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 (D⁸⁹A) or glutamic acid at position 1069 in the ORF1/2 read-through protein, in the RNA-dependent RNA polymerase domain to alanine (E¹⁰⁶⁹A) 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 D⁸⁹ in the coat protein and E¹⁰⁶⁹ 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.

交叉保护是控制黄瓜绿斑驳花叶病毒（CGMMV）的一种有前途的替代方法，这种黄瓜对全球葫芦的生产具有越来越重要的经济意义。交叉保护应用于控制CGMMV面临的主要因素之一是缺乏可用的轻度突变体。本文的目的是筛选减毒的CGMMV突变体，并评估其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启动子的修饰的pCambia0390载体上，获得了CGMMV的感染性cDNA克隆pCGMMV 。通过定点诱变获得了五个pCGMMV衍生的突变体，并将其接种到本氏烟草中用于症状观察的植物。评价减毒的CMGMV突变体在交叉保护中的效率。含内含子的克隆pCGMMV在本氏烟草中诱导出相似的疾病症状，并在病毒中积累了相似的滴度，与野生型CGMMV相似。在RNA依赖性RNA聚合酶结构域中，外壳蛋白中第89位的天冬氨酸突变为丙氨酸（D ⁸⁹ A）或ORF1 / 2读通蛋白中第1069位的谷氨酸突变为丙氨酸（E ¹⁰⁶⁹ A）烟草中pCGMMV的症状显着植物。在交叉保护试验中，两个突变体pCGMMV-CP-D89A和pCGMMV-RdRp-E1069A可以预防CGMMV的重复感染，保护效率分别为91.7％和100％。含内含子的克隆pCGMMV稳定且具有高度感染性。外壳蛋白中的D ⁸⁹和RNA依赖性RNA聚合酶中的E ¹⁰⁶⁹在调节CGMMV的毒力中起重要作用。突变体pCGMMV-CP-D89A和pCGMMV-RdRp-E1069A在通过交叉保护控制CGMMV方面具有巨大潜力。