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

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

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

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

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

Primers used in this study.

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

Expression of genes involved in biosynthesis of secondary metabolites.

<p>Expression level is the average of two replications.</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

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