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: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:Erwinia amylovora causes fire blight. Copper is widely used for fire blight management but there is limited information on the pathogen’s copper homeostasis mechanisms. Upon identifyingE. amylovora strains with unusually high (EaR2, Ea17) and intermediate (Ea19) copper sensitivity, we characterized them phenotypically to find potential correlations with other traits.The highly copper-sensitive strains EaR2 and Ea17 grew slower, showed increased sensitivity to paraquat and cadmium, and developed a characteristic copper-dependent overproduction of amylovoran and levan, with patterns not observed in strain, Ea273, with regular copper tolerance. Copper sensitivity was also associated with higher copper-shock death rates after copper pre-exposure during growth. RNA-Seq analysis revealed similar responses to copper-shock in EaR2 and Ea273 but very different transcriptomic responses during copper adaptation(prolonged growth with copper). EaR2 responded to copper adaptation with earlier activation ofstress responses, exopolysaccharide biosynthesis pathways, and protein quality control systems, while reducing the expression of genes linked to iron uptake. Ea273 mostly displayed an activation of copper homeostasis-related genes, with a characteristic downregulation of histidine catabolism.
Project description:The bacterial pathogen Erwinia amylovora is the causal agent of fire blight, an economically significant disease of apple and pear. Disease initiation by E. amylovora requires the translocation of effector proteins into host cells by the hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS). The alternate sigma factor HrpL positively regulates the transcription of structural and translocated components of the T3SS via hrp promoter elements. To characterize genome-wide hrpL-dependent gene expression in E. amylovora Ea1189, wild-type and Ea1189hrpL strains were cultured in hrp-inducing minimal medium, and total RNA was compared using a custom microarray designed to represent the annotated genes of E. amylovora ATCC 49946. Results revealed 24 genes differentially-regulated in Ea1189hrpL compared to Ea1189 with fold-change expression ratios greater than 1.5; of these, the expression of 19 genes was up-regulated while five genes exhibited negative regulation. To expand our understanding of the HrpL regulon and to elucidate direct versus indirect hrpL-mediated effects on gene expression, the genome of E. amylovora ATCC 49946 was examined in silico using a hidden Markov model assembled from known Erwinia spp. hrp promoters. This technique identified 21 putative type III novel hrp promoters; of these, 8+ were validated with quantitative polymerase chain reaction based on expression analyses. In total, hrpL-regulated genes encode all known components of the hrp T3SS and 5 putative type III effectors. Eight genes displayed apparent indirect hrpL regulation suggesting that the hrpL regulon is connected to downstream signaling networks. Construction of deletion mutants of three novel hrpL-regulated genes has resulted in the identification of additional virulence factors in E. amylovora as well as mutants displaying abnormal motility and biofilm phenotypes.
Project description:ra05-02_erwinia - erwinia - Identification of Arabidopsis genes regulataed by Erwinia amylovora and of a subset of Arabiddopsis genes regulated by the type three secretion system of Erwinia amylovora. Keywords: normal vs disease comparison
Project description:ra06-03_dspa-erwinia - dspa/e of erwinia amylovora - Identification of Arabidopsis genes regulated by the type three effector Dspa/E of Erwinia amylovora. - Regulation of the Arabidopsis transcriptome by the type three effector DspA/E of Erwinia amylovora. 5-week old Arabidopsis plants were leaf infiltrated with Erwinia amylovora wild-type (wt), type three secretion mutant (sec) or dspA/E mutant (dspA/E) strains. Keywords: wt vs mutant comparison
Project description:To investigate the metabolism by edodine treatment in Erwinia amylovora, E. amylovora grown with edodine. Total RNA from the cell pellets of E. amylovora TS3128 that cultured in MGY at 28 ºC with the control group and edodine treated group (IC50) was extracted. Our data provide differentially expression genes, including flagella biosynthesis, nutrient metabolisms, and protein folding, and RNA degradation, in presence of edodine.
Project description:Microarray analysis was used to identify genes that were controlled by AmyR in minimal and on immature pear fruits. Consistent with amylovoran production, an inverse correlation was observed between amyR expression and the expression level of amylovoran biosynthetic genes in liquid media. Interestingly, over-expression of AmyR suppressed the expression of type III secretion system genes including hrpA, hrpN and dspEF after pear fruit infection. Consistent with levan production and swarming motility, levasucrase and flagellar genes were both down-regulated both in the amyR mutant and over-expression strains in liquid media. Together, our results suggest that AmyR plays an important role in regulating bacterial exopolysaccharide production and virulence in E. amylovora. A total of 14 samples were analyzed in two conditions: For the in vitro condition (MBMA medium + 1% sorbitol), Erwinia amylovora wild type strain (2 replicates); E. amylovora amyR mutant strain (3 replicates); E. amylovora amyR over-expression strain (3 replicates); For the in vivo condition (on wounded immature pear fruits), Erwinia amylovora wild type strain (2 replicates); E. amylovora amyR mutant strain (2 replicates); E. amylovora amyR over-expression strain (2 replicates).
Project description:We conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and, for the first time, on immature pear fruit. Our array analyses identified a total of 648 genes differentially regulated by the RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls amylovoran biosynthetic gene expression in vivo, but negatively in vitro. Besides amylovoran biosynthesis and regulatory genes, cell wall and cell envelope (membrane) as well as regulatory genes were the major components of the RcsBC regulon, including many novel genes. In addition, we have also demonstrated that transcripts of rcsA, rcsC and rcsD genes, but not rcsB gene, were up-regulated when grown in minimal medium or after infection of pear fruits compared to LB medium. Furthermore, a hidden Markov model (HMM) has predicted 60 genes with candidate RcsB binding site in the intergenic regions of the E. amylovora ATCC 49946 genome and 18 (28) of them were identified in the microarray assay. Based on our findings, a working model has been proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora. A total of 12 samples were analyzed in two conditions: For in vitro condition (MBMA medium + 1% sorbitol), Erwinia amylovora wild type strain (2 replicates); E. amylovora rcsB mutant strain (2 replicates); E. amylovora rcsC mutant strain (2 replicates): For in vivo condition (on wounded immature pear fruits), Erwinia amylovora wild type strain (2 replicates); E. amylovora rcsB mutant strain (2 replicates); E. amylovora rcsC mutant strain (2 replicates).
Project description:ra06-03_dspa-erwinia - dspa/e of erwinia amylovora - Identification of Arabidopsis genes regulated by the type three effector Dspa/E of Erwinia amylovora. - Regulation of the Arabidopsis transcriptome by the type three effector DspA/E of Erwinia amylovora. 5-week old Arabidopsis plants were leaf infiltrated with Erwinia amylovora wild-type (wt), type three secretion mutant (sec) or dspA/E mutant (dspA/E) strains. Keywords: wt vs mutant comparison 6 dye-swap - CATMA arrays