Length-dependent accumulation of double-stranded RNAs in plastids affects RNA interference efficiency in the Colorado potato beetle
Length-dependent accumulation of double-stranded RNAs in plastids affects RNA interference efficiency in the Colorado potato beetle
复制标题
质体中双链 RNA 的长度依赖性积累影响科罗拉多马铃薯甲虫的 RNA 干扰效率。
DOI：
10.1093/jxb/eraa001
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发表时间：
2020-05-09
影响因子：
6.9
通讯作者：
Zhang, Jiang
中科院分区：
文献类型：
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作者：
He, Wanwan;Xu, Wenbo;Zhang, Jiang
表达针对科罗拉多马铃薯甲虫 (CPB) 必需基因的双链 RNA (dsRNA) 的转质体马铃薯植物可以通过触发 RNA 干扰 (RNAi) 反应而对幼虫致命。质体中 dsRNA 的高积累水平对于赋予昆虫有效的 RNAi 反应至关重要。然而，dsRNA 的长度和序列是否决定 RNAi 的功效和/或影响质体中 dsRNA 积累的水平尚不清楚。我们通过将体外合成的 dsRNA 喂给幼虫，比较了针对 CPB β-肌动蛋白基因 (ACT) 的不同长度 dsRNA 的 RNAi 功效。我们发现，虽然 60 bp dsRNA 在 CPB 中仅诱导相对较低的 RNAi 反应，但 200 bp 及更长的 dsRNA 导致高死亡率和类似的幼虫生长迟缓。当 dsRNA 从马铃薯植物的质体（叶绿体）基因组中表达时，我们发现它们的积累与长度呈负相关。 dsRNA 积累水平与观察到的死亡率、幼虫生长抑制和靶基因表达抑制呈正相关。重要的是，表达 200 bp dsRNA 的转质体马铃薯植物比表达 297 bp dsRNA 的植物能更好地免受 CPB 影响，表达 297 bp dsRNA 是我们之前研究中表现最好的品系。我们的结果表明，dsRNA 的长度是影响其在质体中积累的重要因素，从而决定了杀虫 RNAi 效果的强度。我们的研究结果将有助于设计优化的 dsRNA 表达构建体，通过质体介导的 RNAi 来保护植物。
Transplastomic potato plants expressing double-stranded RNA (dsRNA) targeted against essential genes of the Colorado potato beetle (CPB) can be lethal to larvae by triggering an RNA interference (RNAi) response. High accumulation levels of dsRNAs in plastids are crucial to confer an efficient RNAi response in the insects. However, whether length and sequence of the dsRNA determine the efficacy of RNAi and/or influence the level of dsRNA accumulation in plastids is not known. We compared the RNAi efficacy of different lengths of dsRNA targeted against the CPB beta-Actin gene (ACT) by feeding in vitro-synthesized dsRNAs to larvae. We showed that, while the 60 bp dsRNA induced only a relatively low RNAi response in CPB, dsRNAs of 200 bp and longer caused high mortality and similar larval growth retardation. When the dsRNAs were expressed from the plastid (chloroplast) genome of potato plants, we found that their accumulation were negatively correlated with length. The level of dsRNA accumulation was positively associated with the observed mortality, suppression of larval growth, and suppression of target gene expression. Importantly, transplastomic potato plants expressing the 200 bp dsRNA were better protected from CPB than plants expressing the 297 bp dsRNA, the best-performing line in our previous study. Our results suggest that the length of dsRNAs is an important factor that influences their accumulation in plastids and thus determines the strength of the insecticidal RNAi effect. Our findings will aid the design of optimized dsRNA expression constructs for plant protection by plastid-mediated RNAi.