Identification of apple miRNAs and their potentialrole in Fire Blight resistance
ABSTRACT: 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: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:BACKGROUND: Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. RESULTS: Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. CONCLUSIONS: Rootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.
Project description:BACKGROUND:Although it is known that resistant rootstocks facilitate management of fire blight disease, incited by Erwinia amylovora, the role of rootstock root traits in providing systemic defense against E. amylovora is unclear. In this study, the hypothesis that rootstocks of higher root vigor provide higher tolerance to fire blight infection in apples is tested. Several apple scion genotypes grafted onto a single rootstock genotype and non-grafted 'M.7' rootstocks of varying root vigor are used to assess phenotypic and molecular relationships between root traits of rootstocks and fire blight susceptibility of apple scion cultivars. RESULTS:It is observed that different root traits display significant (p?<?0.05) negative correlations with fire blight susceptibility. In fact, root surface area partially dictates differential levels of fire blight susceptibility of 'M.7' rootstocks. Furthermore, contrasting changes in gene expression patterns of diverse molecular pathways accompany observed differences in levels of root-driven fire blight susceptibility. It is noted that a singular co-expression gene network consisting of genes from defense, carbohydrate metabolism, protein kinase activity, oxidation-reduction, and stress response pathways modulates root-dependent fire blight susceptibility in apple. In particular, WRKY75 and UDP-glycotransferase are singled-out as hub genes deserving of further detailed analysis. CONCLUSIONS:It is proposed that low root mass may incite resource-limiting conditions to activate carbohydrate metabolic pathways, which reciprocally interact with plant immune system genes to elicit differential levels of fire blight susceptibility.
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. Overall design: Actively-growing leaf tissue samples were collected from eighteen apple trees, which includes three biological replicates of inoculated and non-inoculated Gala scions grown on G.30 or M.27 rootstock.
Project description:The apple dwarfing rootstock 'Malling9' ('M9') has been used worldwide both to reduce scion vigour and as a genetic source for breeding new rootstocks. Progeny of 'M9' segregate for rootstock-induced dwarfing of the scion, indicating that this trait is controlled by one or more genetic factors. A quantitative trait locus (QTL) analysis of a rootstock population derived from the cross between 'M9' × 'Robusta5' (non-dwarfing) and grafted with 'Braeburn' scions identified a major QTL (Dw1) on linkage group (LG) 5, which exhibits a significant influence on dwarfing of the scion. A smaller-effect QTL affecting dwarfing (Dw2) was identified on LG11, and four minor-effect QTLs were found on LG6, LG9, LG10 and LG12. Phenotypic analysis indicates that the combination of Dw1 and Dw2 has the strongest influence on rootstock-induced dwarfing, and that Dw1 has a stronger effect than Dw2. Genetic markers linked to Dw1 and Dw2 were screened over 41 rootstock accessions that confer a range of effects on scion growth. The majority of the dwarfing and semi-dwarfing rootstock accessions screened carried marker alleles linked to Dw1 and Dw2. This suggests that most apple dwarfing rootstocks have been derived from the same genetic source.
Project description:BACKGROUND:Grafting is the common propagation method for avocado and primarily benefits orchard production by reducing the time to tree productivity. It also allows use of scions and rootstocks specifically selected for improved productivity and commercial acceptance. Rootstocks in avocado may be propagated from mature tree cuttings ('mature'), or from seed ('juvenile'). While the use of mature scion material hastens early bearing/maturity and economic return, the molecular factors involved in the role of the scion and/or rootstock in early bearing/reduced juvenility of the grafted tree are still unknown. RESULTS:Here, we utilized juvenility and flowering associated miRNAs; miR156 and miR172 and their putative target genes to screen pre-graft and post-graft material in different combinations from avocado. The abundance of mature miR156, miR172 and the miR156 target gene SPL4, showed a strong correlation to the maturity of the scion and rootstock material in avocado. Graft transmissibility of miR156 and miR172 has been explored in annual plants. Here, we show that the scion may be responsible for grafted tree maturity involving these factors, while the rootstock maturity does not significantly influence miRNA abundance in the scion. We also demonstrate that the presence of leaves on cutting rootstocks supports graft success and contributes towards intergraft signalling involving the carbohydrate-marker TPS1. CONCLUSION:Here, we suggest that the scion largely controls the molecular 'maturity' of grafted avocado trees, however, leaves on the rootstock not only promote graft success, but can influence miRNA and mRNA abundance in the scion. This constitutes the first study on scion and rootstock contribution towards grafted tree maturity using the miR156-SPL4-miR172 regulatory module as a marker for juvenility and reproductive competence.
