Selection for resistance to cacao Moniliasis assisted by molecular markers : S04P06

The basidiomycete Moniliophthora rorer, the etiological agent of Moniliasis, reaches cacao plantations (Theobroma cacao) in Central and South Americas, but its occurrence is still not registered in Brazil . The disease causes losses of up to 90 % in the production of cocoa, has difficult to control and is close to the border with Brazil, threatening the Brazilian cacao industry. As part of a larger project of preventive breeding, this study aims to identify plants that have genes involved in the control of resistance to moniliasis , using microsatellite DNA markers associated with them. The material used in this study was collected from parents (clones) and progenies from CEPEC´s breeding program. A total of 74 genotypes were used in this study, being 34 parents used in CEPEC´s breeding program and 40 trees of the cross EET-183 x ( P-7xUF-273). DNA samples from these geotypes was obtained, from leaf tissue, by the method described by Doyle & Doyle (1993). The extracted DNA was amplified by PCR , and after amplification of the SSR loci, the reaction was applied to polyacrylamide gel stained with silver nitrate as described by CRESTE et al. al. (2001) with some modifications. The clones were scored based on the size of alleles at SSR loci previously associated with resistance to moniliasis . Based on results in the loci mtcCir 26 mtcCir 46, mtcCir mtcCir 236 and 240 , it was observed that 26 of the parents used are not suitable to be used in crosses with the source of resistance UF-273, because they had the allele associated with resistance, even though they are not resistant. On the other hand , the parents Ipiranga- 1, PS-13.19, Sca-6, CCN-10, CP-188, CP-49, Salobrinho-3 and CP -111 exhibited different alleles of the source of resistance, being , therefore, suitable to be involved in further crosses with the UF -273. The individuals of the segregating population EET -183 x (P- 7x UF- 273) , were genotyped for the purpose of obtaining individuals who showed the resistance alleles to moniliasis . Out of the 40 individuals used in the study, 16 had alleles associated to resistance. (Texte intégral

Expression of genes involved in cell wall structural components in different genotypes of Theobroma cacao

Resistant in this main way to control witches´ broom resistance, therefore the understanding of pathogen infection and its disease resistance mechanism is very important in order to obtain durable resistance. Through the technique of in situ hybridization, the current study aimed to determine the expression of genes involved in possible mechanisms of resistance of cacao and in which stages of the infection they are acting. Apical meristems of susceptible and resistant genotypes of Theobroma cacao to witches' broom disease were artificially inoculated by placing a drop with a 5x105 basidiospore/mL. Meristems were collected at intervals of 3, 6, 12, 24, 48 and 72 hours, 5 and 15 days after the inoculation day, under free RNAse. The samples were fixed and sent for analysis of gene expression by in Situ Hybridization. HRGP genes (Hidroxyproline-rich glycoprotein) and RGC2 related to cell wall metabolism or plant defense mechanism, were chosen from cDNA libraries available at UESC/ CEPLAC/CEPEC. The analysis showed the localization in the in cells of the vascular system and in parenchymatous cells of the apical meristem in both resistant as well as susceptible genotype. However, no significant variation in the accumulation of HRGP and RGC2 genes were observed between genotypes. Thus, we suggest further investigation with earlier timein order to ascertain whether there is variation in the accumulation of such genes between the genotypes under study. (Texte intégral

MLO and SBP genes from Theobroma cacao are differencially expressed between resistent and suscetible cacao plants infected with Moniliophthora perniciosa

Among sequences previously identified as potentially envolved in the resistance versus susceptibility of Theobroma cacao to the fungus Moniliophthora perniciosa, the MLO (Mildew resistance lócus O) and the SBP (selenium binding protein) genes were found. The MLO gene is characterized as a plant defense and programmed cell death modulator, and the SBP gene was successfully used to increase the rice resistance to Magnaporthe grisea by plant transformation, among other applications. The objective of this work was to evaluate the expression of the MLO and SBP genes from T. cacao in cacao plants infected by M. perniciosa. Varieties of cacao resistant (TSH1188) and suceptible (Catongo) to M. perniciosa were inoculated with a suspension of fungus basidiospores (2.105. ml-1). After inoculation, the plants were kept for 24h at 25±2ºC under 100% of humidity. Apical meristems were harvested in triplicates at 24, 48 and 72 hours after inoculation (hai) and 30, 60 and 90 days after inoculation (dai). Non-inoculated plants (controls) were kept and harvested in the same conditions. Total RNA was extracted using the RNAqueous Kit® (Ambion). First strand cDNA was obtained using the Revertaid Fisrt Strand cDNA Synthesis Kit (Thermo Scientific, Fermentas). Quantitative PCR (qPCR) of MLO and SBP was obtained using the standard settings of the ABI PRISM 7500 and System of Sequence Detection (SDS) software, v.1.6.3 (Applied Biosystems). The expression levels of MLO and SBP was analyzed on triplicates with the comparative Ct method (2-??Ct) using malate dehydrogenase and actin as endogenous reference genes, and non-inoculated plants (control) were used as a calibrator. At the early stages of infection and in the final stage of the disease, the MLO gene was more expressed in Catongo than in TSH1188. In TSH1188, the highest expression of MLO was observed at 30 dai. The SBP gene was highly expressed in TSH1188 at the late stages after infection while in Catongo, the expression was high at the early stages and then constant until the end of the disease. The involvement in the cacao-M. perniciosa interaction of both MLO and SBP genes is discussed. Financial Support: CNPq, BNB, FINEP/Renorbio, Cirad. (Résumé d'auteur

