Structure and expression of two polygalacturonase genes of Claviceps purpurea oriented in tandem and cytological evidence for pectinolytic enzyme activity during infection of rye
Structure and expression of two polygalacturonase genes of Claviceps purpurea oriented in tandem and cytological evidence for pectinolytic enzyme activity during infection of rye
复制标题
紫麦角两个多聚半乳糖醛酸酶基因串联的结构和表达以及黑麦感染过程中果胶分解酶活性的细胞学证据
DOI：
10.1094/phyto-86-1084
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发表时间：
1996
期刊：
影响因子：
3.2
通讯作者：
P. Tudzynski
中科院分区：
文献类型：
--
作者：
K. Tenberge;V. Homann;B. Oeser;P. Tudzynski
以黑曲霉的多聚半乳糖醛酸酶基因(Pgall)为探针，从紫皮杆菌T5菌株中分离到两个可能的多聚半乳糖醛酸酶基因。这两个基因(pg1和pg2)紧密相连，头尾相连。即使在上游非编码序列中，它们也高度同源，在相同的位置各有一个内含子，并有用于加工酶的切割位点。它们可能分别编码343和344个氨基酸的成熟蛋白，并与其他丝状真菌的Endo-PGs有显著的同源性。用逆转录-聚合酶链式反应检测了黑麦在无菌培养和侵染不同阶段中PGL和pg2的表达。推测产物pg1和pg2(聚半乳糖醛酸)的潜在底物首次被证明是黑麦卵巢寄主细胞壁的组成部分。这是通过使用针对果胶非甲酯化表位的单抗(MAb)Jim 5的免疫金透射电子显微镜来实现的。该同源半乳糖醛酸酶与另一株单抗Jim 7沿着正常的感染途径定位于同一细胞壁中。在穿透菌丝与寄主卵巢表皮的交界处，Jim 5的标记密度局部增强，且在菌丝鞘上方很高。在细胞间生长的菌丝附近，标记密度大大增加，金标记不仅出现在中层片区以上，而且遍及整个寄主细胞壁。化学去甲基化和免疫金标记法表明，半乳糖醛酸总含量很高，果胶类化合物在寄主-寄生虫界面发生了转化。在感染后期，没有任何Jim标记，以前出现在细胞内菌丝界面，强调了高半乳糖醛酸与其他植物多糖的充分利用。观察到的寄主细胞壁变化为植物细胞外果胶分解酶的分泌和活性提供了证据。这两个基因在黑麦侵染过程中的表达，以及仅在真菌部位检测到的同型半乳糖醛酸的修饰和降解，都表明果胶分解酶是真菌起源的，这种酶的活性以前已经由B.I.Shaw和P.G.Mantle在感染的卵巢中发现。
Two putative polygalacturonase (PG) genes were isolated from strain T5 of Claviceps purpurea, using the pgall gene of Aspergillus niger as a probe. The two genes (pg1 and pg2) are closely linked and arranged head - to-tail. They are highly homologous even in the upstream noncoding sequences, possess one intron each in the same position, and have cleavage sites for processing enzymes. They probably code for mature proteins of 343 and 344 amino acids, respectively, and share significant homology with endo-PGs of other filamentous fungi. Expression of pgl and pg2 in axenic culture and during various stages of infection of rye was demonstrated using reverse transcription-polymerase chain reaction. The potential substrate of the putative products of pg1 and pg2 (polygalacturonic acid), for the first time, was shown to be a component of the host cell walls in rye ovaries. This was achieved by immunogold transmission electron microscopy with the monoclonal antibody (MAb) JIM 5, specific for nonmethyl-esterified epitopes of pectin. This homogalacturonan was localized along the usual infection path in healthy carpels together with its methyl-esterified galacturonan type in the same cell walls with another MAb, JIM 7. At the interface of the penetrating hyphae and the host ovary epidermis, JIM 5 label density was locally enhanced and very high above hyphal sheaths. In the vicinity of intercellularly growing hyphae, label density was highly increased, and gold label occurred not only above the middle lamella area but also throughout the entire host cell wall. Chemical demethylation and immunogold labeling indicated a high total content of galacturonan and a conversion of pectic compounds at the host-parasite interface. During late infection phases, the lack of any JIM label, which previously occurred at the interface of intracellular hyphae, emphasized the complete utilization of homogalacturonan together with other plant polysaccharides. The observed host wall alterations provide evidence for secretion and activity of extracellular pectinolytic enzymes in planta. Both the expression of the two genes during infection of rye and the modification and degradation of homogalacturonan detected only at fungal sites indicate the fungal origin of pectinolytic enzymes, the activities of which have been documented previously in infected ovaries by B. I. Shaw and P. G. Mantle.