A Small GTPase RHO2 Plays an Important Role in Pre-infection Development in the Rice Blast Pathogen Magnaporthe oryzae

Abstract

The rice blast pathogen Magnaporthe oryzae is a global threat to rice production. Here we characterized RHO2 gene (MGG_02457) that belongs to the Rho GTPase family, using a deletion mutant. This mutant ${\Delta}Morho2$ exhibited no defects in conidiation and germination but developed only 6% of appressoria in response to a hydrophobic surface when compared to the wild-type progenitor. This result indicates that MoRHO2 plays a role in appressorium development. Furthermore, exogenous cAMP treatment on the mutant led to appressoria that exhibited abnormal morphology on both hydrophobic and hydrophilic surfaces. These outcomes suggested the involvement of MoRHO2 in cAMP-mediated appressorium development. ${\Delta}Morho2$ mutation also delayed the development of appressorium-like structures (ALS) at hyphal tips on hydrophobic surface, which were also abnormally shaped. These results suggested that MoRHO2 is involved in morphological development of appressoria and ALS from conidia and hyphae, respectively. As expected, ${\Delta}Morho2$ mutant was defective in plant penetration, but was still able to cause lesions, albeit at a reduced rate on wounded plants. These results implied that MoRHO2 plays a role in M. oryzae virulence as well.

Fig. 1. Domain structure and conserved amino acids of RHO GTPase in M. oryzae. (A) Schematic structure of the small GTP-binding protein domain (IPR005225) in the RHO protein family. The domain structure was predicted using InterProScan. (B) The conserved amino acid sequence alignment of Rho GTPase. G1, G2, G3, G4, G5, switch I, and switch II denote special motifs in the small GTPbinding protein domain. The identity of each protein BLAST search with MoRHO2 is followed by its name.

Fig. 2. Phylogenetic analysis and conserved amino acid sequence alignment of MoRHO2 and homologues from other organisms. (A) Phylogenetic analysis among MoRHO2 and homologues. A phylogenetic tree was generated using a neighbor-joining method based on comparing MoRHO2 and its homologues. (B) The conserved amino acid sequence alignment among MoRHO2 and homologues. The identity of each protein BLAST search with MoRHO2 is followed by its name.

Fig. 3. The expression profile and targeted gene deletion of MoRHO2. (A) The expression profile of MoRHO2 in different developmental stages of M. oryzae. The expression of MoRHO2 was measured during five stages including mycelia (MY), conidia (CO), germinated conidia (GC), appressoria (AP), and infectious hyphae stages in rice leaves (IP). The results were normalized to β-tubulin and presented with a relative value of 1 in MY. (B) The targeted gene knockout of MoRHO2. The knockout strategy used the HPH cassette to replace MoRHO2. (C) The conformation of the MoRHO2 deletion using southern blot analysis. The genomic DNA was digested with HindIII and hybridized with specific probes. (D) Reverse transcription-PCR was used to check the expression of MoRHO2. The total RNA was extracted from wild type, ΔMorho2, and Morho2c samples.

Fig. 4. Appressorium formation on artificial surfaces. (A) Statistical analysis of appressoria formed on the hydrophobic and hydrophilic surface. Appressorium formation was assessed at 48 h after inoculation. (B) The appressorium morphology on a hydrophobic surface. Appressoria were observed after a 6 h incubation. Scale bar = 20 μm.

Fig. 5. An appressorium-like structure (ALS) formed on hyphal tips. Hyphal plugs (5 mm in diameter) of wild type, ΔMorho2, and Morho2c samples were placed on hydrophobic surfaces. Photographs were taken after 24 and 36 h. Scale bar = 50 μm.

Fig. 6. Plant pathogenic assays. (A) The spray assays. The conidial suspension was sprayed onto rice leaves and the leaves were incubated for 7 days. (B) The influence of wounding on disease development. Conidial drops or hyphal plugs (6 mm in diameter) were inoculated onto rice leaves with or without wounding and the leaves were incubated for 2 days.

Fig. 7. Penetration assays. A conidial suspension of the indicated strains was dropped on rice sheath cells. Photographs were taken at 2 days after inoculation. Scar bar = 50 μm.

Table 1. List of primers used in this study.