Heterologous expression of an alternative oxidase from Moniliophthora perniciosa in Saccharomyces cerevisiae: Antioxidant function and in vivo platform for the study of new drugs against witches' broom disease.

The fungus Moniliophthora perniciosa is the causal agent of witches' broom disease (WBD), one of the most devastating diseases of cacao, the chocolate tree. Many strategies to control WBD have been tested so far, including the use of agrochemicals such as the strobilurins. Strobilurins are fungicides of the QoI family, and they are used in the control of a wide array of fungal diseases in many different crops, including cereals, field crops, fruits, tree nuts, and vegetables. These drugs act by specifically inhibiting fungal respiration at the Qo site of complex III, which is a component of the main mitochondrial respiratory chain. However, M. perniciosa is resistant to this family of chemicals. It has been postulated that this resistant phenotype is, at least in part, a result of the strong ability of this fungus to counteract the oxidative stress generated by the impairment of the main mitochondrial respiratory chain, through the activation of an alternative oxidase (Mp-AOX). To test this hypothesis, we expressed functional mitochondria-localized Mp-AOX in the model yeast Saccharomyces cerevisiae. We demonstrated that heterologous expression of Mp-AOX strongly inhibits hydrogen peroxide production by mitochondria. It also diminishes the total cell amount of oxidized glutathione (GSSG), resulting in a fifty-fold higher GSH/GSSG ratio in cells expressing Mp-AOX than in wild type cells. In addition, Mp-AOX activity decreases yeast growth rate and leads to low biomass production. Therefore, we propose the use of this heterologous expression system to direct the development of new inhibitors of fungal AOX by comparing the differences in optical density of Mp-AOX-expressing cells in the presence and absence of potential AOX inhibitors. Together, our results confirm the antioxidant role of Mp-AOX and provide an in vivo platform to be used in the screening of new fungicides based on Mp-AOX inhibition.

Gene expression profiles underlying alternative caste phenotypes in a highly eusocial bee, Melipona quadrifasciata.

To evaluate caste-biased gene expression in Melipona quadrifasciata, a stingless bee, we generated 1278 ESTs using Representational Difference Analysis. Most annotated sequences were similar to honey bee genes of unknown function. Only few queen-biased sequences had their putative function assigned by sequence comparison, contrasting with the worker-biased ESTs. The expression of six annotated genes connected to caste specificity was validated by real time PCR. Interestingly, queens that were developmentally induced by treatment with a juvenile hormone analogue displayed an expression profile clearly different from natural queens for this set of genes. In summary, this study represents an important first step in applying a comparative genomic approach to queen/worker polyphenism in the bee.

Biochemical changes during the development of witches' broom: the most important disease of cocoa in Brazil caused by Crinipellis perniciosa.

Witches' broom disease (WBD) is caused by the hemibiotrophic basidiomycete fungus Crinipellis perniciosa, which is one of the most important diseases of cocoa in the western hemisphere. In this study, the contents of soluble sugars, amino acids, alkaloids, ethylene, phenolics, tannins, flavonoids, pigments, malondialdehyde (MDA), glycerol, and fatty acids were analysed in cocoa (Theobroma cacao) shoots during the infection and development of WBD. Alterations were observed in the content of soluble sugars (sucrose, glucose, and fructose), asparagine and alkaloids (caffeine and theobromine), ethylene, and tannins. Ethylene and tannins increased prior to symptom development and declined with the death of the infected tissues. Furthermore, MDA and glycerol concentrations were higher in infected tissue than in the controls, while fatty acid composition changed in the infected tissues. Chlorophylls a and b were lower throughout the development of the disease while carotenoids and xanthophylls dropped in the infected tissue by the time of symptom development. These results show co-ordinated biochemical alterations in the infected tissues, indicating major stress responses with the production of ethylene. Ethylene levels are hypothesized to play a key role in broom development. Some of the other biochemical alterations are directly associated with ethylene synthesis and may be important for the modification of its effect on the infected tissues.