Rac1 Is Required for Pathogenicity and Chm1-Dependent Conidiogenesis in Rice Fungal Pathogen Magnaporthe grisea

Rac1 is a small GTPase involved in actin cytoskeleton organization and polarized cell growth in many organisms. In this study, we investigate the biological function of MgRac1, a Rac1 homolog in Magnaporthe grisea. The Mgrac1 deletion mutants are defective in conidial production. Among the few conidia generated, they are malformed and defective in appressorial formation and consequently lose pathogenicity. Genetic complementation with native MgRac1 fully recovers all these defective phenotypes. Consistently, expression of a dominant negative allele of MgRac1 exhibits the same defect as the deletion mutants, while expression of a constitutively active allele of MgRac1 can induce abnormally large conidia with defects in infection-related growth. Furthermore, we show the interactions between MgRac1 and its effectors, including the PAK kinase Chm1 and NADPH oxidases (Nox1 and Nox2), by the yeast two-hybrid assay. While the Nox proteins are important for pathogenicity, the MgRac1-Chm1 interaction is responsible for conidiogenesis. A constitutively active chm1 mutant, in which the Rac1-binding PBD domain is removed, fully restores conidiation of the Mgrac1 deletion mutants, but these conidia do not develop appressoria normally and are not pathogenic to rice plants. Our data suggest that the MgRac1-Chm1 pathway is responsible for conidiogenesis, but additional pathways, including the Nox pathway, are necessary for appressorial formation and pathogenicity

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

The interaction between MgRac1 and Nox1/Nox2 and superoxide production in MgRac1 mutants.

<p>(A) Yeast two-hybrid assay with <i>MgRac1-CA</i> or <i>MgRac1-DN</i> as the bait and Nox1 or Nox2 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) and AD (pGADT7) as well as BD (pGBKT7) and AD-Nox1/2 were used as negative controls to rule out self-activation. (B) Bar chart showing mean pixel intensity in hyphal tips and conidia of WT (70-15) and MgRac1 mutants, which quantifies the results in (C) and (D). Increased staining by NBT means reduced pixel intensity. Error bar means standard deviation based on the data of three independent experiments. (C) Detection of superoxide production by 0.6 mM NBT staining in the hyphal tips of WT and MgRac1 mutants. Bar = 10 µm. (D) Detection of superoxide production by 0.3 mM NBT staining in the conidia of WT and MgRac1 mutants. Bar = 10 µm.</p

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

Superoxide production and pathogenicity of <i>Nox</i> over-expression mutants.

<p>(A) Bar chart showing mean pixel intensity in hyphal tips and conidia of 70-15 (wild-type strain), <i>ΔMgrac1-19</i> (the Mgrac1 deletion mutant), <i>NOR1</i> (<i>NOX1</i> over-expressed in <i>ΔMgrac1-19</i>), and <i>NOR2</i> (<i>NOX2</i> over-expressed in <i>ΔMgrac1-19</i>). Error bar means standard deviation based on the data of three independent experiments. Superoxide production was detected by NBT staining. (B) Disease symptoms on the wounded leaf tissues of rice inoculated with mycelial plugs from 70-15, <i>ΔMgrac1-19</i>, <i>NOR1</i>, and <i>NOR2</i>. (C) Detection of superoxide by 0.6 mM NBT staining in the hyphal tips of <i>Nox</i> over-expression mutants. Bar = 10 µm. (D) Detection of superoxide by 0.3 mM NBT staining in the conidia of <i>Nox</i> over-expression mutants. Bar = 10 µm.</p

Primers used in this study.

<p>Primers used in this study.</p

Chm1<i>Δ</i><i>PBD</i> rescues conidiation in <i>Mgrac1</i> deletion mutants.

<p>(A) DIC microscopy of conidia of WT (70-15) and <i>PCA19</i> (Chm1<i>ΔPBD</i> expression in the Mgrac1 deletion mutant) collected after incubation the hydrophobic Gelbond film surface. Bar = 20 µm. (B) Conidia suspensions (about 1,000 in 20 µl) of WT and <i>PCA19</i> were inoculated on strips of onion epidermis. Infectious hyphae were examined at 1 day post-inoculation with DIC microscopy. A = appressorium, C = conidium, H = hypha, IF = infectious hypha. Bar = 20 µm. (C) Leaves of rice cultivar CO39 were sprayed with conidial suspensions (1×10⁵ conidia/ml) from WT and <i>PCA19</i>. Typical leaves were photographed 7 days after inoculation. (D) Disease symptoms on the wounded leaf tissues of rice inoculated with mycelial plugs from WT, <i>ΔMgrac1-19</i>, and <i>PCA19</i>. Typical leaves were photographed 5 days after inoculation. (E) Blast symptoms on rice roots. Arrow indicates necrotic lesions.</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

Cellular localization of tropomyosin-GFP in <i>MgRac1-CA and MgRac1-DN</i> mutants.

<p>Conidia expressing heterologous tropomyosin-GFP from WT (wild-type strain Guy11), <i>MgRac1-CA</i>, and <i>MgRac1-DN</i> were incubated on Gelbond films at 1 h and 24 h and observed by confocal fluorescence microscopy. Arrowhead indicates TpmA-GFP-labeled actin spot, white arrows indicate actin filaments, and red arrows indicate the areas where actin structures accumulate. Bar = 10 µm.</p