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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

Pathogenicity test of transcriptionally upregulated genes using the lesion length test. a Lesion length in Xoo-susceptible rice leaves infected by single-gene knockout Xoo mutants. b Infected rice leaves in multiple experiments
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Fig6: Pathogenicity test of transcriptionally upregulated genes using the lesion length test. a Lesion length in Xoo-susceptible rice leaves infected by single-gene knockout Xoo mutants. b Infected rice leaves in multiple experiments

Mentions: We studied whether the transcriptionally upregulated genes are essential for causing bacterial blight (Additional file 15: Table S8). Pathogenicity tests were performed by measuring lesion length in the leaf of Xoo-susceptible rice Milyang23 [45] after infection with 6 Xoo strains including wild-type and single-gene knockout mutants of oprO− (Xoo1104), hrpX− (Xoo1380), fruA− (Xoo2812), fliC− (Xoo2581) and gumC− (Xoo3178). hrpX (Xoo1380) was essential for Xoo pathogenicity (Fig. 6). In different Xoo strains of KACC10859, the mutants of hrp genes like hrpB, hrpE, hrpD6, hrpD5, as well as hrpG also showed no pathogenicity [46]. In the sugar transport signaling pathway, gumC (Xoo3178) which was upregulated at 5 min, was also strictly required for pathogenicity. In the KACC10859 strain, various gum genes were tested to assess their relation to pathogenicity with a different Xoo-susceptible rice cultivar, IR24 [47]. In addition to gumC, other genes like gumB, gumD, gumE, gumH and gumK were also essential for pathogenicity. The pathways involved in hrp and gum expression could be good target pathways to ify the pathogenicity of Xoo. In our pathogenicity tests, not all the upregulated genes were essential to cause bacterial blight. For instance, upregulated genes such as oprO (Xoo1104), fliC (Xoo2581) and fruA (Xoo2812) were not essential for pathogenicity.Fig. 6


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)

Pathogenicity test of transcriptionally upregulated genes using the lesion length test. a Lesion length in Xoo-susceptible rice leaves infected by single-gene knockout Xoo mutants. b Infected rice leaves in multiple experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Pathogenicity test of transcriptionally upregulated genes using the lesion length test. a Lesion length in Xoo-susceptible rice leaves infected by single-gene knockout Xoo mutants. b Infected rice leaves in multiple experiments
Mentions: We studied whether the transcriptionally upregulated genes are essential for causing bacterial blight (Additional file 15: Table S8). Pathogenicity tests were performed by measuring lesion length in the leaf of Xoo-susceptible rice Milyang23 [45] after infection with 6 Xoo strains including wild-type and single-gene knockout mutants of oprO− (Xoo1104), hrpX− (Xoo1380), fruA− (Xoo2812), fliC− (Xoo2581) and gumC− (Xoo3178). hrpX (Xoo1380) was essential for Xoo pathogenicity (Fig. 6). In different Xoo strains of KACC10859, the mutants of hrp genes like hrpB, hrpE, hrpD6, hrpD5, as well as hrpG also showed no pathogenicity [46]. In the sugar transport signaling pathway, gumC (Xoo3178) which was upregulated at 5 min, was also strictly required for pathogenicity. In the KACC10859 strain, various gum genes were tested to assess their relation to pathogenicity with a different Xoo-susceptible rice cultivar, IR24 [47]. In addition to gumC, other genes like gumB, gumD, gumE, gumH and gumK were also essential for pathogenicity. The pathways involved in hrp and gum expression could be good target pathways to ify the pathogenicity of Xoo. In our pathogenicity tests, not all the upregulated genes were essential to cause bacterial blight. For instance, upregulated genes such as oprO (Xoo1104), fliC (Xoo2581) and fruA (Xoo2812) were not essential for pathogenicity.Fig. 6

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