Project description:To understand the roles of Malus rootstock, scion, and their interaction in Cd accumulation and tolerance, four scion/rootstock combinations consisting of the apple cultivars "Hanfu" (HF) and "Fuji" (FJ) grafted onto M. baccata (Mb) or M. micromalus "qingzhoulinqin" (Mm) rootstocks differing in relative Cd tolerance were exposed either to 0 µM or 50 µM CdCl2 for 18 d. Cd accumulation and tolerance in grafted Malus plants varied within rootstock, scion, and rootstock-scion interaction. Cd-induced decreases in photosynthesis, photosynthetic pigment level, and biomass were lower for HF grafted onto Mb than those for HF grafted onto Mm. Reductions in growth and photosynthetic rate were always the lowest for HF/Mb. Cd concentration, bioconcentration factor (BCF), and translocation factor (Tf ) were always comparatively higher in HF and FJ grafted onto rootstock Mm than in HF and FJ grafted on Mb, respectively. When HF and FJ were grafted onto the same rootstock, the root Cd concentrations were always higher in HF than FJ, whereas the shoot Cd concentrations displayed the opposite trend. The shoot Cd concentrations and Tf were lower for HF/Mb than the other scion/rootstock combinations. Rootstock, scion, and rootstock-scion interaction also affected subcellular Cd distribution. Immobilization of Cd in the root cell walls may be a primary Cd mobility and toxicity reduction strategy in Malus. The rootstock and scion also had statistically significant influences on ROS level and antioxidant activity. Cd induced more severe oxidative stress in HF and FJ grafted onto Mm than it did in HF and FJ grafted onto Mb. Compared with FJ, HF had lower foliar O2 -, root H2O2, and root and leaf MDA levels, but higher ROS-scavenging capacity. The rootstock, scion, and rootstock-scion interaction affected the mRNA transcript levels of several genes involved in Cd uptake, transport, and detoxification including HA7, FRO2-like, NRAMP1, NRAMP3, HMA4, MT2, NAS1, and ABCC1. Hence, the responses of grafted Malus plants to Cd toxicity vary with rootstock, scion, and rootstock-scion interaction.
Project description:Washington State produces about 70% of total fresh market apples in the United States. One of the primary goals of apple breeding programs is the development of new cultivars resistant to devastating diseases such as fire blight. The overall objective of this study was to investigate high-throughput phenotyping techniques to evaluate fire blight disease symptoms in apple trees. In this regard, normalized stomatal conductance data acquired using a portable photosynthetic system, image data collected using RGB and multispectral cameras, and visible-near infrared spectral reflectance acquired using a hyperspectral sensing system, were independently evaluated to estimate the progression of fire blight infection in young apple trees. Sensors with ranging complexity - from simple RGB to multispectral imaging to hyperspectral system - were evaluated to select the most accurate technique for the assessment of fire blight disease symptoms. The proximal multispectral images and visible-near infrared spectral reflectance data were collected in two field seasons (2016, 2017); while, proximal side-view RGB images and multispectral images using unmanned aerial systems were collected in 2017. The normalized stomatal conductance data was correlated with disease severity rating (<i>r</i> = 0.51, <i>P</i> < 0.05). The features extracted from RGB images (e.g., maximum length of senesced leaves, area of senesced leaves, ratio between senesced and healthy leaf area) and multispectral images (e.g., vegetation indices) also demonstrated potential in evaluation of disease rating (|<i>r</i>| > 0.35, <i>P</i> < 0.05). The average classification accuracy achieved using visible-near infrared spectral reflectance data during the classification of susceptible from symptomless groups ranged between 71 and 93% using partial least square regression and quadratic support vector machine. In addition, fire blight disease ratings were compared with normalized difference spectral indices (NDSIs) that were generated from visible-near infrared reflectance spectra. The selected spectral bands in the range 710-2,340 nm used for computing NDSIs showed consistently higher correlation with disease severity rating than data acquired from RGB and multispectral imaging sensors across multiple seasons. In summary, these specific spectral bands can be used for evaluating fire blight disease severity in apple breeding programs and potentially as early fire blight disease detection tool to assist in production systems.
