Plant Virus Laboratory, Department of Biosciences, Jamia Millia Islamia, (A Central University), New Delhi, 110025, India.
Plant Virus Laboratory, Department of Biosciences, Jamia Millia Islamia, (A Central University), New Delhi, 110025, India.
J Biotechnol. 2017 Dec 10;263:21-29. doi: 10.1016/j.jbiotec.2017.10.003. Epub 2017 Oct 7.
Cotton leaf curl disease (CLCuD), a major factor resulting in the enormous yield losses in cotton crop, is caused by a distinct monopartite begomovirus in association with Cotton leaf curl Multan betasatellite (CLCuMB). Micro(mi)RNAs are known to regulate gene expression in eukaryotes, including antiviral defense in plants. In a previous study, we had computationally identified a set of cotton miRNAs, which were shown to have potential targets in the genomes of Cotton leaf curl Multan virus (CLCuMuV) and CLCuMB at multiple loci. In the current study, effect of Gossypium arboreum-encoded miRNAs on the genome of CLCuMuV and CLCuMB was investigated in planta. Two computationally predicted cotton-encoded miRNAs (miR398 and miR2950) that showed potential to bind multiple Open Reading Frames (ORFs; C1, C4, V1, and non- coding intergenic region) of CLCuMuV, and (βC1) of CLCuMB were selected. Functional validation of miR398 and miR2950 was done by overexpression approach in G. hirsutum var. HS6. A total of ten in vitro cotton plants were generated from independent events and subjected to biological and molecular analyses. Presence of the respective Precursor (pre)-miRNA was confirmed through PCR and Southern blotting, and their expression level was assessed by semi quantitative RT-PCR, Real Time quantitative PCR and northern hybridization in the PCR-positive lines. Southern hybridization revealed 2-4 copy integration of T-DNA in the genome of the transformed lines. Remarkably, expression of pre-miRNAs was shown up to 5.8-fold higher in the transgenic (T) lines as revealed by Real Time PCR. The virus resistance was monitored following inoculation of the transgenic cotton lines with viruliferous whitefly (Bemisia tabaci) insect vector. After inoculation, four of the transgenic lines remained apparently symptom free. While a very low titre of viral DNA could be detected by Rolling circle amplification, betasatellite responsible for symptom induction could not be detected in any of the healthy looking transgenic lines. In this study for the first time, efficacy of the host (G. arboreum)-encoded miRNAs against CLCuD symptoms was experimentally demonstrated through overexpression of miR398 and miR2950 in G. hirsutum var. HS6 plants. Computational prediction of miRNAs targeting virus genome and their subsequent implication in translational inhibition or cleavage based suppression of viral mRNA via overexpression could help in generating virus resistant plants.
棉花曲叶病（CLCuD）是导致棉花作物产量巨大损失的主要因素，由一种独特的单分体伴生卫星病毒与棉花曲叶病 Multan 贝塔卫星（CLCuMB）共同引起。微（mi）RNA 已知可调节真核生物中的基因表达，包括植物中的抗病毒防御。在之前的研究中，我们通过计算鉴定了一组棉花 miRNA，这些 miRNA 在多个位点显示出与棉花曲叶病 Multan 病毒（CLCuMuV）和 CLCuMB 基因组的潜在靶标。在当前的研究中，研究了棉属编码 miRNA 对 CLCuMuV 和 CLCuMB 基因组的影响。两种经计算预测的棉花编码 miRNA（miR398 和 miR2950）显示出与 CLCuMuV 的多个开放阅读框（C1、C4、V1 和非编码内含子）和 CLCuMB 的（βC1）结合的潜力。通过在 G. hirsutum var. HS6 中过表达的方法对 miR398 和 miR2950 进行了功能验证。总共从独立事件中生成了十株体外棉花植物，并进行了生物和分子分析。通过 PCR 和 Southern 印迹证实了各自前体（pre）-miRNA 的存在，并通过半定量 RT-PCR、实时定量 PCR 和 Northern 杂交在 PCR 阳性株系中评估了其表达水平。Southern 杂交显示，转化株系基因组中 T-DNA 的整合数为 2-4 个拷贝。值得注意的是，通过实时 PCR 显示，转基因（T）系中 pre-miRNA 的表达水平高达 5.8 倍。在用带毒粉虱（Bemisia tabaci）昆虫载体接种转基因棉花系后监测病毒抗性。接种后，四个转基因系显然无症状。虽然可以通过滚环扩增检测到非常低的病毒 DNA 滴度，但在任何看起来健康的转基因系中都无法检测到负责诱导症状的β卫星。在这项研究中，首次通过在 G. hirsutum var. HS6 植物中过表达 miR398 和 miR2950 来实验证明了宿主（G. arboreum）编码 miRNA 对 CLCuD 症状的功效。通过计算预测靶向病毒基因组的 miRNA，然后通过过表达在翻译抑制或切割基础上抑制病毒 mRNA，可能有助于产生抗病毒植物。
