System-Wide Characterization of MoArf GTPase Family Proteins and Adaptor Protein MoGga1 Involved in the Development and Pathogenicity of Magnaporthe oryzae

Abstract

ADP ribosylation factor (Arf) small GTPase family members are involved in vesicle trafficking and organelle maintenance in organisms ranging from Saccharomyces cerevisiae to humans. A previous study identified Magnaporthe oryzae Arf6 (MoArf6) as one of the Arf proteins that regulates growth and conidiation in the rice blast fungus M. oryzae, but the remaining family proteins remain unknown. Here, we identified six additional Arf proteins, including MoArf1, MoArl1, MoArl3, MoArl8, MoCin4, and MoSar1, as well as their sole adaptor protein, MoGga1, and determined their shared and specific functions. We showed that the majority of these proteins exhibit positive regulatory functions, most notably, in growth. Importantly, MoArl1, MoCin4, and MoGga1 are involved in pathogenicity through the regulation of host penetration and invasive hyphal growth. MoArl1 and MoCin4 also regulate normal vesicle trafficking, and MoCin4 further controls the formation of the biotrophic interfacial complex (BIC). Moreover, we showed that Golgi-cytoplasm cycling of MoArl1 is required for its function. Finally, we demonstrated that interactions between MoArf1 and MoArl1 with MoGga1 are important for Golgi localization and pathogenicity. Collectively, our findings revealed the shared and specific functions of Arf family members in M. oryzae and shed light on how these proteins function through conserved mechanisms to govern growth, transport, and virulence of the blast fungus.IMPORTANCEMagnaporthe oryzae is the causal agent of rice blast, representing the most devastating diseases of rice worldwide, which results in losses of amounts of rice that could feed more than 60 million people each year. Arf (ADP ribosylation factor) small GTPase family proteins are involved in vesicle trafficking and organelle maintenance in eukaryotic cells. To investigate the function of Arf family proteins in M. oryzae, we systematically characterized all seven Arf proteins and found that they have shared and specific functions in governing the growth, development, and pathogenicity of the blast fungus. We have also identified the pathogenicity-related protein MoGga1 as the common adaptor of MoArf1 and MoArl1. Our findings are important because they provide the first comprehensive characterization of the Arf GTPase family proteins and their adaptor protein MoGga1 functioning in a plant-pathogenic fungus, which could help to reveal new fungicide targets to control this devastating disease.

Keywords: ADP ribosylation factor; Golgi; Magnaporthe oryzae; pathogenicity; vesicle trafficking.

FIG 1

Phylogenetic analysis of putative Arf proteins in fungi. The Arf proteins from different fungi were aligned using the Clustal_W program, and the neighbor-joining tree was constructed with 1,000 bootstrap replicates by the use of MEGA 5.05. The sequences were obtained in the following organisms: M. oryzae, F. graminearum, Z. tritici, A. nidulans, N. crassa, C. albicans, and S. cerevisiae.

FIG 2

MoArl1, MoCin4, and MoGga1 are required for full virulence. (A) Pathogenicity assay in rice. Two-week-old rice seedlings were inoculated with related conidial suspensions (ΔMoarl1) or mycelia (ΔMocin4) and photographed at 7 days postinoculation (dpi). (B) Pathogenicity assay in barley. One-week-old detached barley leaves were inoculated with the conidial suspension or mycelia and photographed at 5 dpi. (C) Penetration assay in rice cells at 48 hpi and in barley cells at 36 hpi. The appressorium (in rice) or appressorium-like (in barley) penetration sites (n = 100) were divided into types 1 to 4. Error bars represent standard deviations of results from three replicates. Black asterisks indicate hyphae extended to neighboring cells. Bar, 10 μm. (D) Statistical analysis of appressoria (ΔMoarl1) or appressorium-like structures (ΔMocin4) revealed cytorrhysis in different glycerol concentrations. Asterisks represent significant differences. (E) The localization of Sep5-GFP in appressoria (ΔMoarl1) or appressorium-like structures (ΔMocin4). Bar, 10 μm. DIC, differential inference contrast. (F) Pathogenicity and penetration assays for ΔMogga1 mutant. The criteria of the classification were the same as those described for ΔMoarl1. Error bars represent standard deviations of results from three replicates.

FIG 3

MoArl1 and MoCin4 are required for normal vesicle trafficking. (A) Hyphae of the strains were stained with FM4-64 for different minutes. Bar, 5 μm. (B) The integrated fluorescent density was calculated with ImageJ. Asterisks indicate significant differences compared with Guy11. a.u., arbitrary units. (C and D) Images of BICs and the BIC-accumulating cytoplasmic effector Avr-Pia-GFP and AvrPiz-t-GFP in rice (ΔMoarl1) (C) and barley (ΔMocin4) (C) cells. Arrows indicate BICs. Bar, 10 μm.

