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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

WRKY61 is positively correlated with virus resistance after TCV infection.(a) Comparison of TCV symptoms on Arabidopsis with different backgrounds [WT, WRKY61 deficient (homozygous HM), and overexpression(OE)] at 10 days post inoculation. TCV symptoms were not observed in all the mock control plants. Only TCV infected leaf displayed chlorosis. (b) Statistical analysis of the percentages coverage of chlorosis in each leaf of TCV infected HM, OE and WT plants. Six leaves from each plants (a total of 8 plants from each treatment) were evaluated. The student t test was used to analyze the significant differences between WT and WRKY61-deficient HM lines, as well as WT and WRKY61 OE lines (*p < 0.05, **p < 0.01). Detection of TCV-CP in newly emerged leaves of three types of plants WT, WRKY61 deficient (HM), and overexpression (OE) line at 10 dpi from both RNA (c) and protein (d) levels. The transcript level of TCV-CP gene was analyzed using qRT-PCR. Relative gene transcript levels (CBP20 and Tubulin as internal controls) were analyzed using the 2−∆CT methods. Means of three independent biological repeats were shown by standard deviations. Detection of TCV-CP protein in the WT, WRKY61 deficient (HM) and OE lines were analyzed using western blot. The protein band intensity was quantified using ImageJ software. Ribosome was used as loading control.
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f5: WRKY61 is positively correlated with virus resistance after TCV infection.(a) Comparison of TCV symptoms on Arabidopsis with different backgrounds [WT, WRKY61 deficient (homozygous HM), and overexpression(OE)] at 10 days post inoculation. TCV symptoms were not observed in all the mock control plants. Only TCV infected leaf displayed chlorosis. (b) Statistical analysis of the percentages coverage of chlorosis in each leaf of TCV infected HM, OE and WT plants. Six leaves from each plants (a total of 8 plants from each treatment) were evaluated. The student t test was used to analyze the significant differences between WT and WRKY61-deficient HM lines, as well as WT and WRKY61 OE lines (*p < 0.05, **p < 0.01). Detection of TCV-CP in newly emerged leaves of three types of plants WT, WRKY61 deficient (HM), and overexpression (OE) line at 10 dpi from both RNA (c) and protein (d) levels. The transcript level of TCV-CP gene was analyzed using qRT-PCR. Relative gene transcript levels (CBP20 and Tubulin as internal controls) were analyzed using the 2−∆CT methods. Means of three independent biological repeats were shown by standard deviations. Detection of TCV-CP protein in the WT, WRKY61 deficient (HM) and OE lines were analyzed using western blot. The protein band intensity was quantified using ImageJ software. Ribosome was used as loading control.

Mentions: To verify if WRKY61 is involved in conferring virus resistance, TCV inoculation was carried out in WT, WRKY61 deficient (homozygous: HM), and WRKY61 overexpression (OE) plants. The selected WRKY61 HM deficient line was screened and verified from TAIR seed stock T-DNA insertion mutant (Stock CS459288, individual line CS360187). The primers used for screening HM mutant were listed in Supplementary Table S5. The confirmed WRKY61-deficient HM lines were further analyzed by qRT-PCR which showed that no WRKY61 gene transcripts can be detected. For the WRKY61 overexpression lines, 2 × 35S-WRKY61-6HA construct was successfully transformed into Arabidopsis plants. The overexpression of WRKY61 gene transcript was confirmed using qRT-PCR and the fusion protein of WRKY61-6HA was also detected in 4 lines of WRKY61-6HA transgenic plants tested (Supplementary Fig. S1), indicating successful transformation of WRKY61-6HA to Arabidopsis plants. For virus resistance testing, WRKY61 HM deficient lines, WT and WRKY61 OE lines (3 lines, L2, L3 and L4 were selected for testing) were inoculated with equal amount of TCV in vitro transcripts (400 ng) onto each plant. Compared to WT and WRKY61-deficient mutants, all three WRKY61 OE lines showed similar viral tolerance upon TCV infection. Thus, data from L3 plants were shown as a representative in this study. The results showed that the plants of WRKY61 (HM) deficient mutants displayed the most severe symptoms, compared to that of WT control which appeared to be similar to the symptoms of TCV-infected WRKY61 OE plants (Fig. 5a). The viral symptom comparisons were further demonstrated by the percentages of chlorotic lesions covered in the leaves of TCV-infected plants, which showed TCV-infected WRKY61 (HM) lines contain the highest percentage of chlorosis coverage (Fig. 5b), followed by WRKY61 OE and WT plant control. In addition to the direct symptom observation, the TCV-CP gene transcripts were further assessed by qRT-PCR, which also showed the most abundance TCV-CP gene transcripts were detected in WRKY61 HM deficient mutants (Fig. 5c). Furthermore, these virus tolerance results were also confirmed with Western blot analysis, showing the relative amount of CP expression (Fig. 5d). Although the symptom of WRKY61 OE plants appeared to be similar to that of WT, the amount of CP gene transcript and CP expression is less than that of the WT.


