Background: Wheat yellow dwarf virus disease is infected by barley yellow dwarf virus (BYDV), which causes leaf yellowing and dwarfing symptoms in wheat, thereby posing a serious threat to China's food production. The infection of plant viruses can produce large numbers of vsiRNAs, which can target host transcripts and cause symptom development. However, few studies have been conducted to explore the role played by vsiRNAs in the interaction between BYDV-GAV and host wheat plants.Methods: In this study, small RNA sequencing was conducted to profile vsiRNAs in BYDV-GAV-infected wheat plants. The putative targets of vsiRNAs were predicted by the bioinformatics software psRNATarget. RT-qPCR and VIGS were employed to identify the function of selected target transcripts. To confirm the interaction between vsiRNA and the target, 5’ RACE was performed to analyze the specific cleavage sites.Results: From the sequencing data, we obtained a total of 11,384 detected vsiRNAs. The length distribution of these vsiRNAs was mostly 21 and 22 nt, and an A/U bias was observed at the 5' terminus. We also observed that the production region of vsiRNAs had no strand polarity. The vsiRNAs were predicted to target 23,719 wheat transcripts. GO and KEGG enrichment analysis demonstrated that these targets were mostly involved in cell components, catalytic activity and plant-pathogen interactions. The results of RT-qPCR analysis showed that most chloroplast-related genes were downregulated in BYDV-GAV-infected wheat plants. Silencing of a chlorophyll synthase gene caused leaf yellowing that was similar to the symptoms exhibited by BYDV-GAV-inoculated wheat plants. A vsiRNA from an overlapping region of BYDV-GAV MP and CP was observed to target chlorophyll synthase for gene silencing. Next, 5’ RACE validated that vsiRNA8856 could cleave the chlorophyll synthase transcript in a sequence-specific manner.Conclusion: This report is the first to demonstrate that BYDV-GAV-derived vsiRNAs can target wheat transcripts for symptom development, and the results of this study help to elucidate the molecular mechanisms underlying leaf yellowing after viral infection.
Background: Wheat yellow dwarf virus disease was infected by barley yellow dwarf virus, caused leaf yellowing and dwarfing symptoms in wheat, posing a serious threat to China's food production. Infection of plant viruses can produce a large number of vsiRNAs, which can target host transcripts for symptoms development. However, few studies were conducted to explore the role of vsiRNAs in the interaction between BYDV-GAV and host wheat plants.Methods: In this study, small RNA sequencing was conducted to profile the vsiRNAs in BYDV-GAV infected wheat plants. The putative targets of vsiRNAs were predicted by bioinformatics software. RT-qPCR and VIGS were used to identify the function of selected target transcripts. To confirm the interaction between vsiRNA and the target, 5’RACE was performed to analyze the specific cleavage sites. Results: From the sequencing data, we obtained a total of 11,384 detected vsiRNAs. The length distribution of these vsiRNAs mostly was 21- and 22-nt along with A/U biased at the 5 ' -terminal. We also found the production region of vsiRNAs had no strand polarity. The vsiRNAs were predicted to target 23,719 wheat transcripts. GO and KEGG enrichment analysis revealed that these targets mostly involved in cell parts and catalytic activity and plant-pathogen interaction. The results of RT-qPCR indicted that most chloroplast-related genes showed down-regulation in BYDV-GAV infected wheat plants. Silencing of a chlorophyll synthase caused leaf yellowing that was similar to the symptom of BYDV-GAV inoculated wheat plants. A vsiRNA from an overlap region of BYDV-GAV MP and CP was found that could target the chlorophyll synthase for gene silencing. Then, 5’RACE validated that vsiRNA8856 could cleavage the chlorophyll synthase transcript in a sequence-specific manner.Conclusion: This is the first report demonstrating that BYDV-GAV-derived vsiRNAs can target wheat transcripts for symptom development, and this study provides new insights into the molecular mechanisms underlying leaf yellowing upon viral infection.
