Cucumber mosaic virus strain:I17F
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ABSTRACT:
PROVIDER: PRJNA1076493 | ENA |
REPOSITORIES: ENA
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Project description:We constructed two independent small RNA libraries from leaves of mock and Cucumber mosaic virus (CMV) infected tomatoes, respectively, and sequenced with a high-throughput Illumina Solexa system. Based on sequence analysis and hairpin structure prediction, a total of 50 known miRNAs (32 families) and 568 potentially candidate miRNAs (PC-miRNAs) were firstly identified in tomato, with 12 known miRNAs and 154 PC-miRNAs supported by both the 3p and 5p strands. Comparative analysis revealed 79 miRNAs (including 15 novel tomato miRNAs) and 40 PC-miRNAs were differentially expressed between the two libraries. Among these virus responsive miRNAs, expression patters of some novel tomato miRNAs and PC-miRNAs in mock and in CMV-Fny infected tomatoes were further validated by qRT-PCR. Moreover, we revealed 563 potential targets for 66 tomato miRNAs by the recently developed degradome sequencing approach, including 124 targets for 7 new tomato miRNAs and 97 targets for 24 PC-miRNAs. Target annotation for the newly identified miRNA and PC-miRNAs indicated that they were involved in multiple biological processes, including transcriptional regulation and virus resistance. Gene ontology analysis of these target transcripts demonstrated that stress response- and photosynthesis-related genes were most affected in CMV-Fny infected tomatoes. Examination of small RNAs and their targets in mock and CMV-Fny infected tomatoes.
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Project description:Plant viruses are a major threat for a wide range of host species, causing substantial losses in agriculture. Particularly, Cucumber mosaic virus (CMV) evokes severe symptoms, thus dramatically limiting yield. Activation of plant immunity is associated with changes in the gene expression and consequently, cellular proteome to ensure virus resistance. Proteomics proved to be an extremely valuable tool for discovering multiple targets for the rational design of plant protection strategies. Herein, we studied two cultivars of cucumber (Cucumis sativus) resistant ´Heliana´ and susceptible ´Vanda´. Plant cotyledons were mechanically inoculated with CMV isolate PK1, and systemic leaves were harvested at 33 days post-inoculation. Upon protein extraction and filter-aided sample preparations, peptides were profiled by ultrahigh-performance liquid chromatography and comprehensively quantified by ion mobility enhanced mass spectrometry. From 1,516 reproducibly quantified proteins using label-free approach, 133 were differentially abundant among genotypes or treatments by strict statistic and effect size criteria. Pigments and hydrogen peroxide measurements corroborated proteomic findings. Advanced bioinformatics revealed a modular network of affected host proteins. Direct comparison of both genotypes in the uninfected state highlighted more abundant photosynthetic and development-related proteins in resistant cucumber cultivar. Long-term CMV infection showed worse preservation of energy processes and less robust translation in susceptible cultivar versus resistant genotype. Contrary, susceptible cultivar had numerous more abundant stress and defense-related proteins. We proposed promising targets for functional validation in transgenic lines a step toward durable virus resistance in cucurbits and other crops.
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Project description:RNA silencing has an important role mediating sequence-specific virus resistance. Here, we analyzed in detail the interference of Cucumber Mosaic Virus (CMV) with the RNA silencing machinery of Arabidopsis thaliana. We detected that CMV infection induced the production of viral small interfering RNAs (vsiRNAs) that account for a significant part of the sRNome affecting the levels of other sRNA classes. Furthermore, we analyzed the incorporation of vsiRNAs into the main ARGONAUTE (AGO) proteins with a described antiviral role and the viral RNA silencing suppressor (VRS) 2b, by combining protein immunoprecipitation with sRNA high-throughput sequencing. vsiRNAs accumulated to high levels in AGO2, followed by AGO1, AGO5 and AGO7. Interestingly, vsiRNAs represented a significant percentage of AGO-loaded sRNAs and displaced other endogenous sRNAs. As a countermeasure, the VSR 2b loaded vsiRNAs and mRNA-derived siRNAs, which affected the expression of the genes they derived from. Additionally, we analyzed how vsiRNAs incorporated into the endogenous RNA silencing pathways by exploring their target mRNAs using parallel analysis of RNA end (PARE) sequencing. This strategy allowed us to identify 61 genes with degradome data supporting their vsiRNA-mediated cleavage. This work exemplifies the complex relationship of RNA viruses with the endogenous RNA silencing machinery and the multiple aspects of virus resistance and virulence that this interaction induces.
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