Identification and functional analysis of Botrytis cinerea genes induced during infection of tomato

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 defence 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 Phytophthora 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. This chapter has been published with minor modifications as: Benito, E.P., T.W. Prins and J.A.L. van Kan (1996) Application of differential display RTPCR to the analysis of gene expression in a plant-fungus interaction. Plant Mol Biol 32:947957. The nucleotide sequence data reported are in the EMBL and GENBANK Nucleotide Sequence Databases under the accession numbers Z70214 (ddB-2), Z70215 (ddB-47), Z70217 (ddB-5) and Z70216 (ddT-19).