Table_1_Valsa mali Pathogenic Effector VmPxE1 Contributes to Full Virulence and Interacts With the Host Peroxidase MdAPX1 as a Potential Target.DOCX

The Valsa canker, caused by Valsa mali (V. mali), is a destructive disease of apple in Eastern Asia. Effector proteins are important for fungal pathogenicity. We studied a candidate effector VmPxE1 isolated based on the genome information of V. mali. By using the yeast invertase secretion assay system, VmPxE1 was shown to contain a signal peptide with secretory functions. VmPxE1 can suppress BCL-2-associated X protein (BAX)-induced cell death with a high efficacy of 92% in Nicotiana benthamiana. The expression of VmPxE1 was upregulated during the early infection stage and deletion of VmPxE1 led to significant reductions in virulence on both apple twigs and leaves. VmPxE1 was also shown to target an apple ascorbate peroxidase (MdAPX1) by the yeast two-hybrid screening, bimolecular fluorescence complementation and in vivo co-immunoprecipitation. Sequence phylogenetic analysis suggested that MdAPX1 was an ascorbate peroxidase belonging to a subgroup of heme-dependent peroxidases of the plant superfamily. The ectopic expression of MdAPX1 in the mutant of VmPxE1 significantly enhanced resistance to H2O2, while the presence of VmPxE1 seems to disturb MdAPX1 function. The present results provide insights into the functions of VmPxE1 as a candidate effector of V. mali in causing apple canker.

由苹果腐烂病菌（Valsa mali, V. mali）引起的苹果树腐烂病是东亚地区极具破坏性的苹果病害。效应蛋白（effector proteins）对真菌致病性具有重要作用。本研究基于苹果腐烂病菌的基因组信息，分离得到候选效应蛋白VmPxE1。通过酵母蔗糖酶分泌检测系统（yeast invertase secretion assay system）验证，VmPxE1携带具有分泌功能的信号肽（signal peptide）。在本氏烟草（Nicotiana benthamiana）中，VmPxE1可高效抑制B细胞淋巴瘤2相关X蛋白（BCL-2-associated X protein, BAX）诱导的细胞死亡，抑制效率达92%。VmPxE1的转录水平在侵染早期显著上调，敲除VmPxE1后，病菌在苹果枝条和叶片上的致病力均显著降低。通过酵母双杂交筛选（yeast two-hybrid screening）、双分子荧光互补（bimolecular fluorescence complementation）以及体内免疫共沉淀（co-immunoprecipitation）实验，证实VmPxE1可靶向苹果抗坏血酸过氧化物酶1（MdAPX1）。序列系统发育分析表明，MdAPX1属于植物超家族中血红素依赖性过氧化物酶（heme-dependent peroxidases）亚家族的抗坏血酸过氧化物酶。在VmPxE1敲除突变体中异位表达MdAPX1，可显著增强其对过氧化氢（H2O2）的抗性，而VmPxE1的存在似乎会干扰MdAPX1的功能。本研究结果为阐明VmPxE1作为苹果腐烂病菌候选效应蛋白在引发苹果树腐烂病过程中的功能提供了新的研究视角。