School of Biosciences, University of Nottingham, Loughborough LE12 5RD, UK.
Mol Plant Pathol. 2003 Nov 1;4(6):469-77. doi: 10.1046/j.1364-3703.2003.00192.x.
SUMMARY The rust fungi are obligate biotrophic pathogens that depend on living host tissue for their growth. In compatible interactions they go through a number of developmental stages to form intercellular hyphae and haustoria within host cells, through which they obtain their nutrients. Here we have exploited the cDNA-AFLP technique to isolate fragments of wheat and rust genes that are expressed at specific defined time-points during the infection process. A number of these sequences were used as probes in Northern hybridizations and in RT-PCR to confirm their expression patterns, and were also characterized by PCR analysis and Southern hybridizations to determine whether they are of fungal or wheat origin. A cDNA library was constructed from pooled RNAs extracted from days 5 and 7 after inoculation, and this library was screened to isolate full-length cDNAs of selected sequences. Sequence analysis of these cDNA fragments and clones revealed similarities amongst the fungal genes to a chitinase, a sorbitol utilization protein, an arabinitol dehydrogenase and a proteasome regulatory unit, whilst in wheat, we identified sequences with homology to a katanin and a cell enlargement protein.
摘要 锈菌是专性活体营养病原体，它们的生长依赖于活体宿主组织。在亲和互作中，锈菌经历了多个发育阶段，在宿主细胞内形成细胞间菌丝和吸器，从中获取营养。在这里，我们利用 cDNA-AFLP 技术分离了在侵染过程中特定时间点表达的小麦和锈菌基因的片段。这些序列中的一些被用作 Northern 杂交和 RT-PCR 的探针，以确认它们的表达模式，并用 PCR 分析和 Southern 杂交来确定它们是真菌来源还是小麦来源。从接种后第 5 天和第 7 天提取的混合 RNA 构建了 cDNA 文库，并筛选该文库以分离所选序列的全长 cDNA。对这些 cDNA 片段和克隆的序列分析表明，真菌基因与几丁质酶、山梨糖醇利用蛋白、阿拉伯糖醇脱氢酶和蛋白酶体调节单元具有相似性，而在小麦中，我们鉴定出与katanin 和细胞扩大蛋白同源的序列。
