Project description:Apple leaf spot caused by the Alternaria alternata f. sp. mali (ALT1) fungus is one of the most devastating diseases of apple (Malus × domestica). We identified a hairpin RNA (hpRNA)-mediated small RNAs, MdhpRNA277, from apple (cv. ‘Golden Delicious’) that is induced by infection with ALT1. MdhpRNA277 produces mdm-siR277-1 and mdm-siR277-2, which target five R genes, MdRNL1, MdRNL2, MdRNL3, MdRNL4, and MdRNL5, that are expressed at high levels in the resistant apple variety ‘Hanfu’ and at low levels in the susceptible variety ‘Golden Delicious’ following ALT1 infection. MdhpRNA277 is strongly induced in ‘Golden Delicious’ but was not induced in ‘Hanfu’ following ALT1 inoculation. The promoter activity of MdhpRNA277 was much stronger in ‘Golden Delicious’ than in ‘Hanfu’ after ALT1 inoculation. We identified a single nucleotide polymorphism (SNP) in the MdhpRNA277 promoter region between the susceptible variety ‘Golden Delicious’ (pMdhpRNA277-GD) and resistant variety ‘Hanfu’ (pMdhpRNA277-HF). The transcription factor MdWHy binds to pMdhpRNA277-GD, but not to pMdhpRNA277-HF. Transgenic ‘GL-3’ apple lines expressing pMdhpRNA277-GD: MdhpRNA277 were more susceptible to ALT1 infection than were those expressing pMdhpRNA277-HF:MdhpRNA277 due to induced mdm-siR277 accumulation and low levels of expression of the five target R genes. The failure of MdWHy to bind to pMdhpRNA277-HF might contribute to the low levels of MdhpRNA277 and mdm-siR277-1/-2 expression and the high levels of R gene expression and resistance to Alternaria leaf spot in resistant apple varieties. We confirmed that the SNP in pMdhpRNA277 is associated with Alternaria leaf spot resistance by analyzing the progeny of three additional crosses. The SNP identified in this study could be used as a marker to distinguish between apple varieties that are resistant or susceptible to Alternaria leaf spot.
Project description:RNA sequencing was applied to compare the transcriptome profiles of conidium, appressoruim, infected apple leaf (IL), and cellophane infectious hyphae (CIH)
Project description:We performed Illumina sequencing of sRNA libraries prepared from juvenile and reproductive phase buds from the apple trees. A large number of sRNAs exemplified by 33 previously annotated miRNAs and 6 novel members displayed significant differential expression (DE) patterns in juvenile and reproductive stages. The study provides new insight into our understanding of fundamental mechanism of poorly studied phase transitions in apple and other woody plants and important resource for future in-depth research in the apple development.
Project description:Drought stress is a major problem around the world and although progress in understanding how vegetable crops and model plants adapt to drought have been made, there is still little information about how fruit crops deal with moderate drought stress. In this study, we investigated the response of two apple genotypes: a drought-sensitive genotype (M26) and a drought-tolerant genotype (MBB). Our results of the morphology, physiology and biochemistry under moderate drought stress, indicated that relative water content (RWC) and leaf area (LA) were not significant changes in two genotypes. However, it had larger leaf mass per area (LMA), and accumulated higher free proline (CFP), soluble sugars (CSS) and malonaldehyde (MDA) in the leaves. Thus, it appears that the MBB genotype could produce more osmosis-regulating substances. Phosphoproteomic was analyzed from leaves of both genotypes under moderate drought stress using the isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 595 unique phosphopeptides, 682 phosphorylated sites and 446 phosphoproteins were quantitatively analyzed in the two genotypes. Motif analyses of the phosphorylation sites showed that six motifs including [PxsP], [sP], [sD], [Rxxs], [sxP] and [sxs] were enriched. We identified 12 and 48 PLSC phosphoproteins in M26 and MBB, respectively. Among these, 9 PLSC phosphoproteins were common to both genotypes, perhaps indicating a partial overlaps of the mechanisms to moderate drought stress. Gene ontology analyses revealed that the PLSC phosphoproteins present a unique combination of metabolism, transcription, translation and protein processing, suggesting that the response in apple to moderate drought stress encompasses a new homeostasis of major cellular processes. The basic trend was an increase in protein abundance related to drought and organic substance upon moderate drought stress between two genotypes. These increases were higher in the drought-tolerant genotype (MBB) than in the drought-sensitive genotype (M26). The 23 differentially expressed mRNA encoding phosphoproteins were analysis by quantitative real-time PCR (qRT-PCR). Our study is the first to address the phosphoproteome of a major fruit crop, apple rootstocks, in response to moderate drought stress, and provide insights into the molecular regulation mechanisms of apple rootstock under moderate drought stress.
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: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.