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Xylella fastidiosa gene expression analysis by DNA microarrays.

ABSTRACT: Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM(2) and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants.

SUBMITTER: Travensolo RF

PROVIDER: S-EPMC3036931 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Xylella fastidiosa gene expression analysis by DNA microarrays.

Travensolo Regiane F RF Carareto-Alves Lucia M LM Costa Maria V C G MV Lopes Tiago J S TJ Carrilho Emanuel E Lemos Eliana G M EG

Genetics and molecular biology 20090401 2

Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcriptio ...[more]

PMID: 21637690

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Project description:BACKGROUND: Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines. RESULTS: Cloning of genomic DNA and cDNA revealed that the CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS) in the upstream region and three candidate polyadenylation (PolyA) sites in the downstream region of CYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression. CONCLUSIONS: This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression during growth, development and response to Xf infection.

Project description:Bacterial cold shock-domain proteins are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single-stranded nucleic acid binding activity, little is known about the specific function(s) of Csp1. To further investigate the role(s) of Csp1, we compared phenotypic differences and transcriptome profiles between the wild type and a csp1 deletion mutant (Δcsp1). Csp1 contributes to attachment and long-term survival and influences gene expression. We observed reduced cell-to-cell attachment and reduced attachment to surfaces with the Δcsp1 strain compared to those with the wild type. Transmission electron microscopy imaging revealed that Δcsp1 was deficient in pili formation compared to the wild type and complemented strains. The Δcsp1 strain also showed reduced survival after long-term growth in vitro. Long-read nanopore transcriptome sequencing (RNA-Seq) analysis revealed changes in expression of several genes important for attachment and biofilm formation in Δcsp1 compared to that in the wild type. One gene of interest, pilA1, which encodes a type IV pili subunit protein, was upregulated in Δcsp1. Deleting pilA1 in X. fastidiosa strain Stag's Leap increased surface attachment in vitro and reduced virulence in grapevines. X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show that the impact of Csp1 on virulence may be due to changes in expression of attachment genes. IMPORTANCE Xylella fastidiosa is a major threat to the worldwide agriculture industry. Despite its global importance, many aspects of X. fastidiosa biology and pathogenicity are poorly understood. There are currently few effective solutions to suppress X. fastidiosa disease development or eliminate bacteria from infected plants. Recently, disease epidemics due to X. fastidiosa have greatly expanded, increasing the need for better disease prevention and control strategies. Our studies show a novel connection between cold shock protein Csp1 and pili abundance and attachment, which have not been reported for X. fastidiosa. Understanding how pathogenesis-related gene expression is regulated can aid in developing novel pathogen and disease control strategies. We also streamlined a bioinformatics protocol to process and analyze long-read nanopore bacterial RNA-Seq data, which will benefit the research community, particularly those working with non-model bacterial species.

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Xylella fastidiosa gene expression analysis by DNA microarrays.

Publications

Xylella fastidiosa gene expression analysis by DNA microarrays.

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