Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan.
Frontier Research Laboratories, Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Kanagawa 210-8681, Japan.
J Virol Methods. 2019 Nov;273:113711. doi: 10.1016/j.jviromet.2019.113711. Epub 2019 Aug 9.
Apple latent spherical virus (ALSV) can infect a variety of crops, usually without inducing symptoms. Partial gene sequences can be introduced into ALSV vectors for the induction of virus-induced gene silencing (VIGS). These features are beneficial for the estimation of gene functions in plants, with relatively concise experimental manipulations. Given that the infectability of chili peppers (Capsicum spp.) by ALSV was unknown, an ALSV infectivity test was performed on the highly pungent Capsicum chinense cultivar 'Habanero'. The chili pepper plants were not infected after rub-inoculation with a crude homogenate of ALSV-infected Chenopodium quinoa leaves, whereas inoculating them with a concentrated ALSV virus preparation caused an infection. Inoculation with an ALSV RNA preparation by gold particle bombardment resulted in high infection rates (about 90%). The infection was systemic and the infected plants were symptomless. For the induction of VIGS, 201-nucleotide fragments of the putative aminotransferase (pAMT) gene were introduced into the ALSV vector. These ALSV vectors infected 80-90% of RNA-inoculated chili pepper seedlings. Expression of pAMT-mRNA was repressed in the placenta of immature fruit of infected plants. The silencing of pAMT in the infected plants caused a substantial decrease in capsaicin content and a concomitant moderate accumulation of the non-pungent bioactive metabolite capsiate in these plants. These results showed that ALSV could be used to study gene functions by VIGS and to enhance capsiate accumulation in chili pepper through genetic modification.
苹果潜隐病毒（ALSV）可以感染多种作物，通常不引起症状。部分基因序列可以被引入 ALSV 载体中，诱导病毒诱导的基因沉默（VIGS）。这些特点有利于在植物中估计基因功能，实验操作相对简单。由于不清楚辣椒（Capsicum spp.）是否能被 ALSV 感染，因此对高辣度的辣椒品种‘Habanero’进行了 ALSV 的感染性测试。用感染了 ALSV 的藜叶粗匀浆摩擦接种辣椒植株后，并未感染 ALSV，但用浓缩的 ALSV 病毒制剂接种时，植株会被感染。用金颗粒轰击接种 ALSV RNA 制剂可导致高感染率（约 90%）。感染是系统性的，感染的植株无症状。为了诱导 VIGS，将假定的氨基转移酶（pAMT）基因的 201 个核苷酸片段引入到 ALSV 载体中。这些 ALSV 载体感染了 80-90%的 RNA 接种的辣椒幼苗。在感染植株未成熟果实的胎座中，pAMT-mRNA 的表达受到抑制。在感染植物中 pAMT 的沉默导致辣椒素含量大幅下降，同时这些植物中无辣味的生物活性代谢物辣椒碱的含量适度积累。这些结果表明，ALSV 可用于通过 VIGS 研究基因功能，并通过遗传修饰提高辣椒中的辣椒碱含量。
