Project description:miRNAs are key players in multiple biological processes, therefore analysis and characterization of these small regulatory RNAs is a critical step towards better understanding of animal and plant biology. In apple (Malus domestica) two hundred microRNAs are known, which most probably represents only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species. We performed deep sequencing of twelve small RNA libraries obtained for fire blight resistant and fire blight sensitive trees. In the sequencing results we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop qPCR and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs. In this study we considerably expand the apple miRNAome by identifying and characterizing dozens of novel microRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance.
Project description:miRNAs are key players in multiple biological processes, therefore analysis and characterization of these small regulatory RNAs is a critical step towards better understanding of animal and plant biology. In apple (Malus domestica) two hundred microRNAs are known, which most probably represents only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species. We performed deep sequencing of twelve small RNA libraries obtained for fire blight resistant and fire blight sensitive trees. In the sequencing results we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop qPCR and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs. In this study we considerably expand the apple miRNAome by identifying and characterizing dozens of novel microRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance. Actively-growing shoot tip tissue samples were collected from twelve apple trees, which includes three biological replicates of each following scion-rootstock combinations: B.9, G.30, M.111 and M.27.
Project description:Fire blight (FB) is a bacterial disease affecting plants from Rosaceae family, including apple and pear. FB develops after the infection of Erwinia amylovora, gram-negative enterobacterium, and results in burnt-like damages and wilting, which can affect all organs of the plant. Although the mechanisms underlying disease response in apples are not elucidated yet, it has been well described that FB resistance depends on the rootstock type. The main objective of this work was to identify miRNAs involved in response to bacterial infection in order to better explain apple defense mechanisms against fire blight disease. We performed deep sequencing of eighteen small RNA libraries obtained from inoculated and non-inoculated Gala apple leaves. 233 novel plant mature miRNAs were identified together with their targets and potential role in response to bacterial infection. We identify three apple miRNAs responding to inoculation (mdm-miR168a,b, mdm-miR194C and mdm-miR1392C) as well as miRNAs reacting to bacterial infection in a rootstock-specific manner (miR395 family). Our results provide insights into the mechanisms of fire blight resistance in apple.
Project description:This RNA-seq experiment captures expression data from challenged and mock-inoculated apple flowers (Malus domestica Golden Delicious) to assess the susceptible response of the primary infection court (48h) of apple by the fire blight pathogen Erwinia amylovora (CFBP 1430).
Project description:<p>Cisgenesis, the genetic modification of a plant with genes from a sexually compatible plant, was used to confer fire blight resistance to the cultivar 'Gala Galaxy' by amendment of the resistance gene FB_MR5, resulting in the line C44.4146. To verify whether cisgenesis changed other tree-, flower-, or fruit-related traits, a 5-year field trial was conducted with trees of C44.4.146 and multiple control cultivars, including members of the 'Gala' sports group. None of the 44 investigated tree-, flower-, or fruit-related traits significantly differed between C44.4.146 and at least one of the control cultivars in all observation years. However, fruits of C44.4.146 and its wild-type 'Gala Galaxy' from tissue culture were paler in color than fruits of 'Gala Galaxy' that had not undergone tissue culture. The abundance of no metabolite in the fruit flesh and peel of C44.4.146 was consistently significantly different from the control genotypes in both observation years. Finally, the disease resistance of C44.4.146 was confirmed also when the fire blight pathogen was inoculated through the flowers. We conclude that the use of cisgenesis to confer fire blight resistance to 'Gala Galaxy' in C44.4.146 did not have unintended effects and that the in vitro establishment of 'Gala Galaxy' had a greater effect on C44.4.146 properties than its cisgenic generation.</p>