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Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae.

Kim S, Cho YJ, Song ES, Lee SH, Kim JG, Kang LW - BMC Genomics (2016)

Bottom Line: Genes related to inorganic ion uptake were upregulated within 5 min.We introduced a non-linear regression fit to generate continuous time-resolved gene expression levels and tested the essentiality of the transcriptionally upregulated genes by a pathogenicity assay of lesion length using single-gene knock-out Xoo strains.The in vitro system combined with RNA-Seq generated a genome-wide time-resolved pathogenic gene expression profile within 1 h of initial rice-Xoo interactions, demonstrating the expression order and interaction dependency of pathogenic genes.

View Article: PubMed Central - PubMed

Affiliation: Genomics Division, National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA), Jeonju, 54874, Korea.

ABSTRACT

Background: Plant-pathogen interactions at early stages of infection are important to the fate of interaction. Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is a devastating disease in rice. Although in vivo and in vitro systems have been developed to study rice-Xoo interactions, both systems have limitations. The resistance mechanisms in rice can be better studied by the in vivo approach, whereas the in vitro systems are suitable for pathogenicity studies on Xoo. The current in vitro system uses minimal medium to activate the pathogenic signal (expression of pathogenicity-related genes) of Xoo, but lacks rice-derived factors needed for Xoo activation. This fact emphasizes the need of developing a new in vitro system that allow for an easy control of both pathogenic activation and for the experiment itself.

Results: We employed an in vitro system that can activate pathogenicity-related genes in Xoo using rice leaf extract (RLX) and combined the in vitro assay with RNA-Seq to analyze the time-resolved genome-wide gene expression of Xoo. RNA-Seq was performed with samples from seven different time points within 1 h post-RLX treatment and the expression of up- or downregulated genes in RNA-Seq was validated by qRT-PCR. Global analysis of gene expression and regulation revealed the most dramatic changes in functional categories of genes related to inorganic ion transport and metabolism, and cell motility. Expression of many pathogenicity-related genes was induced within 15 min upon contact with RLX. hrpG and hrpX expression reached the maximum level within 10 and 15 min, respectively. Chemotaxis and flagella biosynthesis-related genes and cyclic-di-GMP controlling genes were downregulated for 10 min and were then upregulated. Genes related to inorganic ion uptake were upregulated within 5 min. We introduced a non-linear regression fit to generate continuous time-resolved gene expression levels and tested the essentiality of the transcriptionally upregulated genes by a pathogenicity assay of lesion length using single-gene knock-out Xoo strains.

Conclusions: The in vitro system combined with RNA-Seq generated a genome-wide time-resolved pathogenic gene expression profile within 1 h of initial rice-Xoo interactions, demonstrating the expression order and interaction dependency of pathogenic genes. This combined system can be used as a novel tool to study the initial interactions between rice and Xoo during bacterial blight progression.

No MeSH data available.


Related in: MedlinePlus

a Schematic representation of the in vitro assay system and sampling for RNA-Seq and qRT-PCR. b Comparison of transcription levels by RNA-Seq and qRT-PCR. The relative transcription levels for 28 randomly selected genes from RNA-Seq data were verified by qRT-PCR. The log2-scaled values from qRT-PCR were plotted against the log2-scaled values from RNA-Seq data. The correlation coefficient (R2) of the datasets was between 0.65 and 0.89
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Fig1: a Schematic representation of the in vitro assay system and sampling for RNA-Seq and qRT-PCR. b Comparison of transcription levels by RNA-Seq and qRT-PCR. The relative transcription levels for 28 randomly selected genes from RNA-Seq data were verified by qRT-PCR. The log2-scaled values from qRT-PCR were plotted against the log2-scaled values from RNA-Seq data. The correlation coefficient (R2) of the datasets was between 0.65 and 0.89

Mentions: We combined an in vitro assay system with RNA-Seq to analyze genome-wide pathogenic gene expression of Xoo in a time-dependent manner (Fig. 1a). The in vitro assay system consists of fresh RLX preparation by grinding the leaves of a Xoo-susceptible rice cultivar (Milyang23) in liquid nitrogen and addition of RLX at the mid-exponential phase of Xoo cell culture in nutrient broth. Samples were harvested for RNA-Seq from the RLX-treated (Pathogenicity-activated; P-activated) and untreated (control) Xoo cells at specific time points. RNA-Seq data were verified by qRT-PCR in separate biological replicates.Fig. 1


Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae.

