Phytopathology. 2004 Nov;94(11):1235-43. doi: 10.1094/PHYTO.2004.94.11.1235.
ABSTRACT In western North America, Douglas-fir (Pseudotsuga menziesii) is the most economically important conifer species susceptible to laminated root rot caused by Phellinus weirii. While attempting to internally sequence an endochitinase found to be up-regulated in P. weirii-infected Douglas-fir roots, we obtained overlapping peptide fragments showing 28% similarity with a PR-5 thaumatin-like protein (TLP) designated PmTLP (Pm for Pseudotsuga menziesi). A rabbit polyclonal antibody was reared against a synthetic peptide composed of a 29-amino-acid-long, conserved, internal sequence of PmTLP and purified by immunoaffinity. Western immunoblot analysis of infected roots of 24-year-old coastalfir showed significantly higher amounts of PmTLP (P < 0.01) closest to the point of P. weirii inoculation and infection than in uninfected regions of the same root. The antibody was also used to screen for PmTLP in roots of 25-year-old interior Douglas-firs naturally infected with a related pathogen, Armillaria ostoyae, and results showed significantly higher levels of PmTLP in bark tissues adjacent to infection (P < 0.05) than in uninfected tissue. Using polymerase chain reaction (PCR)-based cloning, the cDNA of PmTLP was shown to have a 702-bp open reading frame with a signal peptide cleavage site at 155 bp corresponding to a 29-amino-acid-long residue prior to the start of the N-terminal. Based on the deduced amino acid sequence, the molecular mass of the putative PmTLP was calculated to be 21.0 kDa with an isoelectric point of 3.71. Alignment analysis of PmTLP cDNA with a representative genomic DNA PCR sequence showed presence of one intron of variable size, within the coding region. The induction of PmTLP at the site of root infection and its presence in needle tissue suggests a general role for this protein in adaptation to stress and may be part of an integrated defense response initiated by the host to impede further pathogen spread.
摘要 在北美西部，花旗松（Pseudotsuga menziesii）是经济上最重要的针叶树物种，易受由 Phellinus weirii 引起的层状根腐病的影响。在试图对在感染 P. weirii 的花旗松根中上调的一种内壳聚糖酶进行内部测序时，我们获得了与 PR-5 几丁质酶类似蛋白（TLP）具有 28%相似性的重叠肽片段，该蛋白被指定为 PmTLP（Pm 代表 Pseudotsuga menziesii）。针对 PmTLP 的一个 29 个氨基酸长的保守内部序列合成的肽，制备了兔多克隆抗体，并通过免疫亲和法进行了纯化。对 24 年生海岸花旗松感染根的 Western 免疫印迹分析表明，与同一根未感染区域相比，在 P. weirii 接种和感染的最接近点处，PmTLP（P < 0.01）的含量明显更高。该抗体还用于筛选 25 年生内陆花旗松中与相关病原体 Armillaria ostoyae 自然感染的 PmTLP，结果表明，在感染（P < 0.05）附近的树皮组织中 PmTLP 的水平明显更高。使用基于聚合酶链反应（PCR）的克隆，PmTLP 的 cDNA 显示出一个 702 个碱基对的开放阅读框，在起始 N-末端前有一个 155 个碱基对的信号肽切割位点。根据推导的氨基酸序列，推测 PmTLP 的分子质量为 21.0 kDa，等电点为 3.71。与代表基因组 DNA PCR 序列的 PmTLP cDNA 的比对分析表明，在编码区存在一个大小可变的内含子。PmTLP 在根感染部位的诱导及其在针组织中的存在表明该蛋白在适应应激方面具有一般作用，可能是宿主发起的综合防御反应的一部分，以阻止病原体进一步传播。
