Characterization of coffee (coffea sp.) genes induced during coffee leaf miner (leucoptera coffeella) infestation

Orientador: Marcelo Menossi TeixeiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O café é um dos principais produtos agrícolas mundiais. O Brasil é um dos maiores países produtores e consumidores de café. Assim sendo, a cafeicultura possui extrema importância econômica em nosso país. Um dos principais fatores que causam prejuízo à lavoura do café é o ataque do bicho mineiro (Leucoptera coffeella), pois Coffea arabica, principal espécie cultivada do café, é suscetível a essa praga. O Instituto Agronômico de Campinas (IAC) empreende um projeto de melhoramento de Coffea arabica, visando a resistência ao bicho mineiro, através de cruzamentos com C. racemosa, espécie naturalmente resistente a L. coffeella.Neste trabalho isolamos genes diferencialmente expressos durante o ataque do bicho mineiro a plantas de uma progênie híbrida derivada de cruzamentos entre Coffea arabica e C. racemosa. Foram construídos macroarranjos de DNA contendo 1536 ESTs de bibliotecas de subtração enriquecidas em genes preferencialmente expressos em plantas resistentes infestadas. Membranas foram hibridadas com sondas de cDNA obtidas a partir de RNA total de folhas suscetíveis e resistentes ao bicho mineiro, em diferentes momentos da infestação (controle não infestado, pósoviposição e pós-eclosão). Após análises estatísticas e clusterização hierárquica, 21 cDNAs induzidos em pelo menos um tratamento foram selecionados como diferencialmente expressos durante a infestação do bicho mineiro. A expressão diferencial de cinco genes (PR-8, CAX9, SPC25, psaH, BEL) foi confirmada através de RNA blot contendo RNA de um segundo experimento de infestação, demonstrando a eficiência dos macroarranjos de DNA na seleção de genes diferencialmente expressos. O padrão de expressão dos cinco genes citados foi verificado em diferentes órgãos do cafeeiro e durante o desenvolvimento do fruto do café. Nossos resultados sugerem que o mecanismo de resistência ao bicho mineiro é derivado de uma maior expressão basal de genes relacionados a defesa em plantas resistentes do que em plantas suscetíveis, e que plantas resistentes possuem um mecanismo de sinalização de defesa disparado pela oviposição de L. coffeella. Dentre os cDNAs selecionados, destacamos SSH101B04, cuja proteína deduzida é similar a inibidores de protease do tipo Kunitz STI (Soybean Trypsin Inhibitor). Devido a sua alta similaridade com proteínas do tipo miraculina, esse gene foi denominado CoMir (Coffea Miraculin). CoMir foi induzido após a oviposição em plantas resistentes, mas não foi induzido após a eclosão da lagarta do minador em plantas resistentes nem em plantas suscetíveis. Através de ensaios de RNA blot foi verificado que CoMir é expresso em folhas, botões florais verdes e brancos e em frutos verdes imaturos. Ensaios de hibridação in situ demonstraram que CoMir é expresso no metaxilema de folhas, de pétalas e do estigma, e no estômio, endotécio, tapete e feixe vascular da antera. Ensaios de localização subcelular demonstraram que a proteína CoMir localizou-se preferencialmente no apoplasma e no citoplasma de células de epiderme de cebola (Allium cepa). Nossos resultados sugerem que CoMir é uma proteína reguladora de proteólise durante o desenvolvimento do café, que é mobilizada para defesa após a oviposição de L. coffeellaAbstract: 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 ovipositionDoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecula

Homeologous regulation of Frigida-like genes provides insights on reproductive development and somatic embryogenesis in the allotetraploid Coffea arabica

