Antonino de Souza Júnior José Dijair, Ramos Coelho Roberta, Tristan Lourenço Isabela, da Rocha Fragoso Rodrigo, Barbosa Viana Antonio Américo, Lima Pepino de Macedo Leonardo, Mattar da Silva Maria Cristina, Gomes Carneiro Regina Maria, Engler Gilbert, de Almeida-Engler Janice, Grossi-de-Sa Maria Fatima
Graduate Program in Biology Molecular, Universidade de Brasília, Brasília, Distrito Federal, Brazil ; Embrapa Recursos Genéticos e Biotecnologia, Brasília, Distrito Federal, Brazil.
Embrapa Cerrados, Planaltina, Ditrito Federal, Brazil.
PLoS One. 2013 Dec 31;8(12):e85364. doi: 10.1371/journal.pone.0085364. eCollection 2013.
The root-knot nematode Meloidogyne incognita causes serious damage and yield losses in numerous important crops worldwide. Analysis of the M. incognita genome revealed a vast number of proteases belonging to five different catalytic classes. Several reports indicate that M. incognita proteases could play important roles in nematode parasitism, besides their function in ordinary digestion of giant cell contents for feeding. The precise roles of these proteins during parasitism however are still unknown, making them interesting targets for gene silencing to address protein function. In this study we have knocked-down an aspartic (Mi-asp-1), a serine (Mi-ser-1) and a cysteine protease (Mi-cpl-1) by RNAi interference to get an insight into the function of these enzymes during a host/nematode interaction. Tobacco lines expressing dsRNA for Mi-ser-1 (dsSER), Mi-cpl-1 (dsCPL) and for the three genes together (dsFusion) were generated. Histological analysis of galls did not show clear differences in giant cell morphology. Interestingly, nematodes that infected plants expressing dsRNA for proteases produced a reduced number of eggs. In addition, nematode progeny matured in dsSER plants had reduced success in egg hatching, while progeny resulting from dsCPL and dsFusion plants were less successful to infect wild-type host plants. Quantitative PCR analysis confirmed a reduction in transcripts for Mi-cpl-1 and Mi-ser-1 proteases. Our results indicate that these proteases are possibly involved in different processes throughout nematode development, like nutrition, reproduction and embryogenesis. A better understanding of nematode proteases and their possible role during a plant-nematode interaction might help to develop new tools for phytonematode control.
根结线虫南方根结线虫在全球众多重要作物上造成严重损害和产量损失。对南方根结线虫基因组的分析揭示了大量属于五种不同催化类别的蛋白酶。几份报告表明，南方根结线虫蛋白酶除了在消化取食的巨型细胞内容物的常规功能外，还可能在线虫寄生中发挥重要作用。然而，这些蛋白质在寄生过程中的具体作用仍然未知，这使得它们成为通过基因沉默来研究蛋白质功能的有趣靶点。在本研究中，我们通过RNA干扰敲低了一种天冬氨酸蛋白酶（Mi-asp-1）、一种丝氨酸蛋白酶（Mi-ser-1）和一种半胱氨酸蛋白酶（Mi-cpl-1），以深入了解这些酶在宿主/线虫相互作用过程中的功能。构建了表达针对Mi-ser-1（dsSER）、Mi-cpl-1（dsCPL）以及这三个基因的双链RNA（dsFusion）的烟草株系。对虫瘿的组织学分析未显示巨型细胞形态有明显差异。有趣的是，感染表达蛋白酶双链RNA植物的线虫产卵数量减少。此外，在dsSER植物中成熟的线虫后代孵化成功率降低，而来自dsCPL和dsFusion植物的后代感染野生型宿主植物的成功率较低。定量PCR分析证实了Mi-cpl-1和Mi-ser-1蛋白酶转录本的减少。我们的结果表明，这些蛋白酶可能参与线虫发育的不同过程，如营养、繁殖和胚胎发生。更好地了解线虫蛋白酶及其在植物 - 线虫相互作用中的可能作用，可能有助于开发新的植物线虫防治工具。
