含有真菌胞外膜(CFEM)结构域的蛋白质在真菌中独特存在,并在其整个生活史中起着重要作用。在这项研究中,从Marssoninabrunneaf.sp.中鉴定出11种MbCFEM蛋白。多胚珠(MULT),杨树上的一种半营养致病真菌,会导致严重的叶部病害。系统发育分析表明,根据跨膜结构域和CFEM结构域,将11种蛋白质(MbCFEM1-11)分为三个进化枝。CFEM结构域的序列比对和WebLogo分析验证了其中的氨基酸保守性。除了MbCFEM4和MbCFEM11之外,它们都具有8个半胱氨酸,它们各自缺少2个半胱氨酸。具有信号肽且不具有跨膜结构域的六个MbCFEM蛋白被认为是用于进一步功能分析的候选效应子。其CFEM结构域的三维(3D)模型呈现与白色念珠菌的关键毒力因子Csa2同源的螺旋篮结构。之后,成功克隆了六个候选效应子中的四个(MbCFEM1、6、8和9),酵母信号序列陷阱(YSST)测定证实了它们的分泌活性。病原体攻击试验表明,本氏烟草中四种候选MbCFEM效应子的瞬时表达促进了镰刀菌的感染,分别。在N.benthamiana异质表达系统中,MbCFEM1,MbCFEM6和MbCFEM9似乎抑制了BAX/INF1触发的PCD,而MbCFEM8只能击败BAX触发的PCD。此外,亚细胞定位分析表明,四种候选MbCFEM效应子在细胞膜上积累,核,叶绿体,和胞质体。这些结果表明,MbCFEM1,MbCFEM6,MbCFEM8和MbCFEM9是布鲁氏菌的效应子,并为进一步解剖杨树-M的分子机制提供了有价值的靶标。布鲁内呼吸互动。
Proteins containing Common in Fungal Extracellular Membrane (CFEM) domains uniquely exist in fungi and play significant roles in their whole life history. In this study, a total of 11 MbCFEM proteins were identified from Marssonina brunnea f. sp. multigermtubi (MULT), a hemibiotrophic pathogenic fungus on poplars that causes severe leaf diseases. Phylogenic analysis showed that the 11 proteins (MbCFEM1-11) were divided into three clades based on the trans-membrane domain and the CFEM domain. Sequence alignment and WebLogo analysis of CFEM domains verified the amino acids conservatism therein. All of them possess eight cysteines except MbCFEM4 and MbCFEM11, which lack two cysteines each. Six MbCFEM proteins with a signal peptide and without trans-membrane domain were considered as candidate effectors for further functional analysis. Three-dimensional (3D) models of their CFEM domains presented a helical-basket structure homologous to the crucial virulence factor Csa2 of Candida albicans. Afterward, four (MbCFEM1, 6, 8, and 9) out of six candidate effectors were successfully cloned and a yeast signal sequence trap (YSST) assay confirmed their secretion activity. Pathogen challenge assays demonstrated that the transient expression of four candidate MbCFEM effectors in Nicotiana benthamiana promoted Fusarium proliferatum infection, respectively. In an N. benthamiana heterogeneous expression system, MbCFEM1, MbCFEM6, and MbCFEM9 appeared to suppress both BAX/INF1-triggered PCD, whereas MbCFEM8 could only defeat BAX-triggered PCD. Additionally, subcellular localization analysis indicated that the four candidate MbCFEM effectors accumulate in the cell membrane, nucleus, chloroplast, and cytosolic bodies. These results demonstrate that MbCFEM1, MbCFEM6, MbCFEM8, and MbCFEM9 are effectors of M. brunnea and provide valuable targets for further dissection of the molecular mechanisms underlying the poplar-M. brunnea interaction.