Generation of ∆<i>Cshog1</i> strains of <i>Bipolaris sorokiniana</i>.

<p>A, a diagram showing replacement of the <i>CsHOG1</i> gene by a 2.6 kb fragment carrying the <i>E</i>. <i>coli</i> hygromycin phosphotransferase gene (<i>hph</i>) using the split-marker system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128291#pone.0128291.ref017" target="_blank">17</a>]. B, Southern hybridization of <i>Xba</i> I-digested genomic DNA from the wild type and ∆<i>Cshog1</i> strains using probe amplified with primers CsHOG1-F1+CsHOG1-F2. The 1.7 kb fragment in the wild type strain (ND90Pr) was replaced by the 3.3 kb fragment in the ∆<i>Cshog1</i> strains (∆<i>Cshog1</i>-2 and ∆<i>Cshog1</i>-4). <i>ect</i>-<i>Cshog1</i>-3 is an ectopic transformant.</p

Primers used in this study.

<p>*Underlined sequences are complementary to M13F and M13R sequences, respectively.</p><p>Primers used in this study.</p

Fungal development and conidial productivity of knockout mutants.

<p>A, fungal growth and responses to stresses. Colonies were grown on PDA, PDA with 10mM H₂O₂, PDA with 1 M KCl and PDA with 1.5 M sorbitol, respectively, starting with mycelial plugs of uniform size inoculated on the centers of the plates. Photos were taken after growth for 6 days at 25°C. B, Quantitative growth rate of wild-type and mutants grown on PDA, PDA with 1 M KCl, PDA with 1.5 M sorbitol and PDA with 10mM H₂O₂, respectively. The colony diameters were measured 6 days after growth on the media. Error bars indicate standard deviation. C, Conidial productivity of wild-type and mutants grown on minimal medium plates for 6 days at 25°C in a cycle of 14 h of light and 10 h of darkness. Error bars indicate standard deviation. Significant differences (P value = 0.001) are indicated by different letters above the columns under each condition.</p

Infection structure differentiation of the wild type (ND90Pr) (A), <i>∆Csfus3-1</i> (B), <i>∆Csste11-1</i> (C), <i>∆Cshog1-4</i> (D) and <i>∆Csslt2-10</i> (E) strains at 24 hours after inoculation (HAI) on intact leaves of barley cv.

<p><b>Bowman.</b> Fungal and dead plant cells were stained blue with trypan blue. Appressoria are indicated by black arrows. Bars = 60 μm.</p

Pathogenicity test of mutants on barley leaves.

<p>A, Point-inoculation with mycelial plugs of wild type (ND90Pr), <i>∆Csfus3-1</i> and <i>∆Csste11-1</i> mutants on non-wounded (Wt) and wounded (W) leaves of barley cv. Bowman. B, Spray inoculation of conidia of wild type (ND90Pr), <i>∆Cshog1-4</i> and <i>∆Csslt2-10</i> mutants on intact leaves of barley cv. Bowman. C. Quantitative analysis of disease severity based on lesion lengths on inoculated leaves as shown in A. All photographs were taken at 5 days after inoculation (DAI). A syringe needle was used to make wounds on leaves for inoculation with small mycelial plugs (A). Error bars indicate standard deviation. Significant differences (P value = 0.001) are indicated by different letters above the columns.</p

Pathogenicity test on barley roots.

<p>Roots (15cm long) of barley seedlings (cv. Bowman) were inoculated with mycelial plugs (2×2 mm²) of wild type (ND90Pr) and mutants according to the method described by Liljeroth et al. (23). The length of brown discoloration lesions was measured at 9 days after inoculation. Error bars indicate standard deviation. Significant differences (P value = 0.001) are indicated by different letters above the columns.</p

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of <i>in vitro</i> up-regulated genes.

<p>Values with log2 fold change >1 and false discovery rate (<0.01) were considered as differentially expressed. Only pathways having at least two genes up-regulated on either of the population are shown. Detailed information on KEGG pathway analysis is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163803#pone.0163803.s001" target="_blank">S1 Table</a>.</p

Venn diagram of exclusively up-regulated genes in 3ADON population compared to 15ADON population <i>in planta</i>.

<p>HAI: hours after inoculation, 3ADON: population producing 3-acetyl-deoxynivalenol and DON, 15ADON: population producing 15-acetyl-deoxynivalenol and DON. Information of genes corresponding to the Venn diagram are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163803#pone.0163803.s002" target="_blank">S2 Table</a>.</p

Functional analysis of <i>in planta</i> up-regulated genes in the 3ADON and 15ADON populations.

<p>The uniquely upregulated genes either in the 3ADON or the 15ADON population (all time points combined) were used for functional categorization. Total numbers of gene found in MIPS catalogue are listed in parenthesis. The functional categories in which members are significantly enriched compared with the whole genome are marked with asterisks (p < 0.05, FDR < 0.05). Information of these genes and their functional annotation are given on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163803#pone.0163803.s004" target="_blank">S4 Table</a>.</p

Differentially expressed <i>Fusarium graminearum</i> genes in 3ADON population showing at least 5-fold greater expression than 15ADON population <i>in planta</i> or compared to corresponding <i>in vitro</i> expression.

<p>Differentially expressed <i>Fusarium graminearum</i> genes in 3ADON population showing at least 5-fold greater expression than 15ADON population <i>in planta</i> or compared to corresponding <i>in vitro</i> expression.</p