Large-scale analysis of differential gene expression in coffee genotypes resistant and susceptible to leaf miner-toward the identification of candidate genes for marker assisted-selection.

BACKGROUND: A successful development of herbivorous insects into plant tissues depends on coordination of metabolic processes. Plants have evolved complex mechanisms to recognize such attacks, and to trigger a defense response. To understand the transcriptional basis of this response, we compare gene expression profiles of two coffee genotypes, susceptible and resistant to leaf miner (Leucoptera coffella). A total of 22000 EST sequences from the Coffee Genome Database were selected for a microarray analysis. Fluorescence probes were synthesized using mRNA from the infested and non-infested coffee plants. Array hybridization, scanning and data normalization were performed using Nimble Scan® e ArrayStar® platforms. Genes with foldchange values +/-2 were considered differentially expressed. A validation of 18 differentially expressed genes was performed in infected plants using qRT-PCR approach. RESULTS: The microarray analysis indicated that resistant plants differ in gene expression profile. We identified relevant transcriptional changes in defense strategies before insect attack. Expression changes (>2.00-fold) were found in resistant plants for 2137 genes (1266 up-regulated and 873 down-regulated). Up-regulated genes include those responsible for defense mechanisms, hypersensitive response and genes involved with cellular function and maintenance. Also, our analyses indicated that differential expression profiles between resistant and susceptible genotypes are observed in the absence of leaf-miner, indicating that defense is already build up in resistant plants, as a priming mechanism. Validation of selected genes pointed to four selected genes as suitable candidates for markers in assisted-selection of novel cultivars. CONCLUSIONS: Our results show evidences that coffee defense responses against leaf-miner attack are balanced with other cellular functions. Also analyses suggest a major metabolic reconfiguration that highlights the complexity of this response.

The promoter of a gene encoding an isoflavone reductase-like protein in coffee (Coffea arabica) drives a stress-responsive expression in leaves.

A cDNA clone (designated CaIRL) encoding an isoflavone reductase-like protein from coffee (Coffea arabica) was retrieved during a search for genes showing organ/tissue-specific expression among the expressed sequence tags (EST) of the Brazilian coffee EST database. The CaIRL cDNA contains a single open reading frame of 946 nucleotides (nt) encoding 314 amino acids (predicted molecular weight of 34 kDa). Several features identified the predicted CaIRL protein as a new member of the PIP family of NADPH-dependent reductases. Expression studies demonstrated that CaIRL is expressed exclusively in coffee leaves and its transcript level is markedly increased in response to fungal infection and mechanical injury. Analysis of transgenic tobacco plants harboring a CaIRL 5'-flanking region (862 nt) fused to uidA reporter gene (GUS) confirmed the responsiveness of the putative promoter to abiotic stress in wounded leaves. In turn, a 5' deletion to -404 completely abolished promoter activation by abiotic stimulus in transgenic plants. The lack of GUS expression in non-wounded leaf tissues in transgenic tobacco was in contrast to the basal level of CaIRL expression observed in non-stressed healthy coffee leaves.