College of Life Science, Qingdao Agricultural University, Qingdao 266109, China; College of Horticulture, Qingdao Agricultural University, 700 Changcheng Road, Qingdao 266109, China.
Plant Physiol Biochem. 2013 Jan;62:23-32. doi: 10.1016/j.plaphy.2012.10.016. Epub 2012 Nov 7.
Pathogenesis-related protein-4 (PR-4) family is a group of proteins with a Barwin domain in C-terminus and generally thought to be involved in plant defense responses. However, their detailed roles are poorly understood in defense of apple plant against pathogenic infection. In the present study, a new PR-4 gene (designated as MdPR-4) was identified from Malus domestica, and its roles in defense responses of apple were investigated. The open reading frame of MdPR-4 gene is of 447 bp encoding a protein of 148 amino acids with a Barwin domain in C-terminus and a signal peptide of 26 amino acids in N-terminus. Sequence and structural analysis indicated that MdPR-4 protein belongs to class II of PR-4 family. The high-level expression of MdPR-4 was observed in flowers and leaves as revealed by quantitative real time PCR. The temporal expression analysis demonstrated that MdPR-4 expression could be up-regulated by Botryosphaeria dothidea infection and salicylic acid (SA) or methyl jasmonate (MeJA) treatment, but suppressed by diethyldithiocarbamic acid (DIECA). In vitro assays, recombinant MdPR-4 protein exhibited ribonuclease activity specific for single strand RNA and significant inhibition to hyphal growth of three apple pathogenic fungi B. dothidea, Valsa ceratosperma and Glomerella cingulata. Moreover, the inhibition was reduced by the presence of 5'-ADP. Taken all together, the results indicate that MdPR-4 protein is involved in the defense responses of apple against pathogenic attack by directly inhibiting hyphal growth, and the inhibition is correlated with its ribonuclease activity, where as MdPR-4 expression is regulated by both SA and JA signaling pathway.
病程相关蛋白-4（PR-4）家族是一类在 C 末端具有 Barwin 结构域的蛋白，通常被认为参与植物的防御反应。然而，它们在苹果植物抵御病原感染的防御中的确切作用尚不清楚。在本研究中，从苹果中鉴定了一个新的 PR-4 基因（命名为 MdPR-4），并研究了其在苹果防御反应中的作用。MdPR-4 基因的开放阅读框长为 447bp，编码一个 148 个氨基酸的蛋白质，在 C 末端具有 Barwin 结构域和 N 末端 26 个氨基酸的信号肽。序列和结构分析表明，MdPR-4 蛋白属于 PR-4 家族的 II 类。定量实时 PCR 显示，MdPR-4 在花和叶中的表达水平较高。时间表达分析表明，MdPR-4 的表达可被 Botryosphaeria dothidea 感染以及水杨酸（SA）或茉莉酸甲酯（MeJA）处理上调，但被二乙基二硫代氨基甲酸盐（DIECA）抑制。体外试验表明，重组 MdPR-4 蛋白对单链 RNA 具有特异性的核糖核酸酶活性，对三种苹果病原真菌 B. dothidea、Valsa ceratosperma 和 Glomerella cingulata 的菌丝生长有显著抑制作用。此外，5'-ADP 的存在降低了抑制作用。综上所述，结果表明 MdPR-4 蛋白通过直接抑制菌丝生长参与了苹果对病原攻击的防御反应，而抑制作用与核糖核酸酶活性相关，而 MdPR-4 的表达受 SA 和 JA 信号通路的调节。
