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Genome-wide transcriptomic analysis reveals correlation between higher WRKY61 expression and reduced symptom severity in Turnip crinkle virus infected Arabidopsis thaliana.

Gao R, Liu P, Yong Y, Wong SM - Sci Rep (2016)

Bottom Line: Fifteen genes (including upregulated, unchanged and downregulated) were selected for RNA-seq data validation using quantitative real-time PCR, which showed consistencies between these two sets of data.GO enrichment analysis showed that numerous terms such as stress, immunity, defence and chemical stimulus were affected in TCV-infected plants.One putative plant defence related gene named WRKY61 was selected for further investigation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore.

ABSTRACT
Turnip crinkle virus (TCV) is a carmovirus that infects many Arabidopsis ecotypes. Most studies mainly focused on discovery of resistance genes against TCV infection, and there is no Next Generation Sequencing based comparative genome wide transcriptome analysis reported. In this study, RNA-seq based transcriptome analysis revealed that 238 (155 up-regulated and 83 down-regulated) significant differentially expressed genes with at least 15-fold change were determined. Fifteen genes (including upregulated, unchanged and downregulated) were selected for RNA-seq data validation using quantitative real-time PCR, which showed consistencies between these two sets of data. GO enrichment analysis showed that numerous terms such as stress, immunity, defence and chemical stimulus were affected in TCV-infected plants. One putative plant defence related gene named WRKY61 was selected for further investigation. It showed that WRKY61 overexpression plants displayed reduced symptoms and less virus accumulation, as compared to wild type (WT) and WRKY61 deficient lines, suggesting that higher WRKY61 expression level reduced TCV viral accumulation. In conclusion, our transcriptome analysis showed that global gene expression was detected in TCV-infected Arabidopsis thaliana. WRKY61 gene was shown to be negatively correlated with TCV infection and viral symptoms, which may be connected to plant immunity pathways.

No MeSH data available.


Related in: MedlinePlus

Relative gene expression level of WRKY61 in TCV-infected Arabidopsis and nuclear localization of WRKY61-GFP fusion protein in Nicotiana benthamiana leaves.(a) The qRT-PCR was used to validate WRKY61 gene expression. (b) Expression of WRKY61 gene transcript after TCV infection as determined by qRT-PCR. The gene transcript level of WRKY61 was shown at 2, 4, 6, 8, 10, 12, 14 and 16 days post inoculation (dpi). Relative gene transcript levels (CBP20 and Tubulin as internal controls) were analyzed using the 2−∆∆CT method. The values of WRKY61 in TCV-infected plants were calculated by subtracting the values from mock controls which were all set to 1 for standardization. Means of three independent biological repeats were shown with standard deviations. (c) DAPI-stained nuclei (blue-color foci) were superimposed onto the differential interference contrast (DIC) image to form a merged image. N. benthamiana leaves were infiltrated with free GFP and the fluorescent signal was present in the entire cell including nucleus; WRKY61-GFP fusion proteins were only detected in the nucleus. Free GFP represents agro-infiltration with vector lacking of inserted gene. Bar = 5 μm.
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f4: Relative gene expression level of WRKY61 in TCV-infected Arabidopsis and nuclear localization of WRKY61-GFP fusion protein in Nicotiana benthamiana leaves.(a) The qRT-PCR was used to validate WRKY61 gene expression. (b) Expression of WRKY61 gene transcript after TCV infection as determined by qRT-PCR. The gene transcript level of WRKY61 was shown at 2, 4, 6, 8, 10, 12, 14 and 16 days post inoculation (dpi). Relative gene transcript levels (CBP20 and Tubulin as internal controls) were analyzed using the 2−∆∆CT method. The values of WRKY61 in TCV-infected plants were calculated by subtracting the values from mock controls which were all set to 1 for standardization. Means of three independent biological repeats were shown with standard deviations. (c) DAPI-stained nuclei (blue-color foci) were superimposed onto the differential interference contrast (DIC) image to form a merged image. N. benthamiana leaves were infiltrated with free GFP and the fluorescent signal was present in the entire cell including nucleus; WRKY61-GFP fusion proteins were only detected in the nucleus. Free GFP represents agro-infiltration with vector lacking of inserted gene. Bar = 5 μm.

Mentions: Among 238 most significantly expressed genes (>15 FC) after TCV infection, a transcription factor WRKY61 (AT1G18860) was selected for further investigation. The reason for choosing WRKY61 is that it is a transcription factor that displayed significant upregulation and WRKY gene families are reported to be involved in plant immunity48. Using qRT-PCR, the expression level of WRKY61 was validated to be consistent with the transcriptome RNA-seq analysis data (FC: 414.42). (Fig. 4a, Supplementary Table S2). In order to further understand the WRKY61 gene expression level among TCV infection process, a time course of 16 dpi experiments were selected for investigation. The results showed that the gene transcripts of WRKY61 were increased after TCV infection and reached its highest expression level at 8 dpi and declined after that (Fig. 4b). It suggests that the gene expression of WRKY61 responded positively after TCV infection.


