Laboratory of Plant Breeding and Biometry, Faculty of Crop Science, Agricultural University of Athens, Athens 11855, Greece.
Laboratory of Plant Breeding and Biometry, Faculty of Crop Science, Agricultural University of Athens, Athens 11855, Greece.
J Virol Methods. 2023 Nov;321:114805. doi: 10.1016/j.jviromet.2023.114805. Epub 2023 Sep 4.
Zucchini yellow mosaic virus (ZYMV) infects cucurbits causing yellow mosaic in leaves, malformations in fruits, and degradation of the product quality. RNA interference (RNAi) is a cellular mechanism in eukaryotes and it is exploited to protect them against viruses. The artificial micro RNA (amiRNA) mediated approach was employed to develop resistance against ZYMV. Four amiRNAs, amiZYMV_HC-115s and amiZYMV_HC-1162s (sense), amiZYMV_HC-182as and amiZYMV_HC-196as (antisense), were computationally designed and introduced into the AtMIR390a backbone. At four days post agroinfiltration (dpa) of zucchini cotyledons the corresponding pre- and the mature amiRNAs were identified in local tissue. Upon ZYMV inoculation of zucchini, ZYMV titer was significantly lower where amiZYMV_HCs were applied in relation to control starting at two days post inoculation (dpi). Control zucchini plants exhibited symptoms at 5-8 dpi, whereas the amiZYMV_HC-treated zucchini had symptoms at 14 dpi; at 21 dpi treated zucchini exhibited a 16 %, 19 %, 32 %, and 42.5 % protection, respectively. For luffa, we observed a lower protection (0 %, 17 %, 22.5 %, and 31 % at 21 dpi). Nicotiana benthamiana DCL4 knock-down mutants were infected by ZYMV, whereas when the amiZYMV_HC-196as was agroinfiltrated ZYMV was not detected by RT-PCR. These results indicate that amiRNA-mediated resistance could be applied against ZYMV in zucchini.
南瓜黄花叶病毒（ZYMV）感染葫芦科植物，导致叶片出现黄花叶斑驳，果实畸形，并降低产品质量。RNA 干扰（RNAi）是真核生物中的一种细胞机制，可用于保护它们免受病毒侵害。人工 microRNA（amiRNA）介导的方法被用于开发对 ZYMV 的抗性。设计了四个 amiRNA，即 amiZYMV_HC-115s 和 amiZYMV_HC-1162s（正义），amiZYMV_HC-182as 和 amiZYMV_HC-196as（反义），并将其引入 AtMIR390a 骨架中。在南瓜子叶的农杆菌浸润后 4 天（dpa），在局部组织中鉴定出相应的前体和成熟的 amiRNA。在接种 ZYMV 后，与对照相比，应用 amiZYMV_HC 的地方 ZYMV 滴度显著降低，从接种后 2 天（dpi）开始。对照南瓜植物在 5-8dpi 时出现症状，而 amiZYMV_HC 处理的南瓜植物在 14dpi 时出现症状；在 21dpi 时，处理的南瓜植物分别表现出 16%、19%、32%和 42.5%的保护作用。对于丝瓜，我们观察到较低的保护作用（21dpi 时为 0%、17%、22.5%和 31%）。感染了 ZYMV 的尼伯顿烟草 DCL4 敲除突变体，而当 agroinfiltrated 时，ZYMV 则无法通过 RT-PCR 检测到 amiZYMV_HC-196as。这些结果表明，amiRNA 介导的抗性可应用于南瓜中的 ZYMV。
