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Agradecimentos: The authors are grateful for the assistance of Dr Lauro Tatsuo Kubota and Dr Jailson Cardoso Dias in the chromatographic detection and analysis of carbohydrates of cacao apoplastic fluid, and Marcelo Carazzolle, Osvaldo Reis Junior, and Gustavo Gilson Lacerda Costa for the bioinformatics support and management of databases consulted in the conduct of this work. This work was financially supported by CNPq (472710/2008-7) and FAPESP (2006/56942-0, 2009/17507-5, and 2009/50119-9). The authors declare no conflicts of interest Abstract: Witches’ broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant–fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado

Orientadores: Gonçalo Amarante Guimarães Pereira, Gleidson Silva Teixeira Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Avanços biotecnológicos introduziram recentemente mudanças em muitos setores da indústria a fim de promover o uso de recursos renováveis para produzir moléculas de relevância econômica, tais como o etanol. O uso de biomassa vegetal para a produção de combustíveis renováveis tem atraído muita atenção, mas a viabilidade de tal processo depende do desenvolvimento de organismos geneticamente modificados capazes de consumir eficientemente açúcares de 5 carbonos abundantes nesse material e tolerar estresses ambientais decorrentes processo. Para alcançar estes propósitos, a engenharia das vias metabólicas e adaptação das linhagens modificadas às condições industriais irá requerer a regulação precisa da transcrição de genes de interesse através do uso de novas e eficientes combinações de promotores e terminadores. O objetivo deste trabalho foi identificar promotores adequados para a alta expressão de genes durante a fermentação de glicose e xilose e caracterizar terminadores para uma melhor eficiência traducional na linhagem industrial PE-2 de Saccharomyces cerevisiae. No estudo de promotores, os genes que exibiram os dez maiores valores de expressão e a menor variação entre os experimentos estão relacionados com a glicólise, tolerância ao estresse e formação de parede celular. Curiosamente, os resultados da análise de qPCR mostraram que os níveis de transcrição na maioria destes genes selecionados foram diferencialmente regulados durante a fermentação em glicose e xilose, sugerindo bons candidatos para a expressão de genes exclusiva em uma única fonte de carbono ou em ambas (PGK1p, TEF1p, TDH1p, SCW4p e CDC19p), além de possibilitar um melhor controle de vias gênicas heterólogas. Os resultados para terminadores mostraram que três dos terminadores proporcionam uma maior atividade do que o terminador CYC1t (SPG5t, IDP1t e HIS5t) e sugerem que a eficiência dos terminadores é linhagem-dependente Abstract: Biotechnological approaches have recently introduced changes in many industry sectors to promote the use of renewable resources to produce molecules of economical relevance such as ethanol. The use of plant biomass to produce renewable fuels has attracted much attention, but the viability of such process depends on the development of genetically modified organisms able to consume 5-carbon sugars rich in plant material and to tolerate environmental and process-related stresses. To reach these goals, the engineering of metabolic pathways and adaptation of modified strains to industrial conditions will require precise transcriptional regulation of genes of interest through the use of novel and efficient combinations of promoters and terminators. Our goals were to identify promoters suitable for high gene expression during fermentation of glucose and xylose and to characterize terminators for better translational efficiency on the Saccharomyces cerevisiae industrial strain PE-2. In the study of promoters, the genes exhibiting the top ten highest expression values and the lowest variation among experiments are related to glycolysis, stress tolerance and cell wall formation. Interestingly, the qPCR analysis results showed that the transcription levels in the majority of these selected genes were differentially regulated during fermentation of glucose and xylose, suggesting good candidates for exclusive gene expression in one single carbon source or in both (PGK1p, TEF1p, TDH1p, SCW4p e CDC19p), in addition to enabling better control of heterologous gene pathways. Results showed that three terminators have higher terminator activity than CYC1t (SPG5t, IDP1t e HIS5t) and suggest that terminator efficiency is strain-dependent Mestrado Genética de Microorganismos Mestra em Genética e Biologia Molecular FAPESP 2014/05724-0 CNPQ 131261/2014-2