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    Monitoring of the early molecular resistance responses of coffee (Coffea arabica L.) to the rust fungus (Hemileia vastatrix) using real-time quantitative RT-PCR

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    Abstract
    Molecular resistance responses of coffee (Coffea arabica L.) to the orange rust fungus Hemileia vastatrix were monitored by real-time quantitative RT-PCR analysis of gene expression. Significant activation of coffee genes by fungal infection could be observed around 12–16 h post inoculation (hpi) in the incompatible interaction. Microscopic observations indicated that, at this time, only a limited number of fungal germlings already differentiated a penetration hypha through the stomata. Activation of the CaWRKY1 gene, putatively encoding a WRKY transcription factor also occurred in the compatible interaction, but was delayed to 24 hpi. In contrast, activation of the CaR111 gene encoding a protein of unknown function only occurred in the incompatible interaction. The CaNDR1 gene, an homolog of the Arabidopsis non-race specific disease resistance (ndr1) gene was only poorly induced by fungal infection. Wounding and salicylic acid treatment markedly activated CaWRKY1, CaNDR1 and CaR111 gene expression. These results showed that specific transcriptional responses of coffee were detected before penetration of H. vastatrix into the leaf had occurred, and suggest a possible role of activated genes in the molecular resistance responses of coffee to the rust fungus.
    Article Outline
    1. Introduction
    2. Materials and methods
    2.1. Plant variety, fungal strains, and inoculation
    2.2. Wounding and salicylic acid (SA)-treatment
    2.3. Fungal colonization
    2.4. Genomic DNA extraction, restriction endonuclease digestion, electrophoresis and Southern blotting
    2.5. Labelling of probes and hybridization conditions
    2.6. RNA extraction and RT-PCR
    2.7. Real-time quantitative PCR assays
    2.8. Quantitative PCR data analysis
    2.9. Statistical analysis
    3. Results
    3.1. Estimation of gene copy number in the C. arabica genome
    3.2. Coffee gene expression during H. vastatrix infection
    3.3. Coffee gene expression during wounding and SA-treatment
    3.4. Fungal colonization
    4. Discussion
    Acknowledgements
    References
     

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