Abnormal conidial morphology, appressorial formation, and pathogenicity of the MgRac1 dominant mutants.

<p>(A) Differential interference contrast (DIC) microscopy of conidia collected from WT (70-15), <i>MgRac1-CA</i> (constitutively active mutant), and <i>MgRac1-DN</i> (dominant negative mutant), as indicated. Bar = 20 µm. (B) Conidial suspensions of <i>MgRac1-CA and MgRac1-DN</i> were applied on the hydrophobic side of Gelbond film and examined with DIC microscopy. Bar = 20 µm. (C) Conidial suspensions (about 1,000 conidia in 20 µl) of 70-15 and MgRac1 mutants were inoculated on strips of onion epidermis. Infectious hyphae were photographed 2 days after inoculation with DIC microscopy. A = appressorium, C = conidium, H = hypha, IF = infectious hypha. Bar = 20 µm. (D) Leaves of rice cultivar CO39 were sprayed with conidial suspensions (1×10⁵ conidia/ml) from WT, <i>MgRac1-CA</i>, and <i>MgRac1-DN</i>. Typical leaves were photographed at 7 days after inoculation. (E) Disease symptoms on the wounded leaf tissues of rice inoculated with conidia (5×10⁴ conidia/ml) from WT and MgRac1 mutants, as indicated. And unwounded rice leaf tissue was inoculated with the mutant of <i>MgRac1-OE</i>. Typical leaves were photographed 5 days after inoculation.</p

Construction and confirmation of the <i>Mgrac1</i> deletion mutant.

<p>(A) Restriction map of the MgRac1 genomic region and deletion construct pKRA1. Thick arrows indicate orientations of the MgRac1 and hygromycin phosphotransferase (<i>hph</i>) genes. The restriction enzymes are abbreviated as X (<i>Xho</i>I), H (<i>Hin</i>dIII), and Sa (<i>Sac</i>I). The Mgrac1 deletion construct pKRA1 contained the homologous sequences flanking the <i>hph</i> gene to replace the first 525-bp of the MgRac1 ORF. Primers 4F and 4R (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000202#ppat-1000202-t004" target="_blank">Table 4</a>) were used for screening the Mgrac1 deletion mutants. (B) Total genomic DNA samples (5 µg per lane) isolated from WT (wild-type strain 70-15), <i>ΔMgrac1-19</i> (Mgrac1 deletion mutant), <i>ΔMgrac1-21</i> (Mgrac1 deletion mutant), and <i>Ect</i> (Ectopic transformant) were digested with <i>Pst</i>I and subjected to Southern blot analysis. The first probe, a 525-bp PCR fragment amplified from the genomic DNA of wild-type strain 70-15 using primers 10F and 10R (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000202#ppat-1000202-t004" target="_blank">Table 4</a>), is exactly the MgRac1 fragment replaced by the 2.6-kb hph gene and detects only the WT and <i>Ect</i> (top panel). The same blot was then stripped and re-hybridized with a 673-bp probe amplified from the 70-15 genomic DNA by primers 11F and 11R (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000202#ppat-1000202-t004" target="_blank">Table 4</a>) and this probe detects both WT and mutant DNA fragments, with the two deletion mutants showing a larger fragment due to the gene replacement (bottom panel). (C) Total RNA samples (approximately 1 µg per reaction) isolated from mycelia of WT, <i>ΔMgrac1-19</i> and <i>Mgrac1-Com</i> (MgRac1 complementary transformant) were subjected to RT-PCR using MgRac1 gene-specific primers 1F and 1R (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000202#ppat-1000202-t004" target="_blank">Table 4</a>). The RT-PCR product is a 600-bp fragment in WT and <i>Mgrac1-Com</i> as predicted, but is missing in the deletion mutant <i>ΔMgrac1-19</i>.</p

The interaction between MgRac1 and Chm1 or Chm1<i>Δ</i><i>PBD</i> and PAK activity assay.

<p>(A) Yeast two-hybrid assay with MgRac1-CA or MgRac1-DN as the bait and Chm1 or Chm1<i>ΔPBD</i> as the prey. Yeast transformants grown on the SD-Leu-Trp plates were assayed for β-galactosidase activity. The interaction of pGBKT7-53 and pGADT7-T was used as the positive control. The interaction of BD-MgRac1(CA) or BD-MgRac1(DN) and AD (pGADT7) was used as the negative control to rule out self-activation. (B) The indicated yeast transformants diluted to specified concentrations (cell/ml) were plated onto SD-Ade-Leu-Trp-His to examine the <i>HIS3</i> reporter gene expression in the yeast two-hybrid assay. The interaction of pGBKT7-Lam and pGADT7-T was used as the negative control. (C) Model of Chm1 activation and its auto-inhibition by the PBD domain. It involves transition between low-activity (closed) and high-activity (open) conformations. The PBD domain (grey) contains domains that bind MgRac1 and the PAK kinase domain, as indicated. (D) PAK kinase assay showing correlation of MgRac1 and PAK activity in the hyphae of WT and mutants. Total protein preparations were subjected to the kinase assay, which used the HTScan PAK1 kinase assay kit for direct ELISA detection of the product at the absorbance of 450 nm. Means and standard deviation calculated from three replicates were shown on the bar chart.</p

