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iTRAQ-based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation.

Wang B, Hajano JU, Ren Y, Lu C, Wang X - Virol. J. (2015)

Bottom Line: Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis.Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results.The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 100193, Beijing, China. bwang0721@163.com.

ABSTRACT

Background: Rice plants infected by Rice stripe virus (RSV) usually leads to chlorosis and death of newly emerged leaves. However, the mechanism of RSV-induced these symptoms was not clear.

Methods: We used an iTRAQ approach for a quantitative proteomics comparison of non-infected and infected rice leaves. RT-qPCR and Northern blot analyses were performed for assessing the transcription of candidate genes.

Results: As a whole, 681 (65.8% downregulated, 34.2% upregulated infected vs. non-infected) differentially accumulated proteins were identified. A bioinformatics analysis indicated that ten of these regulated proteins are involved in chlorophyll biosynthesis and three in cell death processes. Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis. Three aspartic proteases expressed higher in RSV-infected leaves than those in the control leaves, which were also implicated in RSV-induced cell death. Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results.

Conclusions: The magnesium chelatase and aspartic proteases may be associated with RSV-induced leaf chlorosis and cell death, respectively. The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.

No MeSH data available.


Related in: MedlinePlus

Validation of rice gene expression levels by real time RT-PCR and Northern blotting. a Comparison of protein and mRNA expression levels of mock leaves and RSV-infected leaves using RT-qPCR. Blue represents mock leaves; red represents RSV-infected leaves. The averaged readings from the three biological replicates normalized against endogenous gene OsEF1α; error bar denoted SD. Statistics were analyzed using the Student’s t-test. An asterisk indicated a significant difference from the corresponding control (P < 0.01). b Northern blot of two differentially expressed genes selected for verifying RT-qPCR results. Mock, mock-inoculated leaves; RSV-infected, RSV-infected leaves. Equal loading of total RNA was assessed by staining rRNA with ethidium bromide. Marker contained 2000 bp, 1500 bp, 1000 bp and 750 bp
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Fig6: Validation of rice gene expression levels by real time RT-PCR and Northern blotting. a Comparison of protein and mRNA expression levels of mock leaves and RSV-infected leaves using RT-qPCR. Blue represents mock leaves; red represents RSV-infected leaves. The averaged readings from the three biological replicates normalized against endogenous gene OsEF1α; error bar denoted SD. Statistics were analyzed using the Student’s t-test. An asterisk indicated a significant difference from the corresponding control (P < 0.01). b Northern blot of two differentially expressed genes selected for verifying RT-qPCR results. Mock, mock-inoculated leaves; RSV-infected, RSV-infected leaves. Equal loading of total RNA was assessed by staining rRNA with ethidium bromide. Marker contained 2000 bp, 1500 bp, 1000 bp and 750 bp

Mentions: Based on a proteomics analysis, the proteins differentially accumulated during RSV infection, key proteins for chlorophyll biosynthesis and an aspartic-type endopeptidase were identified as involved in the formation of RSV induced symptoms, and their presence was quantitatively confirmed using RT-qPCR and Northern blot to evaluate the correlation between mRNA and protein levels. Total RNA extracted from RSV-infected and mock leaves was analyzed to measure mRNA transcription levels of putative target proteins. The RT-qPCR results demonstrated that expression of the genes for CHLI and CHLD (magnesium chelatase) in RSV-infected leaves was downregulated more than three times the level of the control (Fig. 6a), and transcription of genes encoding radc1, rap and p0026h03.19 in RSV-infected leaves were upregulated 14, 2, 3 times higher than the level of the control leaves, respectively (Fig. 6a), verifying the iTRAQ results. Similarly, this trend for mRNA levels of the genes for CHLI and p0026h03.19 by Northern blotting analyses also supported the transcription of genes encoding respective protein by RT-qPCR (Fig. 6b). Whereas, elevated levels of five genes were different between transcription and proteins levels that may be due to posttranscription and posttranslational regulatory processes.Fig. 6


iTRAQ-based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation.

