BackgroundThe production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b.ResultsTcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae.ConclusionTo our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively.
The genus Theobroma comprises several trees species native to the Amazon. Theobroma cacao L. plays a key economic role mainly in the chocolate industry. Both cultivated and wild forms are described within the genus. Variations in genome size and chromosome number have been used for prediction purposes including the frequency of interspecific hybridization or inference about evolutionary relationships. In this study, the nuclear DNA content, karyotype and genetic diversity using functional microsatellites (EST-SSR) of seven Theobroma species were characterized. The nuclear content of DNA for all analyzed Theobroma species was 1C = ~ 0.46 pg. These species presented 2n = 20 with small chromosomes and only one pair of terminal heterochromatic bands positively stained (CMA+/DAPI− bands). The small size of Theobroma ssp. genomes was equivalent to other Byttnerioideae species, suggesting that the basal lineage of Malvaceae have smaller genomes and that there was an expansion of 2C values in the more specialized family clades. A set of 20 EST-SSR primers were characterized for related species of Theobroma, in which 12 loci were polymorphic. The polymorphism information content (PIC) ranged from 0.23 to 0.65, indicating a high level of information per locus. Combined results of flow cytometry, cytogenetic data and EST-SSRs markers will contribute to better describe the species and infer about the evolutionary relationships among Theobroma species. In addition, the importance of a core collection for conservation purposes is highlighted.
Summary
Moniliophithora roreri, causal agent of cacao frosty pod rot, is considered one of the most devastating pathogens of this crop. In this study, we report the development of and validation of 28 microsatellite loci from an enriched library. From these, ten loci were demonstrated to be polymorphic in an Ecuadorian population composed of 27 isolates. The mean number of alleles per locus was 3.2, and the observed mean and expected heterozygosity were 0.03 and 0.43, respectively. The polymorphic microsatellites described herein may be used to study the genetic diversity as well as to comprehend other aspects of M. roreri population biology.
Diagnóstico microbiológico e histopatológico de mortalidade em avestruzes (Struthio camelus)[Microbiological and histological diagnosis in mortality of ostrich (Struthio camelus)]
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