Delgado-Martín Josemaría, Ruiz Leticia, Janssen Dirk, Velasco Leonardo
Instituto Andaluz de Investigación y Formación Agraria (IFAPA) Centro de Málaga, Málaga, Spain.
Universidad de Málaga, Málaga, Spain.
Front Plant Sci. 2022 Jun 27;13:895953. doi: 10.3389/fpls.2022.895953. eCollection 2022.
The recurrent emergence of viral diseases in intensive horticultural crops requires alternative control strategies. The topical application of double-stranded RNA (dsRNA) molecules homologous to pathogens has been proposed as a tool for virus control in plants. These dsRNAs induce the silencing mechanism, the RNA interference (RNAi), that degrades homologous dsRNAs. Cucumber green mottle mosaic virus (CGMMV) represents a serious threat to cucurbit crops. Since genetic resistance to the virus is not yet available in commercial varieties, we aimed to control this virus by RNAi. For this purpose, we obtained constructions both for expressing dsRNA in bacteria to treat cucumber plants by topical application and for agroinoculation in experiments done in the growth chamber. Besides, greenhouse tests were performed in spring and in summer when plants were challenged with the virus, and differences in several parameters were investigated, including the severity of symptoms, dry weight, total height, virus accumulation, and virus-derived small interfering RNAs (vsiRNAs). Spraying of plants with dsRNA reduced significatively CGMMV symptoms in the plants in growth chamber tests. Agroinfiltration experiments done under identical conditions were also effective in limiting the progress of CGMMV disease. In the greenhouse assay performed in spring, symptoms were significantly reduced in dsRNA-sprayed plants, and the development of the plants improved with respect to non-treated plants. Virus titers and vsiRNAs were clearly reduced in dsRNA-treated plants. The effect of protection of the dsRNA was less evident in the greenhouse assay carried out in the summer. Besides, we investigated the mobility of long (ds)RNA derived from spraying or agroinfiltrated dsRNA and found that it could be detected in local, close distal, and far distal points from the site of application. VsiRNAs were also detected in local and distal points and the differences in accumulation were compared. In parallel, we investigated the capacity of dsRNAs derived from genes of tomato leaf curl New Delhi virus (ToLCNDV), another economically important virus in cucurbits, to limit the disease in zucchini, both by agroinfiltration or by direct spraying, but found no protective effect. In view of the results, the topical application of dsRNAs is postulated as a promising strategy for CGMMV control in the cucumber.
病毒性疾病在集约化园艺作物中反复出现，需要采用替代控制策略。已有人提出，将与病原体同源的双链RNA（dsRNA）分子进行局部施用，作为一种控制植物病毒的手段。这些dsRNA会诱发沉默机制，即RNA干扰（RNAi），从而降解同源dsRNA。黄瓜绿斑驳花叶病毒（CGMMV）对葫芦科作物构成严重威胁。由于商业品种中尚未获得对该病毒的遗传抗性，我们旨在通过RNAi来控制这种病毒。为此，我们构建了两种载体，一种用于在细菌中表达dsRNA，以便通过局部施用处理黄瓜植株；另一种用于在生长室实验中进行农杆菌介导的接种。此外，在春季和夏季对植株进行病毒挑战时开展了温室试验，研究了包括症状严重程度、干重、总高度、病毒积累以及病毒衍生的小干扰RNA（vsiRNA）等几个参数的差异。在生长室试验中，用dsRNA喷洒植株可显著减轻CGMMV症状。在相同条件下进行的农杆菌浸润实验也有效地限制了CGMMV病害的发展。在春季进行的温室试验中，喷洒dsRNA的植株症状明显减轻，与未处理植株相比，植株生长状况得到改善。dsRNA处理的植株中病毒滴度和vsiRNA明显降低。在夏季进行的温室试验中，dsRNA的保护效果不太明显。此外，我们研究了喷雾或农杆菌浸润dsRNA产生的长链（ds）RNA的移动性，发现可以在施用部位的局部、近端和远端检测到它。在局部和远端也检测到了vsiRNA，并比较了积累量的差异。同时，我们研究了来源于番茄卷叶新德里病毒（ToLCNDV）（葫芦科另一种具有重要经济意义的病毒）基因的dsRNA通过农杆菌浸润或直接喷雾来限制西葫芦病害的能力，但未发现保护作用。鉴于这些结果，局部施用dsRNA被认为是控制黄瓜中CGMMV的一种有前景的策略。
