應用 host-induced gene silencing 探討 OPEL 在疫病菌感染過程的角色

Abstract

疫病菌 (Phytophthoraparasitica)為重要植物病原卵菌，能在許多植物造成嚴 重病害。Chang 等(2015)發現將疫病菌 OPEL 之重組蛋白注射於 Nicotiana tabacum (cv. Samsun NN)葉片可誘發癒傷葡聚醣堆積、活性氧分子生成、誘導防 禦相關基因表現及細胞凋亡; 此外，還導致菸草對 Tobacco mosaic virus, Ralstonia solanacearum 以及 P. parasitica 產生系統性抗性，顯示 OPEL 具有 elicitor 活性，本研究應用寄主誘導基因靜默 (host-induced gene silencing; HIGS) 探討 OPEL 在疫病菌致病過程扮演的角色。為建立 HIGS 系統，首先藉由農桿菌 浸潤暫表現分析雙股 RNA 表現載體 pB7GW1WG2(I)以及 XVE 系統之 pERjahBAR 的表現效率，發現兩者均能於圓葉菸草穩定生成雙股 RNA，但 pERjahBAR 尚須透過雌固醇處理才能誘導雙股 RNA 表現。以 eGFP 為指標比較 不同載體的靜默效率時，發現在浸潤後第 8 天，pERjahBAR 展現較佳之調降 16c 圓葉菸草 eGFP 訊號的能力。為了瞭解於圓葉菸草中，暫表現的雙股 RNA 是否 會導致病原目標基因靜默，於是將表現 eGFP 之疫病菌 (strain 1121)接種於以 pERjahBAR表現eGFP雙股RNA的圓葉菸草，相較於在對照組pERjahBAR(EV) 圓葉菸草，疫病菌 (strain 1121)的病害發展不受影響，但其 eGFP 訊號被顯著調 降，這些結果顯示 HIGS 可應用於研究 OPEL 基因的功能。本研究將 OPEL 基因 分成四個片段分別次選殖至載體 pERjahBAR，逐一導至圓葉菸草以表現雙股 RNA，再進一步接種疫病菌 (strain 1121)，發現表現 OP 300 雙股 RNA 之圓葉菸 草在接種後 30 小時，病斑面積顯著小於以 pERjahBAR (EV)處理的對照組。進一 步進行 qPCR 分析發現，在接種後 30 小時，表現 OP 300 雙股 RNA 的葉片所含 的疫病菌量少於表現 EV 雙股 RNA 的對照組葉片，顯示表現 OP 300 雙股 RNA 可有效抑制疫病菌於接種後 30 小時的病害發展。然而，其他三個雙股 RNA 的處 理組，均未影響疫病菌病害發展。以上結果顯示，表現 OP 300 雙股 RNA 會妨礙 疫病菌 (strain 1121)的侵染，未來還需瞭解此現象是否因為下調 OPEL 或是其同源基因所引起。

Phytophthora parasitica is an oomycete pathogen that causes severe plant diseases worldwide. OPEL, a novel elicitor protein from P. parasitica, caused cell death, callose deposition, reactive oxygen species production, and defense gene expression on Nicotiana tabacum (cv. Samsun NN). Moreover, OPEL conferred plant systemic resistance against several pathogens, including Tobacco mosaic virus, Ralstonia solanacearum, and P. parasitica. However, its role in the infection process of P. parasitica is not clear. Recent studies have shown that the expression in plants of dsRNA targeting on pathogen genes can specifically silence their targets in invading pathogens, which is known as host-induced gene silencing (HIGS). In this study, HIGS was employed to understand the role of OPEL in the infection process of P. parasitica. As a first trial, two vectors were analyzed for their efficiency to express eGFP dsRNA and to induce gene silencing on Nicotiana benthamiana 16c plants, including pB7GW1WG2(I)::eGFP and pERjahBAR::eGFP, the latter involving the use of 17β-estradiol to induce dsRNA expression. Comparison of the GFP signals at 8 days post agroinfiltration indicates pERjahBAR::eGFP showed better silencing efficiency on N. benthamiana 16c than pB7GW1WG2(I)::eGFP. To know whether transiently expressed dsRNA might lead to silencing of target genes in the pathogens, pERjahBAR::eGFP was used to express eGFP dsRNA on N. benthamiana and the plants were then inoculated with P. parasitica (strain 1121), which constitutively expresses GFP. Disease symptom shown on eGFP dsRNA-expressing N. benthamiana looked similar to that with the empty vector control. Nevertheless, the GFP florescence signal emitted by P. parasitica (strain 1121) was significantly downregulated, which indicates the occurrence of HIGS. Thus, this system was further used to uncover the function of OPEL. Four segments of OPEL cDNA sequence were subcloned into pERjahBAR and tested for their respective efficiency to affect the infection of P. parasitica (strain 1121). At 30 hours post inoculation, the size of disease lesions shown on N. benthamiana leaves expressing OPEL 300 dsRNA was smaller than that of the control plants. Analysis by qPCR also detected much less biomass of P. parasitica (strain 1121) than that from the control plants. However, expression of the other three dsRNAs showed no effect on pathogen infection. These results indicate that the expression of OP 300 dsRNA can impede disease development of P. parasitica (strain 1121). More work is required to know whether this effect is indeed caused by downregulation of OPEL and/or its homologs.