Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein
Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein
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DOI：
10.1094/phyto-01-14-0018-r
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发表时间：
2015-01-01
期刊：
影响因子：
3.2
通讯作者：
Qu, Feng
中科院分区：
文献类型：
--
作者：
Chewachong, Godwill M.;Miller, Sally A.;Qu, Feng
许多病毒的温和变种能够通过称为交叉保护的过程保护受感染的植物免受相同病毒的更严重变种的后续入侵。过去，交叉保护性病毒变体通常来源于温和的田间分离株，这些分离株有时具有遗传异质性，提供不同水平的交叉保护。在这里，我们报告了一种新的方法来快速产生交叉保护变种的番茄感染Pepino花叶病毒（PepMV）独立的可用性温和的现场分离。我们的方法试图通过突变大量产生的衣壳蛋白（CP）的保守性较低的氨基酸残基来减毒PepMV。这些保守性较低的氨基酸残基，确定通过多重比对的CP的六个马铃薯X病毒，包括PepMV，并改变系统地产生六个PepMV突变体。随后将这些突变体接种到模式植物本氏烟草（Nicotiana benthamiana）以及番茄上，以评估它们的积累水平、症状严重程度和交叉保护潜力。KD突变体CP 66和67位的苏氨酸（T）和丙氨酸（A）残基分别被赖氨酸（K）和天冬氨酸（D）取代，发现在感染植物中积累到低水平，引起非常轻微的症状，并有效地保护N. benthamiana和番茄对野生型PepMV的继发感染。这些数据表明，我们的方法代表了一种简单，快速，可靠的方法产生能够交叉保护的减毒病毒变体。
Mild variants of many viruses are able to protect infected plants from subsequent invasion by more severe variants of the same viruses through a process known as cross-protection. In the past, the cross-protective viral variants were commonly derived from mild field isolates that were sometimes genetically heterogeneous, providing variable levels of cross-protection. Here, we report a novel approach to rapidly generate cross-protective variants of the tomato-infecting Pepino mosaic virus (PepMV) independently of the availability of mild field isolates. Our approach sought to attenuate PepMV by mutating less conserved amino acid residues of the abundantly produced capsid protein (CP). These less-conserved amino acid residues were identified through multiple alignments of CPs of six potexviruses including PepMV, and were altered systematically to yield six PepMV mutants. These mutants were subsequently inoculated onto the model plant Nicotiana benthamiana, as well as tomato, to evaluate their accumulation levels, symptom severities, and cross-protection potentials. The mutant KD, in which the threonine (T) and alanine (A) residues at CP positions 66 and 67 were replaced with lysine (K) and aspartic acid (D), respectively, were found to accumulate to low levels in infected plants, cause very mild symptoms, and effectively protect both N. benthamiana and tomato against secondary infections by wild-type PepMV. These data suggest that our approach represents a simple, fast, and reliable way of generating attenuated viral variants capable of cross-protection.