Expression analysis of Mildew Resistance Locus O of cacao in resistant and susceptible plants infected by Moniliophthora perniciosa : S01P03

A Mildew Resistance Locus O (MLO) cDNA was identified from a library of Theobroma cacao L. meristems (Catongo varieties) infected by Moniliophthora perniciosa, the fungus responsible for the witches' broom disease. In other plants, the MLO gene is characterized as a defense and programmed cell death (PCD) modulator, and for this reason may be a good candidate for functional studies aiming the increase of plant resistance. An in silico analysis of the cacao MLO (TcMLO) using the BLAST, Pfam, InterProScan and ORF-Finder programs, as well as a search on CocoaGenDB databank were performed. TcMLO belongs to a multigene family of proteins containing 19 sequences present in the cacao genome: 12, 5 and 2 of them showed one, two and three MLO domains, respectively. The complete TcMLO sequence (including UTRs and ORF) is 5712 bp in length with 13 exons and 12 introns, and is located on the chromosome 5. The TcMLO ORF is 1629 bp in length and encodes a protein with 542 amino acids containing 2 MLO domains. The expression of TcMLO was analyzed by quantitative PCR (qPCR) in resistant (TSH1188) and susceptible (Catongo) cacao varieties infected or not by Moniliophthora perniciosa. Plantlets of cacao were inoculated by the droplet method with a basidiospore suspension of M. perniciosa. After inoculation, the plantlets were kept for 24h at 25±2ºC and 100% humidity. Apical meristems were harvested in triplicates at 24, 48 and 72 hours after inoculation (hai), and 8, 15, 30, 45, 60 and 90 days after inoculation (dai). Non-inoculated plants (controls) were kept and harvested in the same conditions. The qPCR of Tc MLO was obtained using the standard settings of the ABI PRISM 7500 and using the System of Sequence Detection software. The TcMLO expression was analyzed with the comparative Ct method (2-??Ct) using malate dehydrogenase and actin as endogenous reference genes, and non-inoculated plants (control) as calibrator. The results showed that TcMLO was more expressed in Catongo than in TSH1188 at the early and final stages of disease. In TSH1188, the highest expression of MLO was observed at 15 dai. The expression of TcMLO at the final stage of the disease in the susceptible infected plants may be related to the PCD events occurring in this variety as a signal for the finalization of the fungus life cycle. Funding Agency: FAPESB, CNPq, CAPES, EMBRAPA, FINEP/Renorbio and CIRAD. (Texte intégral

Classical and "omics" approaches to control witches' broom disease of cacao [W120]

In Theobroma cacao the main biotic stresses are cause by fungi, i.e Moniliophthora perniciosa (Mp), causal agent of witches' broom disease of cacao (WBD). Breeding of Mp-resistant varieties is confronted with two major difficulties at present. First, cacao resistant sources have been identified, but most of them are Scavina 6 descendants. Second, resistance from Scavina sources has shown to be unstable. The OMICS with the classical phytopatological and breeding approaches have allowed identifying genotypes with distinction in relation to WBD resistance, thus, carrying different resistance genes. New microsatellites and SNPs markers, and new QTLs (under natural and artificial inoculations) linked to WBD disease resistance have been identified. In parallel, histopatological studies of the cacao-Mp interaction revealed multiples modes of penetration of the fungus into cacao plants as well different mechanisms of resistance. The adaptability of Mp has also been considered, and advances in the understanding the breakdown of witches´ broom resistance, in Bahia, Brazil, have been achieved. Partial results of these projects and the overall strategy will be presented. (Résumé d'auteur

Selection for cacao resistance to Moniliasis assisted by molecular markers

The basidiomycete Moniliophthora roreri, the causal agent of moniliasis, occurs in many cocoa producer countries in South and Central Americas, but not yet in Brazil. The disease can cause losses of up to 90% in cocoa production, have difficult control and, given its proximity to the border with Brazil, it threatens Brazilian cocoa production. As part of a preventive breeding program, this study aims to identify parental germplasms to be involved in crosses aiming the development of varieties resistant to moniliasis and then select individual trees carrying the QTLs of resistance, assisted by markers. In order to do that, genomic DNA was extracted from 34 clones of CEPEC´s cacao breeding program with good agronomic characteristics and others with known resistance moniliasis and with QTLs mapped. Genomic DNA was extracted according to Doyle & Doyle (1991). Subsequently the DNA was amplified by PCR using primers close to the resistance genes, submitted to the SSR gel and silver stained. It was observed that some parental clones (Sca-6, Salobrinho-3, CP-184, CP-188), even being susceptible, carry the alleles of resistance in the marker loci and so should be avoided as parent in the marker assisted selection program. (Texte intégral

