Liu Jin, Li Xiang-Dong, Xu Shuai
College of Landscape Science and Engineering, Shandong Agriculture and Engineering University, Jinan, 250100, Shandong, China.
Shandong Provincial Key Laboratory for Agricultural University, Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai'an, 271018, Shandong, China.
Virus Genes. 2020 Apr;56(2):228-235. doi: 10.1007/s11262-019-01726-3. Epub 2020 Jan 1.
Cross protection is a promising alternate to control Cucumber green mottle mosaic virus (CGMMV) which is of increasing economic importance to cucurbit production worldwide. One major factor confronting the application of cross protection to control CGMMV is the scarcity of available mild mutants. The objective of this paper was to screen attenuated mutants of CGMMV and evaluate their potential in cross protection. An infectious cDNA clone of CGMMV, pCGMMV, was obtained by cloning intron-containing CGMMV genome to modified pCambia0390 vector with the Cauliflower mosaic virus 35S promoter. Five pCGMMV-derived mutants were obtained via site-directed mutagenesis and inoculated to Nicotiana benthamiana plants for symptom observation. The attenuated CGMMV mutants were evaluated for their efficiency in cross protection. The intron-containing clone pCGMMV induced similar disease symptoms and accumulated similar titres of virus in N. benthamiana plants as wild-type CGMMV. Mutations of aspartic acid at position 89 in the coat protein to alanine (DA) or glutamic acid at position 1069 in the ORF1/2 read-through protein, in the RNA-dependent RNA polymerase domain to alanine (EA) alleviated the symptoms of pCGMMV in N. benthamiana plants significantly. In cross protection assay, the two mutants pCGMMV-CP-D89A and pCGMMV-RdRp-E1069A could prevent the superinfection of CGMMV, with protection efficiency of 91.7% and 100%, respectively. The intron-containing clone pCGMMV was stable and highly infectious. The D in the coat protein and E in the RNA-dependent RNA polymerase played an important role in regulating the virulence of CGMMV. Mutants pCGMMV-CP-D89A and pCGMMV-RdRp-E1069A were of great potential in the control of CGMMV via cross protection.
交叉保护是一种很有前景的控制黄瓜绿斑驳花叶病毒（CGMMV）的替代方法，该病毒对全球葫芦科作物生产的经济重要性日益增加。应用交叉保护来控制CGMMV面临的一个主要因素是可用的温和突变体稀缺。本文的目的是筛选CGMMV的减毒突变体并评估它们在交叉保护中的潜力。通过将含内含子的CGMMV基因组克隆到带有花椰菜花叶病毒35S启动子的修饰pCambia0390载体中，获得了CGMMV的感染性cDNA克隆pCGMMV。通过定点诱变获得了5个pCGMMV衍生的突变体，并接种到本氏烟草植株上进行症状观察。对减毒的CGMMV突变体的交叉保护效率进行了评估。含内含子的克隆pCGMMV在本氏烟草植株中诱导了与野生型CGMMV相似的病害症状，并积累了相似的病毒滴度。外壳蛋白中第89位的天冬氨酸突变为丙氨酸（DA），或ORF1/2通读蛋白中第1069位的谷氨酸在RNA依赖的RNA聚合酶结构域突变为丙氨酸（EA），显著减轻了pCGMMV在本氏烟草植株中的症状。在交叉保护试验中，两个突变体pCGMMV-CP-D89A和pCGMMV-RdRp-E1069A可以防止CGMMV的超感染，保护效率分别为91.7%和100%。含内含子的克隆pCGMMV是稳定且具有高感染性的。外壳蛋白中的D和RNA依赖的RNA聚合酶中的E在调节CGMMV的毒力方面发挥了重要作用。突变体pCGMMV-CP-D89A和pCGMMV-RdRp-E1069A在通过交叉保护控制CGMMV方面具有很大潜力。
