Project description:Six putative ?-galactosidase genes (?-Gals), three acid forms (CsGAL1, CsGAL2, CsGAL3) and three alkaline forms (CsAGA1, CsAGA2, CsAGAL3), were found in the cucumber genome. It is interesting to know the expression pattern and possible function of these ?-Gals in the cucumber plant since it is a stachyose-translocating species. In this study, full-length cDNAs of six ?-Gals were cloned and heterologously expressed. The result showed that all recombinant proteins revealed acid or alkaline ?-Gal activities with different substrate specificities and pH or temperature responding curves, indicating their distinct roles in cucumber plants. Phylogenetic analysis of collected ?-Gal amino acid sequences from different plants indicated that the ancestor of both acid and alkaline ?-Gals existed before monocots and dicots separated. Generally, six ?-Gal genes are universally expressed in different cucumber organs. CsGAL2 highly expressed in fasting-growing leaves, fruits and germinating seeds; CsGAL3 mainly distributes in vacuoles and significantly expressed in cucumber fruits, senescent leaves and seeds during late stage germination; The expression of CsAGA1 increased from leaf 1 to leaf 3 (sink leaves) and then declined from leaf 4 to leaf 7 (source leaves), and this isoform also highly expressed in male flowers and germinating seeds at early stage; CsAGA2 significantly expressed in cucumber leaves and female flowers; CsAGA3 is localized in plastids and also actively expressed in senescent leaves and germinating seeds; The role of CsGAL1 in cucumber plants is now unclear since its expression was relatively low in all organs. According to their expression patterns, subcellular localizations and previously reported functions of these isoforms in other plants, combining the data of soluble sugars contents in different tissues, the possible functions of these ?-Gals were discussed.
Project description:The plant vascular system is essential for the enlarged plant stature and successful colonizzation the land by delivering resources throughout the plants and providing mechanical support. Despite several regulators of vascular patterning have been reported, how vascular system mediates stress resistance remain largely unknown. Here we identified a CsIND transcription factor that is specifically expressed in the xylem and phloem tissues in cucumber. Knock down of CsIND by RNAi lead to dwarf plants with enlarged or disorganized vascular systems in all aerial organs. The content of both auxin and jasmonic acid were increased in the CsIND-RNAi lines. Transcriptome profiling by RNA-Seq hints CsIND-regulated gene networks for defense response and vascular development. Biochemical analyses verified that CsIND directly binds to well-known vascular regulators including CsCCR1, CsMYB116, CsYAB5, CsBP and CsAUX, and physically interacts with dorsiventral patterning genes CsKAN2 and CsYAB5. Further, CsIND-RNAi plants displayed significantly enhanced tolerance to nitrogen dificency and resistance to cucumber downy mildew. Therefore, CsIND regulates vascular formation and resistance to biotic and abiotic stresses in cucumber, through the combinarory interactions with well-known vascular regulaors and hormone metabolism and signaling pathways. Overall design: Leaf veins and fruits four days before anthesis of WT and CsIND-RNAi transgenic cucumber plants were collected for RNA-seq analysis. Two biological replicates were performed for each tissue sample. Please note that  the identifiers in the processed data file are searchable at: http://www.icugi.org/cgi-bin/ICuGI/genome/home.cgi?ver=2&organism=cucumber&cultivar=Chinese-long  the 'M' is changed to 'G', and the isoform information has been deleted. For example, the 'Csa3G002400' corresponds to 'Csa3M002400.1' in the website.
Project description:Transgenic plants are commonly used in breeding programs because of the various features that can be introduced. However, unintended effects caused by genetic transformation are still a topic of concern. This makes research on the nutritional safety of transgenic crop plants extremely interesting. Cucumber (Cucumis sativus L.) is a crop that is grown worldwide. The aim of this study was to identify and characterize differentially expressed genes and regulatory miRNAs in transgenic cucumber fruits that contain the thaumatin II gene, which encodes the sweet-tasting protein thaumatin II, by NGS sequencing. We compared the fruit transcriptomes and miRNomes of three transgenic cucumber lines with wild-type cucumber. In total, we found 47 differentially expressed genes between control and all three transgenic lines. We performed the bioinformatic functional analysis and gene ontology classification. We also identified 12 differentially regulated miRNAs, from which three can influence the two targets (assigned as DEGs) in one of the studied transgenic lines (line 224). We found that the transformation of cucumber with thaumatin II and expression of the transgene had minimal impact on gene expression and epigenetic regulation by miRNA, in the cucumber fruits.
Project description:Powdery mildew (PM) causing fungi are well-known pathogens, infecting over 10.000 plant species, including the economically important crop cucumber (Cucumis sativus L.). Loss-of-function mutations in clade V MLO genes have previously been shown to lead to recessively inherited broad-spectrum resistance to PM in several species. In cucumber, one clade V MLO homolog (CsaMLO8) was previously identified as being a susceptibility factor to PM. Two other closely related homologs (CsaMLO1 and CsaMLO11) were found, but their function was not yet unravelled.CsaMLO1 and CsaMLO11 were cloned from cucumber and overexpressed in a tomato mlo mutant. The transcript abundances of all three CsaMLO genes in different cucumber tissues were quantified using qRT-PCR and RNA-seq, with and without inoculation with the cucumber PM fungus Podosphaera xanthii. Allelic variation of CsaMLO1 and CsaMLO11 was screened in silico in sequenced cucumber germplasm.Heterologous overexpression of all three CsaMLO genes in the tomato mlo mutant restored susceptibility to PM caused by Oidium neolycopersici, albeit to a different extent: whereas overexpression of CsaMLO1 or CsaMLO8 completely restored susceptibility, overexpression of CsaMLO11 was only partially able to restore PM susceptibility. Furthermore, it was observed by qRT-PCR and RNA-seq that CsaMLO8 was significantly higher expressed in non-inoculated cucumber compared to the other two MLO genes. However, inoculation with P. xanthii led to upregulation of CsaMLO1, but not to upregulation of CsaMLO8 or CsaMLO11.Both CsaMLO1 and CsaMLO11 are functional susceptibility genes, although we conclude that based on the transcript abundance CsaMLO8 is probably the major clade V MLO gene in cucumber regarding providing susceptibility to PM. Potential loss-of-function mutations in CsaMLO1 and CsaMLO11 have not been identified. The generation and analysis of such mutants are interesting subjects for further investigation.
Project description:<h4>Background</h4>Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics.<h4>Principal findings</h4>A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147?Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation.<h4>Conclusions</h4>We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.