Coffea arabica is an allotetraploid of high economic importance. C. arabica transcriptome is a combination of the transcripts of two parental genomes (C. eugenioides and C. canephora) that gave rise to the homeologous genes of the species. Previous studies have reported the transcriptional dynamics of C. arabica. In these reports, the ancestry of homeologous genes was identified and the overall regulation of homeologous differential expression (HDE) was explored. One of these genes is part of the FRIGIDA-like family (FRL), which includes the Arabidopsis thaliana flowering-time regulation protein, FRIGIDA (FRI). As nonfunctional FRI proteins give rise to rapid-cycling summer annual ecotypes instead of vernalization-responsive winter-annuals, allelic variation in FRI can modulate flowering time in A. thaliana. Using bioinformatics, genomic analysis, and the evaluation of gene expression of homeologs, we characterized the FRL gene family in C. arabica. Our findings indicate that C. arabica expresses 10 FRL homeologs, and that, throughout flower and fruit development, these genes are differentially transcribed. Strikingly, in addition to confirming the expression of FRL genes during zygotic embryogenesis, we detected FRL expression during direct somatic embryogenesis, a novel finding regarding the FRL gene family. The HDE profile of FRL genes suggests an intertwined homeologous gene regulation. Furthermore, we observed that FLC gene of C. arabica has an expression profile similar to that of CaFRL genes9FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/17544-

Genetic diversity in cultivated carioca common beans based on molecular marker analysis

A wide array of molecular markers has been used to investigate the genetic diversity among common bean species. However, the best combination of markers for studying such diversity among common bean cultivars has yet to be determined. Few reports have examined the genetic diversity of the carioca bean, commercially one of the most important common beans in Brazil. In this study, we examined the usefulness of two molecular marker systems (simple sequence repeats – SSRs and amplified fragment length polymorphisms – AFLPs) for assessing the genetic diversity of carioca beans. The amount of information provided by Roger’s modified genetic distance was used to analyze SSR data and Jaccards similarity coefficient was used for AFLP data. Seventy SSRs were polymorphic and 20 AFLP primer combinations produced 635 polymorphic bands. Molecular analysis showed that carioca genotypes were quite diverse. AFLPs revealed greater genetic differentiation and variation within the carioca genotypes (Gst = 98% and Fst = 0.83, respectively) than SSRs and provided better resolution for clustering the carioca genotypes. SSRs and AFLPs were both suitable for assessing the genetic diversity of Brazilian carioca genotypes since the number of markers used in each system provided a low coefficient of variation. However, fingerprint profiles were generated faster with AFLPs, making them a better choice for assessing genetic diversity in the carioca germplasm

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed2331123137CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP479800/2004-9sem informação03/09361-

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. in situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. in addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. the similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)PBIG-UNICAMPCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Consorcio Brasileiro de Pesquisa e Desenvolvimento do CafeFAEPEX/UNICAMPUniv Estadual Campinas, UNICAMP, Inst Biol, Lab Genoma Func,Dept Genet Evolucao & Bioagentes, BR-13084971 Campinas, SP, BrazilUniv Estadual Campinas, UNICAMP, Ctr Biol Mol & Engn Genet, BR-13083875 Campinas, SP, BrazilUniv Estadual Campinas, UNICAMP, Inst Quim, BR-13084862 Campinas, SP, BrazilUniv São Paulo, Inst Fis Sao Carlos, Grp Cristalog, BR-13566590 Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilLNBio, BR-13083970 Campinas, SP, BrazilUniv Estadual Campinas, UNICAMP, Inst Biol, Dept Biol Vegetal, BR-13084971 Campinas, SP, BrazilIAC, Ctr Cafe Alcides Carvalho, BR-13012970 Campinas, SP, BrazilIAC, Ctr Pesquisa & Desenvolvimento Recursos Genet Veg, BR-13001970 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilFAPESP: 03/09361-4CNPq: 479800/2004-9FAEPEX/UNICAMP: 040504Web of Scienc

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)[03/09361-4]PBIG-UNICAMPCAPES (Conselho de Aperfeicoamento de Pesquisa em Ensino Superior)CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)[479800/2004-9]Consorcio Brasileiro de Pesquisa e Desenvolvimento do CafeFAEPEX/UNICAMP[040504