背景:枯萎病是全世界番茄的一种破坏性土壤传播的真菌病。常规的疾病预防方法,包括使用常见的杀虫剂和土壤日晒法等方法,通常对这种疾病的治疗无效。因此,迫切需要鉴定病原体中的毒力相关基因,这些基因可以作为杀菌剂开发的目标。
结果:本研究中使用的病原体的致病性测试和系统发育分类证实其为尖孢镰刀菌f。sp。lycopersici(Fol)菌株。最近的一项发现表明,EF1α,一种在几个真菌属中具有保守结构相似性的蛋白质,在稻瘟病菌的致病性中起作用,稻瘟病真菌。因此,在这项研究中,我们对EF1α进行了结构和功能分类,以了解其在Fol致病性中的作用。使用酵母延伸因子复合物EEF1A:EEF1BA的模板晶体结构创建了FolEF1α的蛋白质模型,该模型与目标蛋白质具有最大的相似性。使用STRING在线数据库,使用Cytoscape软件鉴定了EF1α的hub基因之间的相互作用信息,并识别了蛋白质-蛋白质相互作用网络。结合功能分析的结果,MCODE,选择包括FolEF1α的CytoNCA和CytoHubba4hub基因用于进一步研究。FolEF1α的三个相互作用物显示出与已知杀菌剂复合的神经孢菌中发现的同源蛋白的最大相似性,环己酰亚胺.通过序列相似性和PDB数据库分析,发现了FolEF1α的同系物:在酵母中与GDPNP复合的EEF1A:EEF1BA和在solfolobussolfaricus中与GDP复合的EF1α。STITCH数据库分析表明,EF1α及其其他相互作用的伴侣与二磷酸鸟苷(GDPNP)和三磷酸鸟苷(GTP)相互作用。
结论:我们的研究为识别枯萎病中的几个枢纽基因网络提供了框架,这些枢纽基因网络可以用作杀菌剂开发的新靶标。EF1α参与核质转运途径表明,它在GTP结合中起作用,因此除了用作生物标志物外,它可以进一步开发作为杀菌剂开发的有效靶标。因为,发现与FolEF1α紧密相关的其他三种蛋白质与与杀菌剂-环己酰亚胺形成复合物的粗糙神经孢菌的同源蛋白质显示出最大的相似性。因此,我们建议环己酰亚胺也可以用于防治番茄枯萎病。也可以使用在酵母和sulfolobussolfataricus中观察到的同源蛋白确定FolEF1α的活性位点腔,以计算筛选杀真菌剂。在此基础上,我们还建议通过STITCH分析鉴定的其他密切相关的基因,它们也可以作为杀菌剂开发的目标。
BACKGROUND: Fusarium wilt is a devastating soil-borne fungal disease of tomato across the world. Conventional method of disease prevention including usage of common pesticides and methods like soil solarisation are usually ineffective in the treatment of this disease. Therefore, there is an urgent need to identify virulence related genes in the pathogen which can be targeted for fungicide development.
RESULTS: Pathogenicity testing and phylogenetic classification of the pathogen used in this study confirmed it as Fusarium oxysporum f. sp. lycopersici (Fol) strain. A recent discovery indicates that EF1α, a protein with conserved structural similarity across several fungal genera, has a role in the pathogenicity of Magnaporthe oryzae, the rice blast fungus. Therefore, in this study we have done structural and functional classification of EF1α to understand its role in pathogenicity of Fol. The protein model of Fol EF1α was created using the template crystal structure of the yeast elongation factor complex EEF1A:EEF1BA which showed maximum similarity with the target protein. Using the STRING online database, the interactive information among the hub genes of EF1α was identified and the protein-protein interaction network was recognized using the Cytoscape software. On combining the results of functional analysis, MCODE, CytoNCA and CytoHubba 4 hub genes including Fol EF1α were selected for further investigation. The three interactors of Fol EF1α showed maximum similarity with homologous proteins found in Neurospora crassa complexed with the known fungicide, cycloheximide. Through the sequence similarity and PDB database analysis, homologs of Fol EF1α were found: EEF1A:EEF1BA in complex with GDPNP in yeast and EF1α in complex with GDP in Sulfolobus solfataricus. The STITCH database analysis suggested that EF1α and its other interacting partners interact with guanosine diphosphate (GDPNP) and guanosine triphosphate (GTP).
CONCLUSIONS: Our study offers a framework for recognition of several hub genes network in Fusarium wilt that can be used as novel targets for fungicide development. The involvement of EF1α in nucleocytoplasmic transport pathway suggests that it plays role in GTP binding and thus apart from its use as a biomarker, it may be further exploited as an effective target for fungicide development. Since, the three other proteins that were found to be tightly associated Fol EF1α have shown maximum similarity with homologous proteins of Neurospora crassa that form complex with fungicide- Cycloheximide. Therefore, we suggest that cycloheximide can also be used against Fusarium wilt disease in tomato. The active site cavity of Fol EF1α can also be determined for computational screening of fungicides using the homologous proteins observed in yeast and Sulfolobus solfataricus. On this basis, we also suggest that the other closely associated genes that have been identified through STITCH analysis, they can also be targeted for fungicide development.