摘要
Insect transmission of plant viruses results in tremendous economic loss within the agricultural sector worldwide. Aphids account for nearly half of insect-borne plant virus transmission. Viruses in the family Luteoviridae are transmitted by aphids in a persistent-circulative manner that requires specific molecular interactions between the aphid and virus. Ingested virions cross the aphid gut and salivary gland epithelial barriers using receptors that have not been identified. We assessed the binding of a model luteovirid, Pea enation mosaic virus (PEMV), to brush border membrane vesicles (BBMV) of the pea aphid, Acyrthosiphon pisum using a two-dimensional far-western blot method. Pea aphid membrane alanyl aminopeptidase N (APN) was identified by mass spectrometry following specific binding to PEMV virions and to a PEMV coat protein-eGFP fusion peptide (CP-P-eGFP). The binding of PEMV to APN was confirmed by multiple methods including a pull-down assay, surface plasmon resonance (SPR) analysis, and by increased binding of CP-P-eGFP to baculovirus-expressed pea aphid APN in Sf9 cells. We also show that a peptide (GBP3.1) that was previously shown to impede uptake of PEMV into the pea aphid also binds to APN. Based on these results, we conclude that APN is a putative gut receptor for PEMV in the pea aphid and if confirmed would be the first insect receptor identified for a plant virus. Interestingly, PEMV appears to bind to a different, as yet unidentified, receptor in a second vector, Myzus persicae, suggesting that different gut receptors may be used by luteoviruses in different vector species.
Luteoviruses are acquired when aphids ingest the phloem sap of an infected plant. Phloem proteins have been shown to associate with luteovirus particles and facilitate aphid transmission in in vitro feeding assays. We showed an increase of virus in the hemocoel of aphids fed on artificial diet containing purified PEMV with bovine serum albumin (BSA) compared to aphids fed on virus in the absence of BSA. Interestingly, BSA reduced the amount of a mutant virus lacking the minor structural protein readthrough domain (RTD) detected in the aphid hemocoel. We also demonstrated that the PEMV RTD binds to multiple aphid proteins. SPR analysis indicated that the CP and RTD both bind to BSA. Based on these data, models are presented to account for the role of the RTD and mechanism by which BSA and plant proteins facilitate virus entry into the aphid hemocoel.
Little is known about the role of glycans in mediating luteovirus-aphid interactions. We used the lectins Concanavalin A (ConA) and Galanthus nivalis agglutinin (GNA) for lectin blot analysis of BBMV and confirmed that pea aphid proteins are glycosylated with mannose and glucose moieties. APN, the PEMV gut receptor, is glycosylated with mannose residues. However, we did not detect any binding of PEMV to a synthesized tri-mannose glycan that is common in insects using both isothermal titration calorimetry or a carbohydrate microarray. These results suggest that mannose by itself is not involved in PEMV-APN binding. ConA bound to PEMV indicating that viral structural proteins are glycosylated. The potential role of virus glycosylation in aphid transmission of luteoviruses is discussed. Taken together, our increased understanding of luteovirus-aphid vector interaction will facilitate research into other plant virus-insect vector systems, and the development of mitigation strategies.
摘要译文
植物病毒的昆虫传播导致了全世界农业部门的巨大经济损失。蚜虫占昆虫传播植物病毒传播的近一半。家庭Luteoviridae中的病毒通过蚜虫以持续循环的方式传播，需要蚜虫和病毒之间的特定分子相互作用。摄入的病毒体使用尚未鉴定的受体穿过蚜虫肠和唾液腺上皮屏障。我们评估了模型luteovirid，豌豆enation花叶病毒（PEMV），使用二维far-western印迹方法刷新豌豆蚜虫，Acyrthosiphon豌豆的边界膜囊泡（BBMV）。豌豆蚜膜丙氨酰氨肽酶N（APN）通过特异性结合PEMV病毒粒子和PEMV外壳蛋白-eGFP融合肽（CP-P-eGFP）后的质谱鉴定。PEMV与APN的结合通过多种方法证实，包括下拉测定，表面等离子体共振（SPR）分析，并且通过在Sf9细胞中增加CP-P-eGFP与杆状病毒表达的豌豆蚜APN的结合。我们还展示了一种肽（GBP3。1）先前显示阻止PEMV摄入豌豆蚜虫也与APN结合。基于这些结果，我们得出这样的结论：APN是PEMV在豌豆蚜虫中的假定肠道受体，并且如果证实将是针对植物病毒鉴定的第一种昆虫受体。有趣的是，PEMV似乎与第二种载体桃蚜Myzus persicae中的另一种不同的受体结合，这表明不同的肠道受体可能被不同的载体物种中的黄疸病毒使用。当蚜虫摄取受感染植物的韧皮部汁液时获得了黄疸病毒。已显示韧皮蛋白与黄体病毒颗粒缔合并促进体外进食测定中的蚜虫传播。我们发现，与牛血清白蛋白（BSA）纯化的PEMV人工饲料喂养的蚜虫hemossel病毒相比，在缺乏BSA病毒饲养的蚜虫病毒增加。有趣的是，BSA减少了缺乏在蚜虫hemocoel中检测到的次要结构蛋白通读域（RTD）的突变病毒的量。我们还证明PEMV RTD结合多种蚜虫蛋白。 SPR分析表明CP和RTD都与BSA结合。根据这些数据，模型是为了说明RTD的作用以及BSA和植物蛋白促进病毒进入蚜虫hemurgel的机制。关于聚糖在调解黄疸病毒 - 蚜虫相互作用中的作用知之甚少。使用凝集素刀豆蛋白A（ConA）和Galanthus nivalis凝集素（GNA）进行BBMV的凝集素印迹分析，并证实豌豆蚜虫蛋白质被甘露糖和葡萄糖部分糖基化。APN（PEMV肠受体）用甘露糖残基糖基化。然而，我们没有使用等温滴定量热法或碳水化合物微阵列检测到PEMV与昆虫常见的合成三甘露聚糖的任何结合。这些结果表明甘露糖本身不参与PEMV-APN结合。 ConA与PEMV结合表明病毒结构蛋白被糖基化。讨论病毒糖基化在黄疸病毒蚜传播中的潜在作用。综合考虑，我们对luteovirus-aphid载体相互作用的了解将会促进对其他植物病毒 - 昆虫载体系统的研究以及缓解策略的开发。
Linz, Lucas Benjamin. Molecular interactions between Pea enation mosaic virus and its pea aphid vector[D]. US: Iowa State University, 2013