Abnormal conidial morphology, appressorial formation, and hyphal branching in the Mgrac1 deletion mutant.

<p>(A) Differential interference contrast (DIC) microscopy of conidia cultured on an oatmeal agar plate at day 10 after incubation. BA = basal appendage where conidia attach to conidiophores. Bar = 20 µm. (B) Conidia incubated on the surface of artificial hydrophobic Gelbond films as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000202#s4" target="_blank">Materials and Methods</a>. Bar = 20 µm. (C) Branching patterns of mycelia on complete media plates at day 3 after incubation. Frequent branching occurs at the terminal mycelia of <i>ΔMgrac1-19</i>. Calcofluor staining of mycelia is used to show the distance of septa. Bar = 20 µm. (D) DAPI staining of mycelia to show the localization of nuclei. Bar = 20 µm.</p

Real-time RT-PCR quantification of the transcripts of <i>Rac1</i>, <i>Cdc42</i>, <i>Chm1</i>, <i>Nox1</i>, and <i>Nox2</i> in different <i>Magnaporthe grisea</i> mutants.

a<p>Relative quantity of the indicated transcripts in the mutant strains, relative to that in the wild-type strain 70-15. A value of greater than 1 indicates increased expression, while a value of smaller than 1 indicates decreased expression. Mean and standard deviation were calculated with the data from three replicates.</p

<i>AtWuschel</i> Promotes Formation of the Embryogenic Callus in <i>Gossypium hirsutum</i>

<div><p>Upland cotton (<i>Gossypium hirsutum</i>) is one of the most recalcitrant species for <i>in vitro</i> plant regeneration through somatic embryogenesis. Callus from only a few cultivars can produce embryogenic callus (EC), but the mechanism is not well elucidated. Here we screened a cultivar, CRI24, with high efficiency of EC produce. The expression of genes relevant to EC production was analyzed between the materials easy to or difficult to produce EC. Quantitative PCR showed that CRI24, which had a 100% EC differentiation rate, had the highest expression of the genes <i>GhLEC1</i>, <i>GhLEC2</i>, and <i>GhFUS3</i>. Three other cultivars, CRI12, CRI41, and Lu28 that formed few ECs expressed these genes only at low levels. Each of the genes involved in auxin transport (<i>GhPIN7</i>) and signaling (<i>GhSHY2</i>) was most highly expressed in CRI24, with low levels in the other three cultivars. WUSCHEL (WUS) is a homeodomain transcription factor that promotes the vegetative-to-embryogenic transition. We thus obtained the calli that ectopically expressed <i>Arabidopsis thaliana Wus</i> (<i>AtWus</i>) in <i>G. hirsutum</i> cultivar CRI12, with a consequent increase of 47.75% in EC differentiation rate compared with 0.61% for the control. Ectopic expression of <i>AtWus</i> in CRI12 resulted in upregulation of <i>GhPIN7</i>, <i>GhSHY2</i>, <i>GhLEC1</i>, <i>GhLEC2</i>, and <i>GhFUS3</i>. <i>AtWus</i> may therefore increase the differentiation potential of cotton callus by triggering the auxin transport and signaling pathways.</p></div

AtWus overexpression results in abnormal development of somatic embryos.

<p><b>A:</b> Many abnormal somatic embryos were produced in 35S:WUS lines, and the somatic embryos were inflated and lacked cotyledons. <b>B:</b> Formation of normal somatic embryos in CK lines at different stages. Scanning electron microscopy: Holistic perspective of somatic embryos in 35S:WUS lines (<b>C</b>) and CK lines (<b>D</b>). <b>E</b>–<b>J:</b> Normal somatic embryos at different stages. <b>E</b>, <b>F:</b> globular embryo. <b>G:</b> heart-shape embryo. <b>H</b>–<b>J:</b> cotyledonary embryo. <b>K</b>–<b>P:</b> Abnormal somatic embryos having various appearance. <b>O:</b> leaf-like embryo. <b>P:</b> multiple-cotyledon embryo. Bar in <b>A</b> or <b>B,</b> 1 cm.</p