Physiology-based differentiation of S H genes and Hemileia vastatrix races is the principal method employed for the characterization of coffee leaf rust resistance. Based on the genefor-gene theory, nine major rust resistance genes (S H 1-9) have been proposed. However, these genes have not been characterized at the molecular level. Consequently, the lack of molecular data regarding rust resistance genes or candidates is a major bottleneck in coffee breeding. To address this issue, we screened a BAC library with resistance gene analogs (RGAs), identified RGAs, characterized and explored for any S H related candidate genes. Herein, we report the identification and characterization of a gene (gene 11), which shares conserved sequences with other S H genes and displays a characteristic polymorphic allele conferring different resistance phenotypes. Furthermore, comparative analysis of the two RGAs belonging to CC-NBS-LRR revealed more intense diversifying selection in tomato and grape genomes than in coffee. For the first time, the present study has unveiled novel insights into the molecular nature of the S H genes, thereby opening new avenues for coffee rust resistance molecular breeding. The characterized candidate RGA is of particular importance for further biological function analysis in coffee.

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Countering the economic hurdle caused by coffee leaf rust disease is most appealing at this time as it has posed a major threat to coffee production around the world. Establishing differential expression profiles at different times following pathogen invasion in both innate and acquired immunities unlocks the molecular components of resistance and susceptibility. Suppression subtractive hybridization (SSH) was used to identify genes differentially over-expressed and repressed during incompatible and compatible interactions between Coffea arabica and Hemileia vastatrix. From 433 clones of expressed sequence tags (ESTs) sequenced, 352 were annotated and categorized of which the proportion of genes expressed during compatible interaction were relatively smaller. The result showed upregulation and downregulation of various genes at 12 and 24 h after pathogen inoculation in both interactions. The use of four different databases in searching for gene homology resulted in different number of similar sequences. BLASTx against EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics Institute) database being with the maximum (100%) hits for all the annotated sequences. RT-qPCR analysis of seven resistance-signaling genes showed similar expression patterns for most of the genes in both interactions, indicating these genes are involved in basal (nonspecific) defense during which immune reactions are similar. Using SSH, we identified different types of resistance related genes that could be used for further studies towards resistant cultivar development. The potential role of some of the resistance related proteins found were also discussed.

The biotrophic fungus Hemileia vastatrix causes coffee leaf rust (CLR), one of the most devastating diseases in Coffea arabica. Coffee, like other plants, has developed effective mechanisms to recognize and respond to infections caused by pathogens. Plant resistance gene analogs (RGAs) have been identified in certain plants as candidates for resistance (R) genes or membrane receptors that activate the R genes. The RGAs identified in different plants possess conserved domains that play specific roles in the fight against pathogens. Despite the importance of RGAs, in coffee plants these genes and other molecular mechanisms of disease resistance are still unknown. This study aimed to sequence and characterize candidate genes from coffee plants with the potential for involvement in resistance to H. vastatrix. Sequencing was performed based on a library of bacterial artificial chromosomes (BAC) of the coffee clone 'Híbrido de Timor' (HdT) CIFC 832/2 and screened using a functional marker. Two RGAs, HdT_ LRR_RLK1 and HdT_LRR_RLK2, containing the motif of leucine-rich repeat-like kinase (LRR-RLK) were identified. Based on the presence or absence of the HdT_LRR_RLK2 RGA in a number of differential coffee clones containing different combinations of the rust resistance gene, these RGAs did not correspond to any resistance gene already characterized (S H 1-9). These genes were also analyzed using qPCR and demonstrated a major expression peak at 24 h after inoculation in both the compatible and incompatible interactions between coffee and H. vastatrix. These results are valuable information for breeding programs aimed at developing CLR-resistant cultivars, in addition to enabling a better understanding of the interactions between coffee and H. vastatrix.