Wang B, Hajano JU, Ren Y, Lu C, Wang X - Virol. J. (2015)

Validation of rice gene expression levels by real time RT-PCR and Northern blotting. a Comparison of protein and mRNA expression levels of mock leaves and RSV-infected leaves using RT-qPCR. Blue represents mock leaves; red represents RSV-infected leaves. The averaged readings from the three biological replicates normalized against endogenous gene OsEF1α; error bar denoted SD. Statistics were analyzed using the Student’s t-test. An asterisk indicated a significant difference from the corresponding control (P < 0.01). b Northern blot of two differentially expressed genes selected for verifying RT-qPCR results. Mock, mock-inoculated leaves; RSV-infected, RSV-infected leaves. Equal loading of total RNA was assessed by staining rRNA with ethidium bromide. Marker contained 2000 bp, 1500 bp, 1000 bp and 750 bp
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4489111&req=5

Fig6: Validation of rice gene expression levels by real time RT-PCR and Northern blotting. a Comparison of protein and mRNA expression levels of mock leaves and RSV-infected leaves using RT-qPCR. Blue represents mock leaves; red represents RSV-infected leaves. The averaged readings from the three biological replicates normalized against endogenous gene OsEF1α; error bar denoted SD. Statistics were analyzed using the Student’s t-test. An asterisk indicated a significant difference from the corresponding control (P < 0.01). b Northern blot of two differentially expressed genes selected for verifying RT-qPCR results. Mock, mock-inoculated leaves; RSV-infected, RSV-infected leaves. Equal loading of total RNA was assessed by staining rRNA with ethidium bromide. Marker contained 2000 bp, 1500 bp, 1000 bp and 750 bp
Mentions: Based on a proteomics analysis, the proteins differentially accumulated during RSV infection, key proteins for chlorophyll biosynthesis and an aspartic-type endopeptidase were identified as involved in the formation of RSV induced symptoms, and their presence was quantitatively confirmed using RT-qPCR and Northern blot to evaluate the correlation between mRNA and protein levels. Total RNA extracted from RSV-infected and mock leaves was analyzed to measure mRNA transcription levels of putative target proteins. The RT-qPCR results demonstrated that expression of the genes for CHLI and CHLD (magnesium chelatase) in RSV-infected leaves was downregulated more than three times the level of the control (Fig. 6a), and transcription of genes encoding radc1, rap and p0026h03.19 in RSV-infected leaves were upregulated 14, 2, 3 times higher than the level of the control leaves, respectively (Fig. 6a), verifying the iTRAQ results. Similarly, this trend for mRNA levels of the genes for CHLI and p0026h03.19 by Northern blotting analyses also supported the transcription of genes encoding respective protein by RT-qPCR (Fig. 6b). Whereas, elevated levels of five genes were different between transcription and proteins levels that may be due to posttranscription and posttranslational regulatory processes.Fig. 6

Bottom Line: Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis.Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results.The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 100193, Beijing, China. bwang0721@163.com.

ABSTRACT

Background: Rice plants infected by Rice stripe virus (RSV) usually leads to chlorosis and death of newly emerged leaves. However, the mechanism of RSV-induced these symptoms was not clear.

Methods: We used an iTRAQ approach for a quantitative proteomics comparison of non-infected and infected rice leaves. RT-qPCR and Northern blot analyses were performed for assessing the transcription of candidate genes.

Results: As a whole, 681 (65.8% downregulated, 34.2% upregulated infected vs. non-infected) differentially accumulated proteins were identified. A bioinformatics analysis indicated that ten of these regulated proteins are involved in chlorophyll biosynthesis and three in cell death processes. Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis. Three aspartic proteases expressed higher in RSV-infected leaves than those in the control leaves, which were also implicated in RSV-induced cell death. Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results.

Conclusions: The magnesium chelatase and aspartic proteases may be associated with RSV-induced leaf chlorosis and cell death, respectively. The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.

No MeSH data available.


Related in: MedlinePlus