Pathogen relative fungal abundance and disease severity scores in each pathosystem.

<p>(A) <i>P</i>. <i>deltoides</i> inoculated with <i>S</i>. <i>musiva</i>. (B) <i>P</i>. <i>balsamifera</i> inoculated with <i>S</i>. <i>populicola</i>. (C) <i>P</i>. <i>tremuloides</i> inoculated with Ston1. Fungal DNA abundance relative to plant DNA measured by qPCR shown in log scale. Each data point represents the mean value of 3 samples. Bars with the same letter are not significant at p < 0.05 using ANOVA and Tukey’s HSD test.</p

Selection of poplar Kunitz-type trypsin inhibitor and WRKY family genes expressed in each poplar species following inoculation with <i>Sphaerulina</i> spp.

<p>Selection of poplar Kunitz-type trypsin inhibitor and WRKY family genes expressed in each poplar species following inoculation with <i>Sphaerulina spp</i>. Numbers in bold show statistical significance at FDR < 0.05.</p><p>Selection of poplar Kunitz-type trypsin inhibitor and WRKY family genes expressed in each poplar species following inoculation with <i>Sphaerulina</i> spp.</p

Transcriptome Analysis of Poplar during Leaf Spot Infection with <i>Sphaerulina</i> spp.

<div><p>Diseases of poplar caused by the native fungal pathogen <i>Sphaerulina musiva</i> and related species are of growing concern, particularly with the increasing interest in intensive poplar plantations to meet growing energy demands. <i>Sphaerulina musiva</i> is able to cause infection on leaves, resulting in defoliation and canker formation on stems. To gain a greater understanding of the different responses of poplar species to infection caused by the naturally co-evolved <i>Sphaerulina</i> species, RNA-seq was conducted on leaves of <i>Populus deltoides</i>, <i>P</i>. <i>balsamifera</i> and <i>P</i>. <i>tremuloides</i> infected with <i>S</i>. <i>musiva</i>, <i>S</i>. <i>populicola</i> and a new undescribed species, Ston1, respectively. The experiment was designed to contain the pathogen in a laboratory environment, while replicating disease development in commercial plantations. Following inoculation, trees were monitored for disease symptoms, pathogen growth and host responses. Genes involved in phenylpropanoid, terpenoid and flavonoid biosynthesis were generally upregulated in <i>P</i>. <i>balsamifera</i> and <i>P</i>. <i>tremuloides</i>, while cell wall modification appears to play an important role in the defense of <i>P</i>. <i>deltoides</i>. Poplar defensive genes were expressed early in <i>P</i>. <i>balsamifera</i> and <i>P</i>. <i>tremuloides</i>, but their expression was delayed in <i>P</i>. <i>deltoides</i>, which correlated with the rate of disease symptoms development. Also, severe infection in <i>P</i>. <i>balsamifera</i> led to leaf abscission. This data gives an insight into the large differences in timing and expression of genes between poplar species being attacked by their associated <i>Sphaerulina</i> pathogen.</p></div

Comparison of gene expression levels between RNA-seq and reverse transcript qPCR by comparing gene expression at 0 and 15 DAI.

<p>(A) <i>P</i>. <i>deltoides</i> infected with <i>S</i>. <i>musiva</i>, (B) <i>P</i>. <i>balsamifera</i> infected with <i>S</i>. <i>populicola</i> and (C) <i>P</i>. <i>tremuloides</i> infected with Ston1. Labels indicate the <i>P</i>. <i>trichocarpa</i> v3 gene ID. Error bars show standard deviation. * represents significant difference in gene expression between 0 and 15 DAI with RT-qPCR measured by paired t-test at p < 0.05 and RNAseq measured by EdgeR at FDR < 0.05.</p

Lignin biosynthesis pathway with heat maps showing <i>P</i>. <i>deltoides</i> gene expression in response to <i>S</i>. <i>musiva</i> at 15 DAI.

<p>Log2 fold ratios are represented by a colour gradient, with blue being upregulated and yellow being downregulated. Graphic regenerated from MapMan 3.5.1R2 lignin synthesis.png mapped to <i>P</i>. <i>trichocarpa</i> v3.0_210_peptide.m02. 4CL, 4 coumaroyl-CoA ligase; CCoAMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; COMT, caffeate O-methyltransferase; CAD, cinnamoyl alcohol dehydrogenase.</p

Significantly expressed genes at 1 DAI that have GO annotations to defense.

<p>Numbers in bold show statistical significance at FDR < 0.05.</p><p>Significantly expressed genes at 1 DAI that have GO annotations to defense.</p

Leaf abscission in <i>P</i>. <i>balsamifera</i> in response to inoculation with <i>S</i>. <i>populicola</i> at 35 DAI.

<p>(A) Control and inoculated trees at 35 DAI. (B) Number of leaves of control and inoculated trees at 35 DAI grouped by leaf harvest time point. (C) Plant height of control and inoculated trees at 35 DAI grouped by leaf harvest time point. Mock indicates non-inoculated control trees at 0 DAI. Error bars show standard deviation. Bars with the same letter are not significant at p < 0.05 using ANOVA and Tukey’s HSD test.</p

Control and inoculated leaves at 30 DAI.

<p>Pictures show the adaxial surface of a fourth fully expanded leaf at time of inoculation. (A) <i>P</i>. <i>deltoides</i> inoculated with <i>S</i>. <i>musiva</i>. (B) <i>P</i>. <i>balsamifera</i> inoculated with <i>S</i>. <i>populicola</i>. (C) <i>P</i>. <i>tremuloides</i> inoculated with Ston1.</p

Differentially regulated secondary metabolite pathways expressed at different time points.

<p>(A) <i>P</i>. <i>deltoides</i> infected with <i>S</i>. <i>musiva</i>, (B) <i>P</i>. <i>balsamifera</i> infected with <i>S</i>. <i>populicola</i> and (C) <i>P</i>. <i>tremuloides</i> infected with Ston1. Log2 fold ratios are represented by a colour gradient, with blue being upregulated and yellow being downregulated. The pathways shown have a p < 0.05 after Benjamini-Hochberg correction. N.S. are not significantly regulated. Partial heat map shown for lignin biosynthesis pathway.</p

Differentially expressed secondary metabolism pathways between control and inoculated poplar trees during infection with <i>Sphaerulina</i> spp.

<p>Analysis performed using Wilcoxon rank sum test with Benjamini-Hochberg correction at p < 0.1. DAI = days after inoculation; P-values in bold are significant at p ≤ 0.1</p><p>Differentially expressed secondary metabolism pathways between control and inoculated poplar trees during infection with <i>Sphaerulina</i> spp.</p