Developmental characteristics of <i>M. oryzae</i> strains.

<p>Notes: The strains (5-mm mycelial blocks) were grown on CM medium for 8 days, and the diameter of colonies was then measured (growth) or conidia were collected and counted (conidiation). 40-µl (1×10⁵ conidia/ml) conidial suspensions were incubated on plastic slides for 4 hpi (conidial germination assay) and 24 hpi (appressorium formation assay).</p><p>*Same capital letters indicate non-significant difference estimated by Duncan's test in each developmental item (<i>P</i>≤0.05).</p><p>The experimental strains were the wild-type strain, the mutants (Δ<i>cca1</i>, Δ<i>conx1</i>, Δ<i>cnf1</i>, Δ<i>cnf2</i>, Δ<i>fzc16</i>, Δ<i>gcc1</i>, Δ<i>gpf1</i>, Δ<i>gta1</i>, Δ<i>Mocod1</i>, Δ<i>Monit4</i> and Δ<i>pcf1</i>) and complemented strains (<i>cca1-c</i>, <i>conx1-c</i>, <i>cnf1-c</i>, <i>cnf2-c</i>, <i>fzc16-c</i>, <i>gcc1</i>-c, <i>gpf1-c</i>, <i>gta1-c</i>, <i>Mocod1-c</i>, <i>Monit4-c</i> and <i>pcf1-c</i>).</p><p>Developmental characteristics of <i>M. oryzae</i> strains.</p

Mycelial appearance and spore-bearing aerial hyphae of the <i>M. oryzae</i> strains.

<p>(<b>A</b>) The mycelial appearance of the wild-type strain, Δ<i>cnf1</i> and its complemented strain <i>cnf1-c</i>. Stars indicate non-sporulating hyphae. Bar = 1 cm. (<b>B</b>) Spore-bearing aerial hyphae of the wild-type strain, Δ<i>cnf1</i> and its complemented strain <i>cnf1-c</i> on CM medium. Bar = 20 µm. (<b>C</b>) Conidial development on conidiophores of the <i>M. oryzae</i> strains Δ<i>cnf1</i> and Δ<i>cca1</i>. The conidiophore pictures of the wild-type, mutants (Δ<i>cnf1</i> and Δ<i>cca1</i>), and their complemented strains (<i>cnf1-c</i> and <i>cca1-c</i>) were taken on the strains grown on CM medium on microscope slides for 1 day. Bar = 40 µm.</p

Overview of the high-throughput gene knockout system in fungus.

<p>(<b>A</b>) Features of a new binary yeast-<i>Escherichia</i>-<i>Agrobacterium</i> shuttle vector, pKO1B. (<b>B</b>) Building of gene-deletion cassettes in pKO1B by yeast recombinational cloning. The 5′ and 3′ flanking fragments of the targeted genes were separately amplified from genomic DNA with primers 5f/5r and 3f/3r. Primers 5r and 3f have 5′ tails homologous to the <i>SUR</i> cassette, whereas those for 5f and 3r are homologous to the vector. The two flanks were cotransformed into yeast along with the <i>SUR</i> cassette and gapped pKO1B. Homologous recombination created the circular knockout vector, and the final knockout vector was subsequently transformed into <i>A. tumefaciens</i>. (<b>C</b>) Deletion of the targeted gene. The gene-deletion cassette was transformed into the fungal cells via ATMT. Homologous recombination created three types of transformants: null mutants, ectopic insertion transformants, and null and ectopic insertion mutants. The <i>GFP</i> gene was discarded in the null mutants. Primers p1/p2 or p3/p4 were used to identify the unique recombinational DNA fragment, indicating a knockout event. (<b>D</b>) The transformants are screened for GFP fluorescence under a microscope. Putative null mutants do have not GFP fluorescence, but ectopic transformants do. (<b>E</b>) The transformants are screened by double PCR for the targeted gene using the <i>β-tubulin</i> gene as a positive control. The wild-type strain or ectopic transformants produced a characteristic band, indicating the targeted gene, while the null mutants did not. (<b>F</b>) The transformants are screened by PCR for a unique recombinational DNA fragment marked as a knockout event. The null mutants have a 1.2–2.0 kb band on an electrophoretic gel, while the wild-type strain and the ectopic transformants do not.</p

Conidia, conidial germination and appressorium formation of the <i>M. oryzae</i> strain Δ<i>cca1</i>.

<p>The conidia (0 h) of the wild-type strain, mutant Δ<i>cca1</i>, and its complemented strain <i>cca1-c</i> were grown in H₂O on plastic cover slides for 4 h (conidial germination) and 24 h (appressorium formation). Bar = 10 µm.</p

Analysis of Zn₂Cys₆ transcription factor mutant phenotypes in fungal development stages.

<p>(<b>A</b>) Number of TFs showing multiple mutant phenotypes. (<b>B</b>) Number of mutants showing mutant phenotypes in each developmental stage. (<b>C</b>) Venn diagram showing the number of mutant phenotypes. The phenotypes included vegetative growth (conidial germination, colony growth, pigmentation and mycelial appearance), conidiation (asexual reproduction), appressorium formation and pathogenicity to rice and barley.</p

Pathogenicity assay of the <i>M. oryzae</i> strains.