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)

WRKY61 is positively correlated with virus resistance after TCV infection.(a) Comparison of TCV symptoms on Arabidopsis with different backgrounds [WT, WRKY61 deficient (homozygous HM), and overexpression(OE)] at 10 days post inoculation. TCV symptoms were not observed in all the mock control plants. Only TCV infected leaf displayed chlorosis. (b) Statistical analysis of the percentages coverage of chlorosis in each leaf of TCV infected HM, OE and WT plants. Six leaves from each plants (a total of 8 plants from each treatment) were evaluated. The student t test was used to analyze the significant differences between WT and WRKY61-deficient HM lines, as well as WT and WRKY61 OE lines (*p < 0.05, **p < 0.01). Detection of TCV-CP in newly emerged leaves of three types of plants WT, WRKY61 deficient (HM), and overexpression (OE) line at 10 dpi from both RNA (c) and protein (d) levels. The transcript level of TCV-CP gene was analyzed using qRT-PCR. Relative gene transcript levels (CBP20 and Tubulin as internal controls) were analyzed using the 2−∆CT methods. Means of three independent biological repeats were shown by standard deviations. Detection of TCV-CP protein in the WT, WRKY61 deficient (HM) and OE lines were analyzed using western blot. The protein band intensity was quantified using ImageJ software. Ribosome was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4834565&req=5

f5: WRKY61 is positively correlated with virus resistance after TCV infection.(a) Comparison of TCV symptoms on Arabidopsis with different backgrounds [WT, WRKY61 deficient (homozygous HM), and overexpression(OE)] at 10 days post inoculation. TCV symptoms were not observed in all the mock control plants. Only TCV infected leaf displayed chlorosis. (b) Statistical analysis of the percentages coverage of chlorosis in each leaf of TCV infected HM, OE and WT plants. Six leaves from each plants (a total of 8 plants from each treatment) were evaluated. The student t test was used to analyze the significant differences between WT and WRKY61-deficient HM lines, as well as WT and WRKY61 OE lines (*p < 0.05, **p < 0.01). Detection of TCV-CP in newly emerged leaves of three types of plants WT, WRKY61 deficient (HM), and overexpression (OE) line at 10 dpi from both RNA (c) and protein (d) levels. The transcript level of TCV-CP gene was analyzed using qRT-PCR. Relative gene transcript levels (CBP20 and Tubulin as internal controls) were analyzed using the 2−∆CT methods. Means of three independent biological repeats were shown by standard deviations. Detection of TCV-CP protein in the WT, WRKY61 deficient (HM) and OE lines were analyzed using western blot. The protein band intensity was quantified using ImageJ software. Ribosome was used as loading control.
Mentions: To verify if WRKY61 is involved in conferring virus resistance, TCV inoculation was carried out in WT, WRKY61 deficient (homozygous: HM), and WRKY61 overexpression (OE) plants. The selected WRKY61 HM deficient line was screened and verified from TAIR seed stock T-DNA insertion mutant (Stock CS459288, individual line CS360187). The primers used for screening HM mutant were listed in Supplementary Table S5. The confirmed WRKY61-deficient HM lines were further analyzed by qRT-PCR which showed that no WRKY61 gene transcripts can be detected. For the WRKY61 overexpression lines, 2 × 35S-WRKY61-6HA construct was successfully transformed into Arabidopsis plants. The overexpression of WRKY61 gene transcript was confirmed using qRT-PCR and the fusion protein of WRKY61-6HA was also detected in 4 lines of WRKY61-6HA transgenic plants tested (Supplementary Fig. S1), indicating successful transformation of WRKY61-6HA to Arabidopsis plants. For virus resistance testing, WRKY61 HM deficient lines, WT and WRKY61 OE lines (3 lines, L2, L3 and L4 were selected for testing) were inoculated with equal amount of TCV in vitro transcripts (400 ng) onto each plant. Compared to WT and WRKY61-deficient mutants, all three WRKY61 OE lines showed similar viral tolerance upon TCV infection. Thus, data from L3 plants were shown as a representative in this study. The results showed that the plants of WRKY61 (HM) deficient mutants displayed the most severe symptoms, compared to that of WT control which appeared to be similar to the symptoms of TCV-infected WRKY61 OE plants (Fig. 5a). The viral symptom comparisons were further demonstrated by the percentages of chlorotic lesions covered in the leaves of TCV-infected plants, which showed TCV-infected WRKY61 (HM) lines contain the highest percentage of chlorosis coverage (Fig. 5b), followed by WRKY61 OE and WT plant control. In addition to the direct symptom observation, the TCV-CP gene transcripts were further assessed by qRT-PCR, which also showed the most abundance TCV-CP gene transcripts were detected in WRKY61 HM deficient mutants (Fig. 5c). Furthermore, these virus tolerance results were also confirmed with Western blot analysis, showing the relative amount of CP expression (Fig. 5d). Although the symptom of WRKY61 OE plants appeared to be similar to that of WT, the amount of CP gene transcript and CP expression is less than that of the WT.

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