Kim S, Cho YJ, Song ES, Lee SH, Kim JG, Kang LW - BMC Genomics (2016)

a Schematic representation of the in vitro assay system and sampling for RNA-Seq and qRT-PCR. b Comparison of transcription levels by RNA-Seq and qRT-PCR. The relative transcription levels for 28 randomly selected genes from RNA-Seq data were verified by qRT-PCR. The log2-scaled values from qRT-PCR were plotted against the log2-scaled values from RNA-Seq data. The correlation coefficient (R2) of the datasets was between 0.65 and 0.89
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862043&req=5

Fig1: a Schematic representation of the in vitro assay system and sampling for RNA-Seq and qRT-PCR. b Comparison of transcription levels by RNA-Seq and qRT-PCR. The relative transcription levels for 28 randomly selected genes from RNA-Seq data were verified by qRT-PCR. The log2-scaled values from qRT-PCR were plotted against the log2-scaled values from RNA-Seq data. The correlation coefficient (R2) of the datasets was between 0.65 and 0.89
Mentions: We combined an in vitro assay system with RNA-Seq to analyze genome-wide pathogenic gene expression of Xoo in a time-dependent manner (Fig. 1a). The in vitro assay system consists of fresh RLX preparation by grinding the leaves of a Xoo-susceptible rice cultivar (Milyang23) in liquid nitrogen and addition of RLX at the mid-exponential phase of Xoo cell culture in nutrient broth. Samples were harvested for RNA-Seq from the RLX-treated (Pathogenicity-activated; P-activated) and untreated (control) Xoo cells at specific time points. RNA-Seq data were verified by qRT-PCR in separate biological replicates.Fig. 1

Bottom Line: Genes related to inorganic ion uptake were upregulated within 5 min.We introduced a non-linear regression fit to generate continuous time-resolved gene expression levels and tested the essentiality of the transcriptionally upregulated genes by a pathogenicity assay of lesion length using single-gene knock-out Xoo strains.The in vitro system combined with RNA-Seq generated a genome-wide time-resolved pathogenic gene expression profile within 1 h of initial rice-Xoo interactions, demonstrating the expression order and interaction dependency of pathogenic genes.

View Article: PubMed Central - PubMed

Affiliation: Genomics Division, National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA), Jeonju, 54874, Korea.

ABSTRACT

Background: Plant-pathogen interactions at early stages of infection are important to the fate of interaction. Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is a devastating disease in rice. Although in vivo and in vitro systems have been developed to study rice-Xoo interactions, both systems have limitations. The resistance mechanisms in rice can be better studied by the in vivo approach, whereas the in vitro systems are suitable for pathogenicity studies on Xoo. The current in vitro system uses minimal medium to activate the pathogenic signal (expression of pathogenicity-related genes) of Xoo, but lacks rice-derived factors needed for Xoo activation. This fact emphasizes the need of developing a new in vitro system that allow for an easy control of both pathogenic activation and for the experiment itself.

Results: We employed an in vitro system that can activate pathogenicity-related genes in Xoo using rice leaf extract (RLX) and combined the in vitro assay with RNA-Seq to analyze the time-resolved genome-wide gene expression of Xoo. RNA-Seq was performed with samples from seven different time points within 1 h post-RLX treatment and the expression of up- or downregulated genes in RNA-Seq was validated by qRT-PCR. Global analysis of gene expression and regulation revealed the most dramatic changes in functional categories of genes related to inorganic ion transport and metabolism, and cell motility. Expression of many pathogenicity-related genes was induced within 15 min upon contact with RLX. hrpG and hrpX expression reached the maximum level within 10 and 15 min, respectively. Chemotaxis and flagella biosynthesis-related genes and cyclic-di-GMP controlling genes were downregulated for 10 min and were then upregulated. Genes related to inorganic ion uptake were upregulated within 5 min. We introduced a non-linear regression fit to generate continuous time-resolved gene expression levels and tested the essentiality of the transcriptionally upregulated genes by a pathogenicity assay of lesion length using single-gene knock-out Xoo strains.

Conclusions: The in vitro system combined with RNA-Seq generated a genome-wide time-resolved pathogenic gene expression profile within 1 h of initial rice-Xoo interactions, demonstrating the expression order and interaction dependency of pathogenic genes. This combined system can be used as a novel tool to study the initial interactions between rice and Xoo during bacterial blight progression.

No MeSH data available.


Related in: MedlinePlus