DOI: 10.1094/MPMI-19-1240
The Application of Laser Microdissection to In Planta Gene Expression
Profiling of the Maize Anthracnose Stalk Rot Fungus Colletotrichum
graminicola. Weihua Tang (1), Sean Coughlan (2), Edmund Crane (1), Mary
Beatty (1), and Jon Duvick (1). (1) Pioneer Hi-Bred International, A DuPont
Company, Johnston IA 50131-1004 U.S.A.; (2) Agilent Technologies, Wilmington, DE
19808 U.S.A. MPMI 19:1240-1250. Submitted 30 April 2006. Accepted 26 June 2006.
Copyright 2006 The American Phytopathological Society.
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. Additional keywords:
oligonucleotide microarray, phytase.
Supplemental Fig. 1 is a representative picture of a mature breakout area in
the infected maize stalk at 2 days after inoculation. The picture was taken
under GFP filter.
Supplemental Fig. 2 shows an evaluation of the fidelity of 2-round RNA
amplification versus 1-round amplification.
Supplemental Fig. 3 shows TIFF images giving a global view of slides 9 and 10.
Supplemental Fig. 4 shows the categories of putative proteins encoded by in
planta-upregulated C. graminicola genes.
Supplemental Fig. 5 depicts the protein subcellular localization prediction of
up-regulated C. graminicola genes.
Supplemental Table 1 shows an assessment of RNA yields for different fixation
methods.
