Application of differential display RT-PCR to the analysis of gene expression in a plant-fungus interaction

Abstract

Establishment of a plant-pathogen interaction involves differential gene expression in both organisms. In order to isolate Botrytis cinerea genes whose expression is induced during its interaction with tomato, a comparative analysis of the expression pattern of the fungus in planta with its expression pattern during in vitro culture was performed by differential display of mRNA (DDRT-PCR). Discrimination of fungal genes induced in planta from plant defense genes induced in response to the pathogen was attempted by including in this comparative analysis the expression patterns of healthy tomato leaves and of tomato leaves infected with two different pathogens, either Rhytophthora infestans or tobacco necrosis virus (TNV). Using a limited set of primer combinations, three B. cinerea cDNA fragments, ddB-2, ddB-5 and ddB-47, were isolated representing fungal genes whose expression is enhanced in planta. Northern blot analysis showed that the transcripts detected with the cDNA clones ddB-2 and ddB-5 accumulated at detectable levels only at late time points during the interaction. The cDNA clone ddB-47 detected two different sizes of transcripts displaying distinct, transient expression patterns during the interaction. Sequence analysis and database searches revealed no significant homology to any known sequence. These results show that the differential display procedure possesses enough sensitivity to be applied to the detection of fungal genes induced during a plant-pathogen interaction. Additionally, four cDNA fragments were isolated representing tomato genes induced in response to the infection caused by B. cinerea, but not by P. infestans.

Induction of New Defensin Genes in Tomato Plants via Pathogens-Biocontrol Agent Interaction

“…Our results are partially in agreement with those of Benito et al [10], who studied the differential display expression in tomato plants induced by Botrytis cinerea. They also studied the different plant genes induced by fungal infection, through different patho-genes.…”

“…The trial was conducted twice and the experiment was arranged in a completely randomized block design. After transplanting, samples from treatments (C, RB, RT, FB and FT) were taken at different times (2,4,6,8, and 24 h), to detect the induced defense genes using differential display technique and real-time PCR [10]. Three plants of each treatment were carefully harvested (Three weeks after inoculation with the pathogen), washed under running water to remove soil particles, and evaluated for the following growth parameters: shoot fresh and dry weights, and root fresh and dry weights.…”

Differential Expression of Pine and Cronartium quercuum f. sp. fusiforme Genes in Fusiform Rust Galls

“…Our yield of 0.4 differentially expressed cDNAs per primer combination compares favorably to other studies that have used differential display to analyze plantfungus interactions. In these cases, yields have ranged from 0.08 to 0.31 cDNAs per tested primer combination (9,50,55). Our protocol of replicating each display three or four times before candidate bands were isolated may have contributed to our relatively high confirmation rate.…”

Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology

“…A cDNA product homologous to chitinase was found to be induced in a plant root after infection with the fungus Azorhizobium caulinodans ORS571 (40). Benito et al identified differentially expressed transcripts from both the fungal pathogen Botrytis cinerea and its host, the tomato, that were induced during the plant-fungus interaction (6). Of course, it is important to confirm the source of the DNA.…”

“…Of course, it is important to confirm the source of the DNA. Benito et al discriminated induced B. cinerea genes from plant defense genes by comparing expression patterns from healthy tomatoes and tomatoes infected with two nonrelated pathogens (6). The initial interactions between host and pathogen have also been studied in normal symbiotic organisms, e.g., mycorrhizial fungi (69,89).…”