Choi Jiyeong, Browning Scottie, Schmitt-Keichinger Corinne, Fuchs Marc
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science College of Agriculture and Life Sciences, Cornell University, Cornell AgriTech at the New York State Agricultural Experiment Station, Geneva, NY, United States.
CNRS, IBMP UPR 2357, Université de Strasbourg, Strasbourg, France.
Front Microbiol. 2024 Aug 12;15:1451285. doi: 10.3389/fmicb.2024.1451285. eCollection 2024.
Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine ( spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1B, as well as their fused form (1AB). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1B in their systemic RNA silencing suppression ability by co-infiltrating 16c line plants with a silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity , and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1AB abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate expression by 1AB. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of , , and expression by 1B. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection . Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate and expression. Finally, protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology.
葡萄扇叶病毒（GFLV）是葡萄（葡萄属）最具经济影响的病毒之一，其编码的RNA沉默病毒抑制子（VSRs）最近被鉴定出来。GFLV VSRs包括RNA1编码的蛋白1A和假定的解旋酶蛋白1B，以及它们的融合形式（1AB）。GFLV VSRs抑制功能的关键特征尚不清楚。在本研究中，我们通过将沉默构建体与野生型或突变型GFLV VSR共同浸润16c系植物，探讨了蛋白1A中保守的色氨酸-甘氨酸（WG）基序和蛋白1B中甘氨酸-色氨酸（GW）基序在其系统性RNA沉默抑制能力中的作用。我们使用分子和生化分析以及诸如AlphaFold2等生物信息学工具，分析并比较了野生型和突变型GFLV VSRs在以下方面的表现：（i）抑制RNA沉默的效率，（ii）限制小干扰RNA（siRNA）积累的能力，（iii）对六个参与RNA沉默的宿主基因表达的调节作用，（iv）对病毒感染性的影响，以及（v）预测蛋白结构的变化。将蛋白1A和1AB的WG基序中的色氨酸（W）突变为丙氨酸（A），消除了它们诱导系统性RNA沉默抑制、限制siRNA积累以及下调1AB表达的能力。GFLV基因组中的这种突变导致了一种非感染性病毒。将蛋白1BHel和1ABHel的GW基序中的W突变为A，降低了它们抑制系统性RNA沉默的能力，并消除了1B对、和表达的下调作用。GFLV基因组中的这种突变在局部水平延迟了感染并抑制了系统性感染。蛋白1ABHel的WG和GW基序中的W双重突变为A消除了其诱导RNA沉默抑制、限制siRNA积累以及下调和表达的能力。最后，蛋白质结构预测表明，W到A的替换可能改变三种GFLV VSRs的结构和理化性质。总之，本研究不仅深入了解了WG/GW在GFLV VSR功能中的具体作用，还了解了其在GFLV生物学中的作用。
