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| Author(s): |
Jorge Mauricio Costa Mondego
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2005-11-22 |
| Examining board members: |
Marcelo Menossi;
Paulo Mazzafera;
Gonçalo Amarante Guimarães Pereira;
Luiz Gonzaga Esteves Vieira;
Carlos Augusto Colombo
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| Advisor: | Marcelo Menossi |
| Abstract | |
Coffee is one of the most important crops in the world. Brazil is one of the biggest coffee producer and consumer countries. Therefore, coffee plantations have great relevance in our country. One of the main factors that affect coffee plantations is the attack of the coffee leaf miner (Leucoptera coffeella). This is due to the susceptibility of Coffea arabica, the main cultivated species. The Agronomic Institute of Campinas (IAC) develops a Coffea arabica breeding program aiming the resistance to the infestation of coffee leaf miner, using crosses with C. racemosa, a resistant species. In this work, we have isolated differentially expressed genes during L. coffeella attack to plants of a hybrid progenie between C. arabica and C. racemosa. We have produced cDNA arrays containing ESTs from subtracted cDNA libraries enriched in genes preferentially expressed in infested resistant plants. Arrays were probed with samples from susceptible and resistant leaves, in different treatments (control noninfested, after oviposition and after caterpillar eclosion). After statistical analysis and hierarchical clustering, 21 cDNA clones induced in at least one treatment were selected as differentially expressed during coffee leaf miner infestation. The differential expression of five genes (PR-8, CAX9, SPC25, psaH, BEL) was confirmed by RNA blot containing samples from a second infestation experiment, demonstrating the efficiency of DNA arrays in the identification of differentially expressed genes. The expression profile of these five genes was verified in different organs of coffee plants and during coffee fruit development. Our results suggest that the resistance mechanism against coffee leaf miner is derived from a higher basal expression of defense/stress genes in resistant plants, and that resistant plants have a defense signaling mechanism triggered by L. coffeella oviposition. Among the selected cDNAs, we identified SSH101B04, which deduced protein is similar to Kunitz STI (Soybean Trypsin Inhibitor) protease inhibitors. The gene was named CoMir due to its high similarity to miraculin-like proteins. CoMir was induced after oviposition in resistant plants, but it was not induced after larval eclosion in susceptible and resistant plants. RNA-blot experiments showed that CoMir was expressed in leaves, green flower buds, white flower buds and early green fruits. In situ hybridization showed that CoMir is expressed in the metaxylem vessels of leaves, petals and stigma and in the stomium, endothecium and vascular bundles of anthers. Subcellular localization assays demonstrated that CoMir was localized in the apoplasm and citoplasm of onion (Allium cepa) epidermal cells. Our results suggest that CoMir is a protein that regulates proteolysis during coffee development that is mobilized to defense after L. coffeella oviposition (AU) | |