Genome-wide transcriptomic analysis reveals correlation between higher WRKY61 expression and reduced symptom severity in Turnip crinkle virus infected Arabidopsis thaliana.

Gao R, Liu P, Yong Y, Wong SM - Sci Rep (2016)

Relative gene expression level of WRKY61 in TCV-infected Arabidopsis and nuclear localization of WRKY61-GFP fusion protein in Nicotiana benthamiana leaves.(a) The qRT-PCR was used to validate WRKY61 gene expression. (b) Expression of WRKY61 gene transcript after TCV infection as determined by qRT-PCR. The gene transcript level of WRKY61 was shown at 2, 4, 6, 8, 10, 12, 14 and 16 days post inoculation (dpi). Relative gene transcript levels (CBP20 and Tubulin as internal controls) were analyzed using the 2−∆∆CT method. The values of WRKY61 in TCV-infected plants were calculated by subtracting the values from mock controls which were all set to 1 for standardization. Means of three independent biological repeats were shown with standard deviations. (c) DAPI-stained nuclei (blue-color foci) were superimposed onto the differential interference contrast (DIC) image to form a merged image. N. benthamiana leaves were infiltrated with free GFP and the fluorescent signal was present in the entire cell including nucleus; WRKY61-GFP fusion proteins were only detected in the nucleus. Free GFP represents agro-infiltration with vector lacking of inserted gene. Bar = 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4834565&req=5

f4: Relative gene expression level of WRKY61 in TCV-infected Arabidopsis and nuclear localization of WRKY61-GFP fusion protein in Nicotiana benthamiana leaves.(a) The qRT-PCR was used to validate WRKY61 gene expression. (b) Expression of WRKY61 gene transcript after TCV infection as determined by qRT-PCR. The gene transcript level of WRKY61 was shown at 2, 4, 6, 8, 10, 12, 14 and 16 days post inoculation (dpi). Relative gene transcript levels (CBP20 and Tubulin as internal controls) were analyzed using the 2−∆∆CT method. The values of WRKY61 in TCV-infected plants were calculated by subtracting the values from mock controls which were all set to 1 for standardization. Means of three independent biological repeats were shown with standard deviations. (c) DAPI-stained nuclei (blue-color foci) were superimposed onto the differential interference contrast (DIC) image to form a merged image. N. benthamiana leaves were infiltrated with free GFP and the fluorescent signal was present in the entire cell including nucleus; WRKY61-GFP fusion proteins were only detected in the nucleus. Free GFP represents agro-infiltration with vector lacking of inserted gene. Bar = 5 μm.
Mentions: Among 238 most significantly expressed genes (>15 FC) after TCV infection, a transcription factor WRKY61 (AT1G18860) was selected for further investigation. The reason for choosing WRKY61 is that it is a transcription factor that displayed significant upregulation and WRKY gene families are reported to be involved in plant immunity48. Using qRT-PCR, the expression level of WRKY61 was validated to be consistent with the transcriptome RNA-seq analysis data (FC: 414.42). (Fig. 4a, Supplementary Table S2). In order to further understand the WRKY61 gene expression level among TCV infection process, a time course of 16 dpi experiments were selected for investigation. The results showed that the gene transcripts of WRKY61 were increased after TCV infection and reached its highest expression level at 8 dpi and declined after that (Fig. 4b). It suggests that the gene expression of WRKY61 responded positively after TCV infection.

Bottom Line: Fifteen genes (including upregulated, unchanged and downregulated) were selected for RNA-seq data validation using quantitative real-time PCR, which showed consistencies between these two sets of data.GO enrichment analysis showed that numerous terms such as stress, immunity, defence and chemical stimulus were affected in TCV-infected plants.One putative plant defence related gene named WRKY61 was selected for further investigation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore.

ABSTRACT
Turnip crinkle virus (TCV) is a carmovirus that infects many Arabidopsis ecotypes. Most studies mainly focused on discovery of resistance genes against TCV infection, and there is no Next Generation Sequencing based comparative genome wide transcriptome analysis reported. In this study, RNA-seq based transcriptome analysis revealed that 238 (155 up-regulated and 83 down-regulated) significant differentially expressed genes with at least 15-fold change were determined. Fifteen genes (including upregulated, unchanged and downregulated) were selected for RNA-seq data validation using quantitative real-time PCR, which showed consistencies between these two sets of data. GO enrichment analysis showed that numerous terms such as stress, immunity, defence and chemical stimulus were affected in TCV-infected plants. One putative plant defence related gene named WRKY61 was selected for further investigation. It showed that WRKY61 overexpression plants displayed reduced symptoms and less virus accumulation, as compared to wild type (WT) and WRKY61 deficient lines, suggesting that higher WRKY61 expression level reduced TCV viral accumulation. In conclusion, our transcriptome analysis showed that global gene expression was detected in TCV-infected Arabidopsis thaliana. WRKY61 gene was shown to be negatively correlated with TCV infection and viral symptoms, which may be connected to plant immunity pathways.

No MeSH data available.


Related in: MedlinePlus