Biphenyl and dibenzofuran levels in transition zones.

<p>Concentrations were determined in the transition zones of fire blight-infected stems of 'Conference' and 'Harrow Sweet' at two time points post-inoculation (dpi). Data are average values ± SD of three independent experiments. Compounds are numbered according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158713#pone.0158713.g004" target="_blank">Fig 4</a>.</p

GC-MS analysis of biphenyls and dibenzofurans.

<p>The methanolic extracts studied were prepared from shoots of 'Harrow Sweet' 28 d after either infection with <i>E</i>. <i>amylovora</i> (a) or mock-inoculation (b). Compounds were separated as trimethylsilyl (TMS) derivatives named 1a–6a. IS, internal standard (4-phenylphenol), ^†sugar derivatives, *fatty acid derivatives.</p

Neighbour-joining tree of PcBISs and MdBISs.

<p>The amino acid sequences were used for tree construction by MEGA 6.0. Numbers at the forks are percentage values of the bootstrap test (500 replicates). The accession numbers of the genes are: <i>MdBIS1</i>, JQ390521; <i>MdBIS2</i>, JQ390522; <i>MdBIS3</i>, JQ390523; <i>MdBIS4</i>, JQ390524.</p

Array of phytoalexins in 'Conference' and 'Harrow Sweet'.

<p>Methanolic extracts stem from shoots collected 28 d after inoculation with <i>E</i>. <i>amylovora</i>.</p

Chemical structures of biphenyls and dibenzofurans.

<p>The compounds depicted were detected in <i>E</i>. <i>amylovora</i>-inoculated shoots of 'Conference' and 'Harrow Sweet'.</p

Phytopathological changes and <i>PcBIS</i> expression.

<p>Morphological changes (A) and expression of <i>PcBIS1</i> and <i>PcBIS2</i> (B-E) were studied in three pear cultivars after inoculation with <i>E</i>. <i>amylovora</i>. B: healthy leaf, C: leaf 1 (collectively necrotic leaves), D: leaf 2 (next leaf with a necrotic principal vein), E: transition zone (4-cm-stem segment between the necrotic and healthy parts).</p

Expression of Biphenyl Synthase Genes and Formation of Phytoalexin Compounds in Three Fire Blight-Infected <i>Pyrus communis</i> Cultivars

<div><p>Pear (<i>Pyrus communis</i>) is an economically important fruit crop. Drops in yield and even losses of whole plantations are caused by diseases, most importantly fire blight which is triggered by the bacterial pathogen <i>Erwinia amylovora</i>. In response to the infection, biphenyls and dibenzofurans are formed as phytoalexins, biosynthesis of which is initiated by biphenyl synthase (BIS). Two PcBIS transcripts were cloned from fire blight-infected leaves and the encoded enzymes were characterized regarding substrate specificities and kinetic parameters. Expression of <i>PcBIS1</i> and <i>PcBIS2</i> was studied in three pear cultivars after inoculation with <i>E</i>. <i>amylovora</i>. Both <i>PcBIS1</i> and <i>PcBIS2</i> were expressed in ‘Harrow Sweet’, while only PcBIS2 transcripts were detected in ‘Alexander Lucas’ and ‘Conference’. Expression of the <i>PcBIS</i> genes was observed in both leaves and the transition zone of the stem; however, biphenyls and dibenzofurans were only detected in stems. The maximum phytoalexin level (~110 μg/g dry weight) was observed in the transition zone of ‘Harrow Sweet’, whereas the concentrations were ten times lower in ‘Conference’ and not even detectable in ‘Alexander Lucas’. In ‘Harrow Sweet’, the accumulation of the maximum phytoalexin level correlated with the halt of migration of the transition zone, whereby the residual part of the shoot survived. In contrast, the transition zones of ‘Alexander Lucas’ and ‘Conference’ advanced down to the rootstock, resulting in necrosis of the entire shoots.</p></div

Biosynthesis of biphenyls and dibenzofurans.

<p>The biosynthetic pathway was proposed for phytoalexin formation in elicitor-treated <i>S</i>. <i>aucuparia</i> cell cultures [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158713#pone.0158713.ref013" target="_blank">13</a>]. Solid arrows, established reactions; broken arrows, postulated reactions. BIS, biphenyl synthase; OMT, <i>O</i>-methyltransferase; B4H, biphenyl 4-hydroxylase.</p

Primers used for RT-PCR (a) and qRT-PCR (b).

<p>Primers used for RT-PCR (a) and qRT-PCR (b).</p

Transcript levels of PcBISs.

<p>Using RT-qPCR, relative transcript levels of PcBIS1 and PcBIS2 were determined in leaf samples and transition zones of three pear cultivars at day 5, 8, and 12 after inoculation with <i>E</i>. <i>amylovora</i>. Control mRNA was obtained from leaves and stems of mock-inoculated shoots (0 dpi). Leaf 1, collectively necrotic leaves; leaf 2, next leaf with a necrotic principal vein; transition zone, 4-cm-stem segment between the necrotic and healthy parts.</p