Project description:RNA interference (RNAi) is a conserved, RNA-mediated, regulatory mechanism in eukaryotes. In plants, it plays an important role in growth, development and resistance against viral infections. As a counter-defence, plant viruses, e.g. geminiviruses, encode RNAi suppressors, such as AC2, AC4 and AV2. To obtain Nicotiana tabacum virus resistant plants against Tomato leaf curl New Delhi virus (ToLCNDV), we employ the biogenesis pathway of a class of endogenous siRNAs, the trans-acting siRNAs (ta-siRNAs), by engineering artificial ta-siRNAs (ata-siRNAs) targeting the AC2 (TRiV-AC2) and AC4 (TRiV-AC4) RNAi suppressors using miRNA390 dual target sites. The mode of action of ta-siRNAs comprises of the cleavage of the target (similar to the miRNA targeting). Using degradome approaches, the abundance of the resulting 3' fragment of the cleaved transcript can be quantified and the precise localization of the cleavage on the target mRNA can be identified. We sequenced degradome libraries of Nicotiana tabacum plants infected with ToLCNDV which were treated with the ata-siRNA-AC2 construct; mock-treated plants were used as controls. Following quality checks, the abundance distributions of the degradation fragments were normalized. The transcripts with different cleavage patterns was the AC2, supporting the conclusion that an efficient cleavage of the target occurred, without significant off-target effects.
Project description:RNA interference (RNAi) is a conserved, RNA-mediated, regulatory mechanism in eukaryotes. In plants, it plays an important role in growth, development and resistance against viral infections. As a counter-defence, plant viruses, e.g. geminiviruses, encode RNAi suppressors, such as AC2, AC4 and AV2. To obtain Nicotiana tabacum virus resistant plants against Tomato leaf curl New Delhi virus (ToLCNDV), we employ the biogenesis pathway of a class of endogenous siRNAs, the trans-acting siRNAs (ta-siRNAs), by engineering artificial ta-siRNAs (ata-siRNAs) targeting the AC2 (TRiV-AC2) and AC4 (TRiV-AC4) RNAi suppressors using miRNA390 dual target sites. The mode of action of ta-siRNAs comprises of the cleavage of the target (similar to the miRNA targeting). Using degradome approaches, the abundance of the resulting 3' fragment of the cleaved transcript can be quantified and the precise localization of the cleavage on the target mRNA can be identified. We sequenced degradome libraries of Nicotiana tabacum plants infected with ToLCNDV which were treated with the ata-siRNA-AC2 construct; mock-treated plants were used as controls. Following quality checks, the abundance distributions of the degradation fragments were normalized. The transcripts with different cleavage patterns was the AC2, supporting the conclusion that an efficient cleavage of the target occurred, without significant off-target effects.
Project description:Jiashi melon and 86-1 melon were inoculated with Alternaria alternata, and the difference of gene expression was analyzed after 0, 6, 12, 18 and 24 days storage.
Project description:Viruses are among the most destructive and difficult to control plant pathogens. Melon (Cucumismelo L.) has become the model species for the agriculturally important Cucurbitaceae family. Approaches that take advantage of recently developed genomic tools in melon are being extremely useful for understanding viral pathogenesis and can contribute to the identification of target genes to breed new resistant cultivars. In this work, we have used a recently described melon microarray for transcriptome profiling of two melon cultivars infected with two strains of Melon necrotic spot virus (MNSV) that only differ on their 3´-untranslated regions. Tissues of melon plants from cultivars Out of 7566 and 7074 genes deregulated by MNSV-Mα5 and MNSV-Mα5/3’264, 1851 and 1356, respectively, were strain-specific. Likewise, MNSV-Mα5/3’264 specifically deregulated 2925 and 1618 genes in Planters Jumbo and Tendral, respectively. Thus, significantly affected GO categories were clearly different for the different virus/host combinations. Grouping genes according to their patterns of expression allowed the identification of two groups specifically deregulated by MNSV-Mα5/3’264 with respect to MNSV-Mα5 in Tendral, and one group antagonistically regulated in Planters Jumbo vs. Tendral after MNSV-Mα5/3’264 infection. Genes in these three groups belong to a diversity of functional classes, and no obvious regulatory commonalities were identified. When data on MNSV-Mα5/Tendral infections were compared to equivalent data on cucumber mosaic virus or watermelon mosaic virus infections, cytokinin-O-glucosyltransferase2 was identified as the only gene deregulated by the three viruses, with infections dynamics correlating with the amplitude of transcriptome remodeling. Both common and strain-specific changes, as well as common but also cultivar-specific changes, have been identified by profiling transcriptomes of plants from two melon cultivars infected with two MNSV strains. No obvious regulatory features shared among deregulated genes have been identified, pointing toward regulation through differential functional implications. Viruses are among the most destructive and difficult to control plant pathogens. Melon (Cucumismelo L.) has become the model species for the agriculturally important Cucurbitaceae family. Approaches that take advantage of recently developed genomic tools in melon are being extremely useful for understanding viral pathogenesis and can contribute to the identification of target genes to breed new resistant cultivars. In this work, we have used a recently described melon microarray for transcriptome profiling of two melon cultivars infected with two strains of Melon necrotic spot virus (MNSV) that only differ on their 3´-untranslated regions. Tissues of melon plants from cultivars Out of 7566 and 7074 genes deregulated by MNSV-Mα5 and MNSV-Mα5/3’264, 1851 and 1356, respectively, were strain-specific. Likewise, MNSV-Mα5/3’264 specifically deregulated 2925 and 1618 genes in Planters Jumbo and Tendral, respectively. Thus, significantly affected GO categories were clearly different for the different virus/host combinations. Grouping genes according to their patterns of expression allowed the identification of two groups specifically deregulated by MNSV-Mα5/3’264 with respect to MNSV-Mα5 in Tendral, and one group antagonistically regulated in Planters Jumbo vs. Tendral after MNSV-Mα5/3’264 infection. Genes in these three groups belong to a diversity of functional classes, and no obvious regulatory commonalities were identified. When data on MNSV-Mα5/Tendral infections were compared to equivalent data on cucumber mosaic virus or watermelon mosaic virus infections, cytokinin-O-glucosyltransferase2 was identified as the only gene deregulated by the three viruses, with infections dynamics correlating with the amplitude of transcriptome remodeling. Both common and strain-specific changes, as well as common but also cultivar-specific changes, have been identified by profiling transcriptomes of plants from two melon cultivars infected with two MNSV strains. No obvious regulatory features shared among deregulated genes have been identified, pointing toward regulation through differential functional implications.
