<em>Erwinia amylovora</em> Expresses Fast and Simultaneously <em>hrp</em>/<em>dsp</em> Virulence Genes during Flower Infection on Apple Trees

<div><h3>Background</h3><p>Pathogen entry through host blossoms is the predominant infection pathway of the Gram-negative bacterium <em>Erwinia amylovora</em> leading to manifestation of the disease fire blight. Like in other economically important plant pathogens, <em>E. amylovora</em> pathogenicity depends on a type III secretion system encoded by <em>hrp</em> genes. However, timing and transcriptional order of <em>hrp</em> gene expression during flower infections are unknown.</p> <h3>Methodology/Principal Findings</h3><p>Using quantitative real-time PCR analyses, we addressed the questions of how fast, strong and uniform key <em>hrp</em> virulence genes and the effector <em>dspA/E</em> are expressed when bacteria enter flowers provided with the full defense mechanism of the apple plant. In non-invasive bacterial inoculations of apple flowers still attached to the tree, <em>E. amylovora</em> activated expression of key type III secretion genes in a narrow time window, mounting in a single expression peak of all investigated <em>hrp/dspA/E</em> genes around 24–48 h post inoculation (hpi). This single expression peak coincided with a single depression in the plant <em>PR-1</em> expression at 24 hpi indicating transient manipulation of the salicylic acid pathway as one target of <em>E. amylovora</em> type III effectors. Expression of <em>hrp/dspA/E</em> genes was highly correlated to expression of the regulator <em>hrpL</em> and relative transcript abundances followed the ratio: <em>hrpA</em>><em>hrpN</em>><em>hrpL</em>><em>dspA/E</em>. Acidic conditions (pH 4) in flower infections led to reduced virulence/effector gene expression without the typical expression peak observed under natural conditions (pH 7).</p> <h3>Conclusion/Significance</h3><p>The simultaneous expression of <em>hrpL</em>, <em>hrpA</em>, <em>hrpN</em>, and the effector <em>dspA/E</em> during early floral infection indicates that speed and immediate effector transmission is important for successful plant invasion. When this delicate balance is disturbed, e.g., by acidic pH during infection, virulence gene expression is reduced, thus partly explaining the efficacy of acidification in fire blight control on a molecular level.</p>