Classical and "omics" approaches to control witches' broom (Moniliopthora perniciosa) disease of cacao [P0997]

In Theobroma cacao the main biotic stresses are cause by fungi, i.e Moniliophthora perniciosa (Mp), causal agent of witches' broom disease of cacao (WBD). Breeding of Mp-resistant varieties is confronted with two major difficulties at present. First, cacao resistant sources have been identified, but most of them are Scavina 6 descendants. Second, resistance from Scavina sources has shown to be unstable. The OMICS with the classical phytopatological and breeding approaches have allowed identifying genotypes with distinction in relation to WBD resistance, thus, carrying different resistance genes. New microsatellites and SNPs markers, and new QTLs (under natural and artificial inoculations) linked to WBD disease resistance have been identified. In parallel, histopatological studies of the cacao-Mp interaction revealed multiples modes of penetration of the fungus into cacao plants as well different mechanisms of resistance. The adaptability of Mp has also been considered, and advances in the understanding the breakdown of witches´ broom resistance, in Bahia, Brazil, have been achieved. Partial results of these projects and the overall strategies to control WBD will be presented. (Texte intégral

In silico characterization and expression analysis of a Selenium-Binding Protein gene from cacao : S01P01

Witches' broom disease, caused by the fungus Moniliophthora perniciosa, is one of the main diseases of cacao (Theobroma cacao L.) and is responsible to severe economic losses in the production areas. Recently, expressed sequence tags (ESTs) from cacao-M. perniciosa interaction were obtained and differentially defense-related genes expressed during the cacao-M. perniciosa interaction were identified. Among them, a Selenium-Binding Protein (TcSBP) was found. In other organisms, SBP genes are related to the increase of plant defenses against abiotic and biotic stresses; in rice the SBP gene was successfully used to increase the plant resistance to Magnaporthe grisea by plant transformation. Here, in silico characterization and expression analysis of TcSBP were developed. Search on the Cacao Genome Database revealed the presence of only one SBP sequence of 4774 pb in length located on the chromosome 4. The TcSBP ORF is 1431 bp in length and encodes a protein of 476 amino acids which does not contain any signal peptide. Prediction of possible post-translational events allowed the identification of several glycosylation, phosphorylation and acetylation sites. The comparison of TcSBP sequence with SBP from other organisms using the BLASTP tool revealed identity from 62% to 91% and allowed the identification of specific conserved regions. The expression analysis of TcSBP in meristems of cacao plantlets varieties Catongo (susceptible) and TSH1188 (resistant to M. perniciosa), inoculated or not with M. perniciosa, was obtained by RT-qPCR using 3 biological and 3 experimental replicates. qPCR analysis of TcSBP gene was conducted using the standard settings of the ABI PRISM 7500 and the System of Sequence Detection software. The TcSBP relative expression was analyzed with the comparative Ct method (2-??Ct) using malate dehydrogenase and actin as endogenous reference genes, and noninoculated plants (control) as calibrator. The relative expression of TcSBP was significantly increased 8 and 15 days after inoculation in the resistant variety TSH1188 compared to susceptible Catongo. These data suggest the possible role of TcSBP in cacao resistance to M. perniciosa. This study is the first step to better understand the role of TcSBP in cacao resistance as well as for the development of control strategies of the witches' broom disease (e.g. using plant transformation). Work supported by FAPESB, CAPES, EMBRAPA, CNPq, FINEP/Renorbio and CIRAD. (Texte intégral

The pathogen Moniliophthora perniciosa promotes differential proteomic modulation of cacao genotypes with contrasting resistance to witches´ broom disease

Background: Witches' broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction. Results: A total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions. Conclusions: We present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype

Bioinformatic analysis of glutathione peroxidase family from theobroma cacao and gene expression during Moniliophthora perniciosa infection.[Poster-B185]

Glutathione peroxidases (GPXs) are enzymes which are part of the antioxidant system of the cell. Mammalian GPXs are known as selenoproteins because containing the selenocysteine (Sec) amino acid. In plants, these proteins are less known. Here, were analyzed the protein structure and the gene expression of five GPXs from Theobroma cacao . The three-dimensional structure of the TcGPXs showed that the catalytic site of Tc PHGPX and TcGPX ( 2,4 and 5) contain a cysteine while the GPX8 contain a tryptophan. Interestingly, the T. cacao GPX did not show any selenocysteine in their structure. Docking analysis revealed that TcGPXs can bind to selenium. Phylogenetic analysis split plant and mammalian GPXs in two distinct branches. RT-qPCR analysis of TcGPXs during the T. cacao - Moniliophthora perniciosa interaction showed that TcGPX8 gene is overexpressed in the green broom phase of the susceptible cacao variety. In the resistant variety, the TcGPX5 was significantly more expressed in the final stages of the interaction. This study shows that TcGPXs are important targets for the understanding of the T. cacao - M. perniciosa interaction but also for the functionality of these proteins