Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

Abstract

DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.<br>DNA Microarray foi desenvolvida para monitorar a expressão de muitos genes de Xylella fastidiosa, permitindo a comparação de duas situações distintas em um único experimento. Os experimentos foram feitos utilizando células de X. fastidiosa cultivada em dois meios de cultura: BCYE e XDM2. Pares de oligonucleotídeos iniciadores foram sintetizados, depositados em lâminas de vidro e o arranjo foi hibridizado contra cDNAs marcados fluorescentemente. Os sinais emitidos foram quantificados, normalizados e os dados foram estatisticamente analisados para verificar os genes diferencialmente expressos. De acordo com nossos dados, 104 genes foram diferencialmente expressos para o meio de cultura XDM2 e 30 genes para o BCYE. No presente estudo, nós demonstramos que a técnica de DNA microarrays eficientemente diferencia genes expressos sob diferentes condições de cultivo

From Nucleotides to Satellite Imagery: Approaches to Identify and Manage the Invasive Pathogen Xylella fastidiosa and Its Insect Vectors in Europe

“…Gene expression profiling of Xf, together with genomic comparisons and mutagenesis (see Section 2.5), clarified the genetic basis and mechanisms of colonization and pathogenicity, although several processes remain elusive largely due to uncharacterized or nonexclusive genetic elements [17,56,106]. Hybridization-based microarray, quantitative PCR (qPCR) and RNAseq studies revealed that differences in genomic sequences between strains are reflected and amplified in gene expression, with a conserved core gene pool linked to survival in plant xylem and a flexible set including genes that favors adaptation in different hosts and putative pathogenicity factors: Xf plastically adapts its survival strategy to the environment [111,[148][149][150][151][152]. They also showed that plasmids and prophages are transcriptionally active and modulated independently from the rest of the genome [111,149].…”

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

“…The location and identity of each gene on the slide was defined in a text file, created with the aid of the Clone-Tracker2 program (Biodiscovery) by Travensolo et al (2008) and Travensolo et al (2009). The signal was quantified through ImaGene software (v.4.1, BioDisco-very), in which two images from the Cy3 and Cy5 fluorescent dyes were overlapped and the spots classified according to morphology and intensity.…”

“…We focused on the transcriptional activity of some genes related to biochemical processes involved in heat shock and antioxidant responses, toxins and detoxification processes as well as other possible mechanisms involved in bacterial survival. For this purpose, we used microarray analysis to identify global changes in gene expression patterns using a DNA microarray previously standardized by the Laboratory of Biochemistry of Microorganisms and Plants, by Travensolo et al (2008) and Travensolo et al (2009), including all genes identified in the X. fastidiosa genome.…”

Estudos bioanalíticos envolvendo a Xylella fastidiosa

“…As 11 proteínas identificadas foram distribuídas em diferentes categorias como: componente de membrana,estruturas de superfície, metabolismo de energia, proteínas hipotéticas e conservada, relacionadas ao fago, transporte e toxinas.Gene relacionado com as proteínas hipotéticas não apresentou homologia com sequências depositadas no banco de dados. Gene relacionado com transporte referese ao transporte de ânions, cátions, carboidratos, peptídeos, proteínas e de substâncias que são relacionadas com as vias de secreção(TRAVENSOLO et al, 2009b).A proteína TolC, codificada pelo gene tolC, é essencial na secreção do tipo-I de uma variedade de enzimas degradativas e também da virulência, algumas das quais são antibióticos e outras estão envolvidas na patogenicidade de animais e também em plantas. Em uvas atacadas pela X. fastidiosa, o gene tolC apresentou ser uma vantagem seletiva, e provavelmente ser um meio de sobrevivência essencial da bactéria no xilema de plantas de uvas(REDDY et al, 2007; CHATTERJEE et al, Algumas proteínas podem estar localizadas na membrana como é o caso das proteínas hipotéticas, e também estar envolvidas na adesão da bactéria à planta, assim proteínas que não apresentam homologia com o banco de dados são classificadas como proteínas hipotéticas.…”