Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31755
標題: 胡瓜嵌紋病毒之MP 及2b 基因能夠互補馬鈴薯Y 病毒屬HC-Pro 協同性基因造成過敏性反應及毒力能力之缺失
The MP and 2b genes of Cucumber mosaic virus complement the mutated potyviral HC-Pro gene defective in hypersensitive reaction and virulence
作者: 林愉瑄
Lin, Yu-Hsiuan
關鍵字: Cucumber mosaic virus
胡瓜嵌紋病毒
CMV
Zucchini yellow mosaic virus
ZYMV
movement protein
MP
矮南瓜黃化嵌紋病毒
移動性蛋白
出版社: 植物病理學系
摘要: 胡瓜嵌紋病毒(Cucumber mosaic virus , CMV)寄主範圍非常廣泛,可感染超過一千種以上的植物。在自然界中,此病毒可與矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV)共同感染產生協力作用造成嚴重危害,另外,此病毒亦可以藉由數十種蚜蟲以非永續性的方式傳播,因其具有上述特性,所以目前尚未有快速而有效率的防治方法被發展出來。 本實驗乃是藉由修飾之重症型(p35ZYMVBam)或突變之輕症型(p35ZYMVACBam)矮南瓜黃化嵌紋病毒載體攜帶各種與胡瓜嵌紋病毒之轉移相關的移動性蛋白(movement protein, MP)基因,鞘蛋白(coat protein, CP)基因,或者後轉錄基因沉寂作用抑制子(post-transcriptional gene silencing suppressor)2b蛋白基因,觀察重組病毒在奎藜(Chenopodium quinoa)與矮南瓜(Cucurbita pepo L. var. Zucchini)上的表現情形,藉以探討CMV各項蛋白基因之功能及其在病毒協力作用上之影響。CMV各項蛋白基因分別插入至ZYMV協同性蛋白 (HC-Pro) 之胺基端 (N-terminal) 或NIb蛋白之羧基端(C-terminal),藉由機械接種重組病毒至植物體內而表現外來蛋白。分別感染的罹病植物可經由西方墨點法(western blotting)以及反轉錄聚合酶鏈鎖反應(reverse transcription-polymerase chain reaction, RT-PCR)測得重組病毒帶有CMV各片段基因。本實驗結果顯示,攜帶CMV MP之重症型重組病毒ZYMVcMP與ZYMVNcMP在四天內即能在奎藜上形成單斑,較野生型(ZYMV TW-TN3)提早三天,其單斑型態為較小型、壞疽狀、外圍具黃色暈環之斑點。攜帶CMV CP之重症型重組病毒ZYMVcCP與攜帶CMV 2b之重症型重組病毒ZYMVc2b在奎藜上七天形成單斑,單斑型態與野生型相似,在系統性寄主矮南瓜上,重症型重組病毒都會造成葉片斑駁、黃化、嵌紋以及葉片變形等病徵。攜帶CMV MP之弱系重組病毒ZYMVACcMP以及攜帶CMV 2b之弱系重組病毒ZYMVACc2b有回覆突變之協同性蛋白基因(HC-Pro gene)不產生單班的特性,在奎藜上造成單斑反應,且ZYMVACcMP在四天內即能形成單斑,較野生型(ZYMV TW-TN3)提早三天,其單斑型態與重症型重組病毒ZYMVcMP或ZYMVNcMP相似,同為較小型、壞疽狀、外圍具黃色暈環之斑點。ZYMVACc2b在奎藜上七天形成單斑,與野生型(ZYMV TW-TN3)時間相近,型態為較大之黃色褪綠單斑。攜帶CMV CP之弱系重組病毒ZYMVACcCP則無法在奎藜上回復單斑反應的形成,在系統性寄主矮南瓜上,ZYMVACcMP與ZYMVACcCP只會造成葉片輕微斑駁的病徵,但在ZYMVACc2b感染的矮南瓜葉片上,則亦造成嵌紋、變形等類似重症型ZYMV所產生之病徵。由本實驗結果可知,CMV MP可回覆原本弱系病毒ZYMVAC在奎藜上不產生單斑之特性 ,在奎藜上造成單斑,在系統性寄主上則不會增加ZYMV之毒力(Virulence)而僅產生輕微斑駁的病徵。另外,ZYMVAC表現後轉錄基因消寂作用抑制子CMV 2b可回復單斑反應,在奎藜上形成透化斑點,但在系統性寄主矮南瓜上,ZYMVAC ZYMVACc2b可增加ZYMVAC之毒力,造成嚴重病徵。
Synergistic effects of mixed infection of Cucumber mosaic virus (CMV), a member of Cucumovirus, with Zucchini yellow mosaic virus (ZYMV), a member of Potyvirus, cause severe losses on production of cucurbits worldwide. Enhancement of systemic translocation of CMV plays an important role in disease severity. In order to analyze the possible functions of the individual genes of CMV on synergistic effects in mixed infection, in vivo infectious clone of ZYMV was used to express the MP, CP, or 2b ORF of CMV in zucchini squash and local lesion hosts. The individual genes were in frame inserted into the N-terminal region of the HC-Pro coding sequence, or the C-terminal region of the NIb coding sequence of the ZYMV vector, with additional six histidine residues and an NIa protease cleavage site to facilitae the purification and processing of expressed proteins. The recombinant ZYMVcMP and ZYMVNcMP that carried the entire MP ORF of CMV at the N-terminal region of HC-Pro and the C terminal region of NIb coding sequence, respectively, induced local lesions on Chenopodium quinoa 3-4 days earlier than those induced by the wild type ZYMV TW-TN3. ZYMVcCP carrying the CP ORF of CMV and ZYMVc2b carrying the 2b ORF of CMV both induced local lesions on C. quinoa 7 days after inoculation similar to those induced by the wild type ZYMV TW-TN3. On squash, ZYMVcMP, ZYMVNcMP, ZYMVcCP and ZYMVc2b induced severe yellow mosaic symptoms similar to those induced by the parental ZYMV. The individual ORFs of CMV were also constructed in the in vivo infectious clone of an engineered mild strain ZYMVAC, which contained two amino acid changes in the HC-Pro protein and caused infection on C. quinoa without formation of local lesions. Interestingly, the mild recombinant ZYMVACcMP expressing the entire MP ORF of CMV restored the hypersensitive reaction and induced tiny and necrotic lesions on C. quinoa 3-4 days earlier than those induced by the wild type ZYMV. ZYMVACcCP expressing the entire CP ORF of CMV did not induce local lesions on C. quinoa. ZYMVACc2b expressing the gene-slicing suppressor 2b ORF of CMV induced hypersensitive reaction and caused chlorotic lesions on C. quinoa 7 days after inoculation. On squash plants, ZYMVACcMP and ZYMVACcCP caused mild symptoms similar to those induced by ZYMVAC, but ZYMVACc2b caused yellow mosaic and leaf distortion on leaves similar to those caused by the wild type ZYMV TW-TN3. Our data demonstrates that on C. quinoa, the expressed CMV MP complements the mutated HC-Pro protein defective in inducing hypersensitive reaction and fasten the development of local lesions but does not enhance the virulence of ZYMV. We conclude that the expressed CMV 2b protein restore the ZYMV's ability to form local lesions without necrosis and is a virulence enhancer for ZYMV infection.
URI: http://hdl.handle.net/11455/31755
Appears in Collections:植物病理學系

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