Instituto de Microbiologia Paulo de Goes, UFRJ, Ilha do Fundao, Rio de Janeiro, Rio de Janeiro, Brazil.
PLoS One. 2012;7(10):e48170. doi: 10.1371/journal.pone.0048170. Epub 2012 Oct 31.
Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa.
植物质体是鞭毛门原生动物门的植物寄生虫，通过食草昆虫传播。一些植物质体对农业造成重大损害。半翅目昆虫 Oncopeltus fasciatus 是几种锥虫的天然和实验宿主，包括植物质体。昆虫唾液腺的入侵是植物质体生命周期中的最重要事件之一。在本研究中，我们通过扫描电子显微镜显示植物质体 serpens 在 O. fasciatus 唾液腺外表面的结合，并通过配体印迹分析显示活寄生虫与唾液腺总蛋白的体外相互作用。这种结合主要通过与 130 kDa 唾液腺蛋白的相互作用发生。该蛋白的胰蛋白酶消化肽的质谱分析通过 16 个消化肽与人类层粘连蛋白-5β3 链前体序列匹配 23%。通过 O. fasciatus 胚胎转录组的蛋白质序列搜索显示出与人层粘连蛋白β3 亚基具有 51%相似性的部分序列。抗人层粘连蛋白-5β3 链多克隆抗体通过免疫印迹识别 130 kDa 蛋白。寄生虫与唾液腺的结合强烈受到人层粘连蛋白-5 的抑制，受纯化的 130 kDa 昆虫蛋白和针对人层粘连蛋白-5β3 链的多克隆抗体的抑制。这是第一个证明来自 O. fasciatus 唾液腺的层粘连蛋白样分子作为植物质体结合的受体的报告。本研究中的结果是重要的发现，将支持进一步的研究，旨在开发防止植物质体从昆虫传播到植物和反之亦然的新方法。
