The study of NbGAI involved in the accumulation of Bamboo mosaic virus in Nicotiana benthamiana

Abstract

竹嵌紋病毒(Bamboo mosaic virus, BaMV)是單一正股RNA病毒，隸屬於Flexiviridae科Potexvirus屬。宿主基因的表現可能會因為受到病毒的感染而產生改變，這些具有差異性表現的基因可能扮演著幫助或是抵抗病毒的角色。在先前的研究中，本實驗室利用cDNA-amplified fragment length polymorphism (cDNA-AFLP) 分離出90個在BaMV感染後具有差異性表現的菸草(Nicotiana benthamiana) 基因片段。其中一條具有差異性表現的基因片段ACCT7-1，在病毒感染後表現量有增加的趨勢，並且利用病毒引發基因靜默技術(virus-induced gene silencing, VIGS)使ACCT7-1表現量下降，發現BaMV的外鞘蛋白在菸草的葉子上累積量有明顯的下降。接著進一步探討ACCT7-1是影響在病毒複製還是移動的層面，將處理過VIGS的原生質體接種病毒RNA，結果顯示BaMV的外鞘蛋白的累積量也有顯著的下降，從這兩個結果可以推測ACCT7-1可能扮演幫助病毒複製的角色。為了做更進一步的研究，以Rapid Amplification of cDNA Ends (RACE) 技術延長ACCT7-1上下游的序列，並經過National Center for Biology Information (NCBI)資料庫比對，推測ACCT7-1應該是GA-INSENSITIVE (GAI)，並將ACCT7-1命名為NbGAI。接著利用得到的全長大量表達於菸草後感染病毒，發現BaMV的外鞘蛋白的累積量有明顯的上升，這個結果不僅能更確定ACCT7-1可能扮演幫助病毒的角色並且也驗證了病毒引發基因靜默技術的結果。NbGAI是DELLA家族的成員之一，同時也是植物生長賀爾蒙吉貝素(gibberellin, GA)的抑制因子，除此之外，DELLA也參與並調節許多植物賀爾蒙路徑，包括茉莉酸(Jasmonates, JA)，已有很多研究指出JA跟植物的防禦有關。本篇研究將進一步探討是否NbGAI會透過JA而對BaMV而產生影響。

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. The RNA genome comprises 6366 nts with a 5 cap and a 3 poly (A) tail. In general, the expression profile of host genes could be altered when infected by viral pathogens. These differentially expressed genes might play positive or negative roles in regulating BaMV infection cycle. In a previous study, our lab isolated 90 differentially expressed genes from BaMV-inoculated N. benthamiana plants using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. One of the upregulated genes post BaMV infection ACCT7-1 was found to involve in BaMV replication since the accumulation of BaMV coat protein was reduced when the expression of ACCT7-1 was knocked down by virus-induced gene silencing (VIGS) in the inoculated leaves and protoplasts. Furthermore, the full-length cDNA was cloned using rapid amplification of cDNA ends technique and sequenced. The identity of ACCT7-1 was revealed as a homolog of GA-INSENSITIVE (GAI), a member of DELLA family, when compared to the databases of National Center for Biology Information (NCBI). Therefore, we then designated this gene as NbGAI. To localize NbGAI in cell, I subcloned NbGAI into pEpyon vector with which can produce the fusion protein of NbGAI with Orange fluorescent protein (OFP) when expressed in plant cells. Under the circumstance of the fusion protein expression, the accumulation of BaMV coat protein is increased compared to the control plants with the expression of OFP only. The results indicate that NbGAI plays a positive role in assisting BaMV accumulation. Since NbGAI is one of the DELLA proteins that regulate plant hormones including gibberellin (GA) and Jasmonate (JA), the involvement of regulating in BaMV replication could be through the JA pathway. In applying JA or GA in plants and followed BaMV inoculation revealed that JA has either no effect or negative on the accumulation of BaMV, whereas the GA plays a positive role on that of BaMV. These results are conflict on the effect of NbGAI in BaMV. Therefore, we conclude that NbGAI, although it is a DELLA protein, may not go through the signaling pathway.