Project description:Viruses are among the most destructive and difficult to control plant pathogens. Melon (Cucumismelo L.) has become the model species for the agriculturally important Cucurbitaceae family. Approaches that take advantage of recently developed genomic tools in melon are being extremely useful for understanding viral pathogenesis and can contribute to the identification of target genes to breed new resistant cultivars. In this work, we have used a recently described melon microarray for transcriptome profiling of two melon cultivars infected with two strains of Melon necrotic spot virus (MNSV) that only differ on their 3´-untranslated regions. Tissues of melon plants from cultivars Out of 7566 and 7074 genes deregulated by MNSV-Mα5 and MNSV-Mα5/3’264, 1851 and 1356, respectively, were strain-specific. Likewise, MNSV-Mα5/3’264 specifically deregulated 2925 and 1618 genes in Planters Jumbo and Tendral, respectively. Thus, significantly affected GO categories were clearly different for the different virus/host combinations. Grouping genes according to their patterns of expression allowed the identification of two groups specifically deregulated by MNSV-Mα5/3’264 with respect to MNSV-Mα5 in Tendral, and one group antagonistically regulated in Planters Jumbo vs. Tendral after MNSV-Mα5/3’264 infection. Genes in these three groups belong to a diversity of functional classes, and no obvious regulatory commonalities were identified. When data on MNSV-Mα5/Tendral infections were compared to equivalent data on cucumber mosaic virus or watermelon mosaic virus infections, cytokinin-O-glucosyltransferase2 was identified as the only gene deregulated by the three viruses, with infections dynamics correlating with the amplitude of transcriptome remodeling. Both common and strain-specific changes, as well as common but also cultivar-specific changes, have been identified by profiling transcriptomes of plants from two melon cultivars infected with two MNSV strains. No obvious regulatory features shared among deregulated genes have been identified, pointing toward regulation through differential functional implications. Viruses are among the most destructive and difficult to control plant pathogens. Melon (Cucumismelo L.) has become the model species for the agriculturally important Cucurbitaceae family. Approaches that take advantage of recently developed genomic tools in melon are being extremely useful for understanding viral pathogenesis and can contribute to the identification of target genes to breed new resistant cultivars. In this work, we have used a recently described melon microarray for transcriptome profiling of two melon cultivars infected with two strains of Melon necrotic spot virus (MNSV) that only differ on their 3´-untranslated regions. Tissues of melon plants from cultivars Out of 7566 and 7074 genes deregulated by MNSV-Mα5 and MNSV-Mα5/3’264, 1851 and 1356, respectively, were strain-specific. Likewise, MNSV-Mα5/3’264 specifically deregulated 2925 and 1618 genes in Planters Jumbo and Tendral, respectively. Thus, significantly affected GO categories were clearly different for the different virus/host combinations. Grouping genes according to their patterns of expression allowed the identification of two groups specifically deregulated by MNSV-Mα5/3’264 with respect to MNSV-Mα5 in Tendral, and one group antagonistically regulated in Planters Jumbo vs. Tendral after MNSV-Mα5/3’264 infection. Genes in these three groups belong to a diversity of functional classes, and no obvious regulatory commonalities were identified. When data on MNSV-Mα5/Tendral infections were compared to equivalent data on cucumber mosaic virus or watermelon mosaic virus infections, cytokinin-O-glucosyltransferase2 was identified as the only gene deregulated by the three viruses, with infections dynamics correlating with the amplitude of transcriptome remodeling. Both common and strain-specific changes, as well as common but also cultivar-specific changes, have been identified by profiling transcriptomes of plants from two melon cultivars infected with two MNSV strains. No obvious regulatory features shared among deregulated genes have been identified, pointing toward regulation through differential functional implications.
Project description:Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21â24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analyzed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. This analysis provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melonâvirus interactions.