Background: Wheat yellow dwarf virus disease is infected by barley yellow dwarf virus (BYDV), which causes leaf yellowing and dwarfing symptoms in wheat, thereby posing a serious threat to China's food production. The infection of plant viruses can produce large numbers of vsiRNAs, which can target host transcripts and cause symptom development. However, few studies have been conducted to explore the role played by vsiRNAs in the interaction between BYDV-GAV and host wheat plants.Methods: In this study, small RNA sequencing was conducted to profile vsiRNAs in BYDV-GAV-infected wheat plants. The putative targets of vsiRNAs were predicted by the bioinformatics software psRNATarget. RT-qPCR and VIGS were employed to identify the function of selected target transcripts. To confirm the interaction between vsiRNA and the target, 5’ RACE was performed to analyze the specific cleavage sites.Results: From the sequencing data, we obtained a total of 11,384 detected vsiRNAs. The length distribution of these vsiRNAs was mostly 21 and 22 nt, and an A/U bias was observed at the 5' terminus. We also observed that the production region of vsiRNAs had no strand polarity. The vsiRNAs were predicted to target 23,719 wheat transcripts. GO and KEGG enrichment analysis demonstrated that these targets were mostly involved in cell components, catalytic activity and plant-pathogen interactions. The results of RT-qPCR analysis showed that most chloroplast-related genes were downregulated in BYDV-GAV-infected wheat plants. Silencing of a chlorophyll synthase gene caused leaf yellowing that was similar to the symptoms exhibited by BYDV-GAV-inoculated wheat plants. A vsiRNA from an overlapping region of BYDV-GAV MP and CP was observed to target chlorophyll synthase for gene silencing. Next, 5’ RACE validated that vsiRNA8856 could cleave the chlorophyll synthase transcript in a sequence-specific manner.Conclusion: This report is the first to demonstrate that BYDV-GAV-derived vsiRNAs can target wheat transcripts for symptom development, and the results of this study help to elucidate the molecular mechanisms underlying leaf yellowing after viral infection.
Background: P. huashanica ( Psathyrostachys huashanica ), known as an important resistance resource reservoir, is a rare and endangered plant growing suitably in Huashan mount region and would be urgently exploited in wheat genetic improvements sooner. During the utilization process, different IRGs (internal reference genes) need to be appropriately selected as standards based on biotic and abiotic stress conditions. It is crucial that Real-time RT-qPCR with combination of bioinformatics were adopted to explore the reliable IRGs from transcriptome of P . huashanica.Results: The present work reported new 3 species of IRGs, UBC2 , UBC17, 18S rRNA , which were screened from transcriptome of P. huashanica under biotic and abiotic stress conditions, using RT-qPCR and four algorithms, including geNorm, NormFinder, BestKeeper, and RefFinder, to analyse expression of sixteen candidate reference genes. These genes appear as following 18S rRNA (18S ribosomal RNA), EF1-α (eukaryotic elongation factor 1 alpha), UBC2 (ubiquitin-conjugating enzyme E2-2), UBC17 (ubiquitin-conjugating enzyme E2-17), α-TUB2A (alpha tubulin-2A), β-TUB3 (beta tubulin 3), ADF4 (Actin-depolymerising factor 4), ACTIN (actin), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), 60SARP (60S acidic ribosomal protein), UBQ (polyubiquitin), SamDC (S-Adenosylmethionine decarboxylase), EIF4A (eukaryotic initiation factor 4A), ARF (ADP-ribosylation factor), HIS1 (histone H1), and HIS2B (histone H2B). Analysis of gene expression demonstrated that the expression of UBC2 gene was most stable under ABA hormone stress, low temperature stress and high temperature stress, similarly, UBC17 gene under IAA hormone stress, salinity stress and drought stress, both UBC17 genes and 18S rRNA genes under abiotic and biotic stress, respectively. The most stable gene was UBC2 gene in the root, UBC17 gene in stem and leaf. In this study, α-TUB2A , UBC and ACTIN genes were verified as the suitable reference genes across all tested samples. To further validate the suitability of the selected reference genes, we evaluated the relative expression of PsaCPK3 (Calcium-dependent protein kinase) and PsaHSP70-1 (heat shock protein 70-1), which are stress-related genes that may be involved in response to adversity.Conclusions: This study has identified a set of the most stable IRGs suiting for RT-qPCR detection of a few target gene expressions from P . huashanica in different experimental conditions. In addition, this study should provide the accuracy information for gene expression analysis in P . huashanica .
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