Tang Weihua, Coughlan Sean, Crane Edmund, Beatty Mary, Duvick Jon
Pioneer Hi-Bred International, A DuPont Company, Johnston IA 50131-1004, USA.
Mol Plant Microbe Interact. 2006 Nov;19(11):1240-50. doi: 10.1094/MPMI-19-1240.
Laser microdissection (LM) offers a potential means for deep sampling of a fungal plant-pathogen transcriptome during the infection process using whole-genome DNA microarrays. The use of a fluorescent protein-expressing fungus can greatly facilitate the identification of fungal structures for LM sampling. However, fixation methods that preserve both tissue histology and protein fluorescence, and that also yield RNA of suitable quality for microarray applications, have not been reported. We developed a microwave-accelerated acetone fixation, paraffin-embedding method that fulfills these requirements and used it to prepare mature maize stalk tissues infected with an Anemonia majano cyan fluorescent protein-expressing isolate of the anthracnose stalk rot fungus Colletotrichum graminicola. We successfully used LM to isolate individual maize cells associated with C. graminicola hyphae at an early stage of infection. The LM-derived RNA, after two-round linear amplification, was of sufficient quality and quantity for global expression profiling using a fungal microarray. Comparing replicated LM samples representing an early stage of stalk cell infection with samples from in vitro-germinated conidia, we identified 437 and 370 C. graminicola genes showing significant up- or downregulation, respectively. We confirmed the differential expression of several representative transcripts by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and documented extensive overlap of this dataset with a PCR-subtraction library enriched for C. graminicola transcripts in planta. Our results demonstrate that LM is feasible for in planta pathogen expression profiling and can reveal clues about fungal genes involved in pathogenesis. The method in this report may be advantageous for visualizing a variety of cellular features that depend on a high degree of histochemical preservation and RNA integrity prior to LM.
激光显微切割（LM）为利用全基因组DNA微阵列在感染过程中对真菌植物病原体转录组进行深度取样提供了一种潜在方法。使用表达荧光蛋白的真菌能够极大地便于识别用于LM取样的真菌结构。然而，尚未有既能保存组织组织学结构又能保持蛋白质荧光，且能产生适用于微阵列应用的高质量RNA的固定方法的报道。我们开发了一种满足这些要求的微波加速丙酮固定、石蜡包埋方法，并将其用于制备被表达海葵绿色荧光蛋白的炭疽病茎腐病菌禾生炭疽菌分离株感染的成熟玉米茎组织。我们成功地使用LM在感染早期分离出与禾生炭疽菌菌丝相关的单个玉米细胞。经过两轮线性扩增后，LM获得的RNA在质量和数量上足以用于使用真菌微阵列进行全局表达谱分析。将代表茎细胞感染早期的重复LM样本与体外萌发分生孢子的样本进行比较，我们分别鉴定出437个和370个禾生炭疽菌基因显示出显著上调或下调。我们通过定量逆转录聚合酶链反应（RT-PCR）证实了几种代表性转录本的差异表达，并记录了该数据集与一个富含植物中禾生炭疽菌转录本的PCR扣除文库有广泛重叠。我们的结果表明，LM对于植物病原体表达谱分析是可行的，并且可以揭示参与致病过程的真菌基因的线索。本报告中的方法对于在LM之前可视化依赖于高度组织化学保存和RNA完整性的各种细胞特征可能具有优势。
