Gene expression analysis of plant host-pathogen interactions by SuperSAGE

Abstract

The type III restriction endonuclease EcoP15I was used in isolating fragments of 26 bp from defined positions of cDNAs. We call this substantially improved variant to the conventional serial analysis of gene expression (SAGE) procedure "SuperSAGE." By applying SuperSAGE to Magnaporthe grisea (blast)-infected rice leaves, gene expression profiles of both the rice host and blast fungus were simultaneously monitored by making use of the fully sequenced genomes of both organisms, revealing that the hydrophobin gene is the most actively transcribed M. grisea gene in blast-infected rice leaves. Moreover, SuperSAGE was applied to study gene expression changes before the so-called hypersensitive response in INF1 elicitor-treated Nicotiana benthamiana, a "nonmodel" organism for which no DNA database is available. Again, SuperSAGE allowed rapid identification of genes up- or down-regulated by the elicitor. Surprisingly, many of the down-regulated genes coded for proteins involved in photosynthesis. SuperSAGE will be especially useful for transcriptome profiling of two or more interacting organisms like hosts and pathogens, and of organisms, for which no DNA database is available.

Fig. 1.

Flowchart of SuperSAGE. For details see Materials and Methods.

Fig. 2.

(A) RT-PCR of M. grisea genes encoding hydrophobin, nucleoside diphosphate kinase, and 60S ribosomal protein, from cDNA template prepared from M. grisea-infected rice cv. Norin 1. (B) RT-PCR of M. grisea genes from cDNA templates prepared from rice cv. Kakehashi and cv. Himenomochi either mock-inoculated (–) or inoculated with M. grisea race 007 (+).

Fig. 3.

(A) 3′ RACE-PCR of genes identified by SuperSAGE to be differentially expressed in flooded and INF1-treated N. benthamiana leaves. Oligonucleotides corresponding to the 26-bp tag sequences were used as PCR primers. (B) Expression kinetics of four N. benthamiana genes encoding chlorophyll a/b binding protein, photosystem II protein, phosphoglycerate kinase, and ATP synthase after flooding and INF1 treatments as revealed by 3′ RACE-PCR. Numbers above indicate minutes after treatment.

Fig. 4.

Application of SuperSAGE for functional genomics of eukaryotic organisms.