<p>(<b>A</b>) Pathogenicity assay of 5 mutants and their complementation strains on rice. These 5 mutants are Δ<i>gpf1</i>, Δ<i>gta1</i>, Δ<i>cnf2</i>, Δ<i>cnf1</i>, Δ<i>cca1</i> and their complementation strains <i>gpf1-c</i>, <i>gta1-c</i>, <i>cnf2-c</i>, <i>cnf1-c</i>, and <i>cca1-c</i>. The rice seedlings were sprayed with a conidial suspension of <i>M. oryzae</i> strains and cultured for 7 days. (<b>B</b>) Pathogenicity assay of the mutants on rice leaf explants. The mycelial agar plugs of the mutants Δ<i>Mocod1</i> and Δ<i>conx1</i>, their complementation strains <i>Mocod1-c</i> and <i>conx1-c</i>, and the wild-type strain 70-15 were placed on intact rice leaves for 4 days. (<b>C</b>) Penetration assay. 10-or 20-µl (5×10⁴ conidia/ml) conidial suspensions were inoculated on onion cuticle or barley leaves (intact or abraded) and incubated for 24 or 48 h. The experimental strains were the wild-type strain 70-15, mutants (Δ<i>gpf1</i>, Δ<i>gta1</i>, Δ<i>cnf2</i>, Δ<i>cnf1</i> and Δ<i>cca1</i>) and complemented strains (<i>gpf1-c</i>, <i>gta1-c</i>, <i>cnf2-c</i>, <i>cnf1-c</i> and <i>cca1-c</i>). Same capital letters in same treatment item indicate non-significant difference estimated by Duncan's test (<i>P</i>≤0.05). (<b>D</b>) Penetration of Δ<i>gpf1</i>. 20 µl (5×10⁴ conidia/ml) conidial suspensions of Δ<i>gpf1</i>, complemented strain <i>gpf1-c</i> or wild-type strain were inoculated on barley leaf explants (intact or abraded) and incubated for 24 or 48 h. Arrows indicate the <i>in planta</i> hyphae invaded. Bar = 25 µm.</p

Growth of the <i>M. oryzae</i> strains under stress conditions.

<p>(<b>A</b>) Growth inhibition rate. 5-mm mycelial blocks of <i>M. oryzae</i> strains were inoculated on CM medium and medium with stress conditions for 8 days, and the diameter of colonies was then measured to calculate the growth inhibition rate. The experimental strains were the wild-type strain, mutants (Δ<i>Monit4</i>, Δ<i>gpf1</i>, Δ<i>gcc1</i>, Δ<i>tas1</i> and Δ<i>gta1</i>), and complemented strains (<i>Monit4-c</i>, <i>gpf1-c</i>, <i>gcc1-c</i>, <i>tas1-c</i> and <i>gta1-c</i>). Same capital letters in same stress item indicate non-significant difference estimated by Duncan's test (<i>P</i>≤0.05). (<b>B</b>) Mycelial growth of Δ<i>Monit4</i> on MM medium. The wild-type strain, Δ<i>Monit4</i>, and complemented strain <i>Monit4-c</i> were grown on MM medium at 25°C for 8 days. Bar = 5 mm.</p

Colony growth and conidiophores of <i>M. oryzae</i> strains.

<p>(<b>A</b>) Colony growth of the wild-type strain 70-15, mutants (Δ<i>gcc1</i>, Δ<i>gpf1</i> and Δ<i>gta1</i>), and complemented strains (<i>gcc1</i>-c, <i>gta1</i>-c, <i>gta1</i>-c) on CM medium for 8 days. Bar = 1 cm. (<b>B</b>) Conidiophores of the wild-type, mutants (Δ<i>cnf1</i>, Δ<i>cnf2</i>, Δ<i>pcf1</i>, Δ<i>cca1</i>, Δ<i>conx1</i>, Δ<i>gcc1</i> and Δ<i>Mocod1</i>), and complemented strains (<i>cnf1-c</i>, <i>cnf2-c</i>, <i>pcf1-c</i>, <i>cca1-c</i>, <i>conx1-c</i>, <i>gcc1-c</i> and <i>Mocod1-c</i>). Bar = 20 µm.</p

<i>GPF1</i>- or <i>CNF2</i>-dependent genes encoding known virulence factors.

<p>Note:</p>a<p>) The expression of genes increased or decreased in Δ<i>gpf1</i> and Δ<i>cnf2</i>.</p><p>The number means the fold change, and the symbol “−” means no significant change (FDR<0.05) afterwards compared with the wild-type strain.</p><p><i>GPF1</i>- or <i>CNF2</i>-dependent genes encoding known virulence factors.</p

Comparison of differentially expressed genes between Δ<i>gpf1</i> and Δ<i>cnf2</i>.

<p>Green or red arrows are the directions of gene expression regulation in Δ<i>gpf1</i> or Δ<i>cnf2</i>.</p