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. 2020 Dec 13;22(2):255–270. doi: 10.1111/mpp.13024

FIGURE 6.

FIGURE 6

In vivo activation of RprA during host infection. (a) Confocal observation of rprA promoter activity in Erwinia amylovora Ea1189(pNptII‐gfp‐rprA‐mCherry) in LB medium and in flesh and ooze of inoculated immature pears. Gfp (ex/em = 488 nm/510 nm) is expressed constitutively, whereas mCherry (ex/em = 587 nm/610 nm) is expressed under the control of the promoter of rprA in Ea1189(pNptII‐gfp‐rprA‐mCherry) cultures. Images were captured through sequential scanning using a FluoView 1000 (Olympus) laser scanning confocal microscope. (b) Expression of rprA and several virulence factor genes in E. amylovora Ea1189 cells emerged in ooze from the inoculated immature pears and from cells that were grown overnight in LB medium. To ensure representativity of gene expression levels in E. amylovora cells from pear ooze, ooze from groups of six of the 18 inoculated pears were pooled together as one biological replication (labelled as “Ooze 1”, “Ooze 2”, and “Ooze 3”). Gene expression levels were quantified through quantitative reverse transcriptionPCR and fold changes were calculated using the 2−ΔΔ C t formula. The housekeeping gene recA was used as an endogenous control. Error bars indicate standard deviations of the mean within each biological replication