respectively, were detected from the total RNA extracted from WSMV-infected tissue of Nicotiana benthamiana, indicated the M RNA uses an ambisense coding strategy. The 1.0 kb and 3.4 kb RNAs were also detected in the viral RNAs extracted from purified nucleocapsids, suggesting that the putative messages of the M RNA of WSMV can also be encapsidated by the nucleocapsid protein. In the chapter 3 of the thesis, the complete nucleotide sequence of the L segment of WSMV was determined. The L RNA is 8917 nucleotides long and of negative polarity, with a predicted Mr of 331.8. L protein shares lower amino acid identities (22.3-46.5 %) with those of TSWV and INSV, but with higher amino acid identity (91.3 %) with that of PBNV. At the amino acid level, the L protein is the most conserved gene product among the tospoviruses compared. Comparison of the deduced L protein of WSMV with those of the L proteins of other members of the family Bunyaviridae indicated that its amino acid sequence includes the conserved motifs of RNA-dependent RNA polymerases. A phylogenetic dendrogram of the RNA polymerase indicated that the genus Tospovirus has a closer relationship with the genus Bunyavirus than with the genera Phlebovirus and Hantavirus. In the chapter 4, the NP of WSMV was purified from local lesion host Chenopodium quinoa by Triton X-100 treatment, differential centrifugation and gel electrophoresis and used for animal immunization. A rabbit polyclonal antibody (PCA) and mouse monoclonal antibodies (MAbs) against the NP of WSMV were produced. The titer of the PCA to WSMV NP determined by indirect ELISA using crude antigens from WSMV -infected tissue of Nicotiana benthamiana was 16000X. The titers of MAbs in culture medium were 400X and those of ascitic fluids were 106 as determined by indirect ELISA. The MAbs of the culture medium and the ascitic fluid reacted strongly with both crude sap of WSMV-infected plants and purified WSMV NP. In indirect ELISA tests, the background values of the PCA to the healthy control was twice higher than those of the MAbs, indicating that the MAbs can more precisely differentiate the infected plants from healthy plants. In indirect ELISA and western blot tests using the produced PCAs and MAbs tasted with the crude antigens from N. benthamiana infected with individual tospoviruses, the results clearly indicated that the NP of WSMV was serologically distinct from those of TSWV, GRSV and INSV. Moreover, the strong cross reactions between TSWV and GRSV when tested against the PCAs to their NPs suggested that these two viruses should be reclassified in the same serogroup. Finally, as sources of natural resistance to WSMV remain unavailable, there is an urgent need for novel forms of resistance involving genetic modifications of host plants. Thus, the WSMV N-gene was transferred into tobacco. After selection on the kanamycin medium, 27 putative transgenic tobacco lines were obtained. Three resistant lines 12-3, 12-4, 12-12 were found resistant to WSMV infection. All the three R1 lines have a segregation ratio of 3:1 for the resistance, indicating that the transgene was inherited as a single dominant nuclear trait in each line. The line 12-12 was self-pollinated three times to obtain homozygous progenies. The R4 homozygous progenies were further hybridized with nontransformed tobacco to obtain hemizygous progenies. All homozygous plants were symptomless after mechanical inoculation with WSMV. However, the hemizygous plants showed only a delay of 2-3 days in development of systemic symptoms and fewer local lesions on inoculated leaves were noticed. All homozygous and hemizygous progenies were not resistant to other tospoviruses tested, including TSWV, GRSV, INSV, and a gloxinia tospovirus HT-1 (high temperature-recovered tospovirus). The successful production of the WSMV N-gene transgenic tobacco resistant to WSMV infection provides a model system for the construction of WSMV N-gene transgenic cucurbit and tomato plants for control of WSMV.
|Appears in Collections:||植物病理學系|
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