Expression and antiviral application of exogenous lectin (griffithsin) in sweetpotatoes

Shuai Liu, Yang Yu, Ke Guo, Qian Zhang, Zhaodong Jia, Morales Rodríguez Alfredo, Peiyong Ma, Hao Xie, X. Bian
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Abstract

Griffithsin (GRFT) is a highly effective, broad-spectrum, safe, and stable viral inhibitor used to suppress a variety of viruses. However, little information is available on whether GRFT can prevent plant viral diseases. In this study, we constructed a GRFT overexpression vector containing the sweetpotato storage cell signal peptide and generated exogenous GRFT overexpression lines through genetic transformation. The transgenic plants showed notable resistance to sweetpotato virus disease in the virus nursery. To verify the antiplant virus function of GRFT, transient expression in tobacco leaves showed that GRFT inhibited the sweetpotato leaf curl virus (SPLCV). The replication of SPLCV was entirely inhibited when the concentration of GRFT reached a certain level. The results of pulldown and BIFC assays showed that GRFT did not interact with the six components of SPLCV. In addition, the mutated GRFTD/A without the binding ability of carbohydrate and anticoronavirus function, in which three aspartate residues at carbohydrate binding sites were all mutated to alanine, also inhibited SPLCV. Quantitative reverse-transcription PCR analyses showed that the tobacco antiviral-related genes HIN1, ICS1, WRKY40, and PR10 were overexpressed after GRFT/GRFTD/A injection. Furthermore, HIN1, ICS1, and PR10 were more highly expressed in the leaves injected with GRFTD/A. The results suggest that sweetpotato is able to express GRFT exogenously as a bioreactor. Moreover, exogenous GRFT expression inhibits plant viruses by promoting the expression of plant antiviral genes.
外源凝集素(griffithsin)在甘薯中的表达和抗病毒应用
Griffithsin (GRFT) 是一种高效、广谱、安全且稳定的病毒抑制剂,可用于抑制多种病毒。然而,有关 GRFT 能否预防植物病毒病的信息却很少。在本研究中,我们构建了含有甘薯贮藏细胞信号肽的 GRFT 过表达载体,并通过遗传转化产生了外源 GRFT 过表达株系。在病毒苗圃中,转基因植株对甘薯病毒病表现出显著的抗性。为了验证 GRFT 的抗植物病毒功能,在烟草叶片中的瞬时表达表明,GRFT 对甘薯卷叶病毒(SPLCV)有抑制作用。当 GRFT 的浓度达到一定水平时,SPLCV 的复制完全被抑制。pulldown 和 BIFC 检测结果表明,GRFT 与 SPLCV 的六种成分没有相互作用。此外,没有碳水化合物结合能力和抗oronavirus功能的突变GRFTD/A(其中碳水化合物结合位点的三个天冬氨酸残基全部突变为丙氨酸)也对SPLCV有抑制作用。定量反转录 PCR 分析表明,注射 GRFT/GRFTD/A 后,烟草抗病毒相关基因 HIN1、ICS1、WRKY40 和 PR10 表达过高。此外,HIN1、ICS1 和 PR10 在注射 GRFTD/A 的叶片中的表达量更高。这些结果表明,甘薯能够作为生物反应器外源表达 GRFT。此外,外源表达 GRFT 可通过促进植物抗病毒基因的表达来抑制植物病毒。
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