Project description:Fast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in samples. In this study, LAMP assay that gives more consistent results for on-site diagnosis of fire blight than the previous developed LAMP assays was developed. Primers for new LAMP assay (named as DS-LAMP) were designed from a histidine-tRNA ligase gene (EAMY_RS32025) of E. amylovora CFBP1430 genome. The DS-LAMP amplified DNA (positive detection) only from genomic DNA of E. amylovora strains, not from either E. pyrifoliae (causing black shoot blight) or from Pseudomonas syringae pv. syringae (causing shoot blight on apple trees). The detection limit of DS-LAMP was 10 cells per LAMP reaction, equivalent to 104 cells per ml of the sample extract. DS-LAMP successfully diagnosed the pathogens on four fire-blight infected apple and pear orchards. In addition, it could distinguish black shoot blight from fire blight. The Bühlmann-LAMP, developed previously for on-site diagnosis of fire blight, did not give consistent results for specificity to E. amylovora and on-site diagnosis; it gave positive reactions to three strains of E. pyrifoliae and two strains of P. syringae pv. syringae. It also, gave positive reactions to some healthy sample extracts. DS-LAMP, developed in this study, would give more accurate on-site diagnosis of fire blight, especially in the Republic of Korea, where fire blight and black shoot blight coexist.
Project description:High-throughput amplicon sequencing spanning conserved portions of microbial genomes (16s rRNA and ITS) was used in the present study to describe the endophytic microbiota associated with three apple varieties, "Royal Gala," "Golden Delicious," and "Honey Crisp," and two rootstocks, M.9 and M.M.111. The objectives were to (1) determine if the microbiota differs in different rootstocks and apple varieties and (2) determine if specific rootstock-scion combinations influence the microbiota composition of either component.Results indicated that Ascomycota (47.8%), Zygomycota (31.1%), and Basidiomycota (11.6%) were the dominant fungal phyla across all samples. The majority of bacterial sequences were assigned to Proteobacteria (58.4%), Firmicutes (23.8%), Actinobacteria (7.7%), Bacteroidetes (2%), and Fusobacteria (0.4%). Rootstocks appeared to influence the microbiota of associated grafted scion, but the effect was not statistically significant. Pedigree also had an impact on the composition of the endophytic microbiota, where closely-related cultivars had a microbial community that was more similar to each other than it was to a scion cultivar that was more distantly-related by pedigree. The more vigorous rootstock (M.M.111) was observed to possess a greater number of growth-promoting bacterial taxa, relative to the dwarfing rootstock (M.9).The mechanism by which an apple genotype, either rootstock or scion, has a determinant effect on the composition of a microbial community is not known. The similarity of the microbiota in samples with a similar pedigree suggests the possibility of some level of co-evolution or selection as proposed by the "holobiont" concept in which metaorganisms have co-evolved. Clearly, however, the present information is only suggestive, and a more comprehensive analysis is needed.