FIG 4

MoCin4 is involved in the scavenging of reactive oxygen species. (A and B) DAB was used to stain the sheaths injected with related conidial suspensions for ΔMoarl1 cells and the stained cells were statistically analyzed. (C and D) DAB was used to stain the barleys infected with related mycelia for ΔMocin4 cells and statistically analyzed. Asterisks indicate a significant difference. Bar, 10 μm.

FIG 5

MoArl1 is localized to the Golgi and the cytoplasm. MoArl1 partially colocalizes with MoSft2 in conidia, germ tubes, appressoria, and vegetative and invasive hyphae. MoSft2 was expressed as a Golgi marker, and images were observed with confocal fluorescence microscopy (Zeiss LSM710 laser scanning microscope; 63× oil). Arrowheads show the representative colocalized areas. Bar, 5 μm.

FIG 6

The localization of MoArl1 is nucleotide dependent. (A and B) Localization patterns of different forms of MoArl1 in hyphae (A) and conidia (B). Arrowheads show the areas used for determinations of fluorescence intensity profiles by line-scan analysis. Green lines stand for the fluorescence intensity of related MoArl1-GFP results and red for MoSft2-RFP. (C) Model of the association/disassociation of MoArl1 with the Golgi membrane. Bar, 5 μm.

FIG 7

The GTP/GDP binding motifs are important for the functions of MoArl1 and MoCin4. (A) Colony morphology of ΔMoarl1-related strains after 7 days of incubation with CM plates. (B) Colony morphology of ΔMocin4-related strains after 7 days of incubation with CM plates. (C) Rice spraying assay of the ΔMoarl1-related strains. (D) Detached rice leaf assay of the ΔMocin4-related strains.

FIG 8

The N-myristoylated motif is essential for functions and Golgi localization of MoArl1. (A) Colony morphology of ΔMoarl1-related strains after 7 days of incubation with CM plates. (B) Rice spraying assay of the ΔMoarl1-related strains. (C) Localization pattern of different forms of MoArl1 in conidia and hyphae. Arrowheads show the areas used for determinations of fluorescence intensity profiles by line-scan analysis. Green lines stand for the fluorescence intensity of related MoArl1-GFP results and red for MoSft2-RFP. Bar, 5 μm.

FIG 9

MoGga1 interacts with both MoArl1 and MoArf1. (A and B) Y2H assay for the interaction between the constitutively active (A) and dominant negative (B) forms of MoArf proteins with MoGga1. The yeast transformants expressing the bait and prey constructs were incubated on SD-Leu-Trp plates. The β-galactosidase activity was assayed on SD-Ade-His-Leu-Trp plates with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). (C and D) BiFC assays for the interaction of MoArl1 (C) or MoArf1 (D) with MoGga1 in vivo. The transformants coexpressing MoGga1-YFP^N and MoArl1-YFP^C or MoArf1-YFP^C were observed in different developmental stages with confocal fluorescence microscopy (Zeiss LSM710 laser scanning microscope; 63× oil). Bar, 5 μm. (E) Co-IP assays for the interactions of MoArl1, MoGga1, and MoArf1. The coexpressing proteins (lanes 1, MoArl1-S/MoGga1-GFP; lanes 2, MoArl1-S/GFP; lanes 3, MoGga1-S/MoArf1-GFP; lanes 4, MoGga1-S/GFP; lanes 5, MoArl1-S/MoArf1-GFP) were extracted individually as the total proteins (T). Total proteins were eluted from the anti-GFP beads (E) and analyzed by Western blotting with anti-S and anti-GFP antibodies.

FIG 10

Localization and function of MoGga1 are dependent on its interaction with MoArf1 and MoArl1. (A) The localization of MoGga1-GFP in the Guy11 and ΔMoarl1 mutant strains. The averaged GFP punctate numbers determined for 50 conidia were counted and analyzed. Images were observed with confocal fluorescence microscopy (Zeiss LSM710 laser scanning microscope; 63× oil). Bar, 5 μm. (B) Localization of MoGga1-GFP in Guy11 following BFA treatment. The averaged GFP punctate numbers determined for 50 conidia were counted and analyzed. Asterisks indicate significant differences. Images were observed with confocal fluorescence microscopy (Zeiss LSM710 laser scanning microscope; 63× oil). Bar, 5 μm. (C) Structure and domain prediction of MoGga1. Regions of the domains are indicated by amino acid numbers. The asterisk indicates the conserved leucine or isoleucine residue of MoGga1 relative to that in ScGgas and HsGgas. (D and E) Y2H assay for interactions between the point mutation MoGga1^I208N or GAT domain with the constitutively active forms of MoArf1 (D) and MoArl1 (E). (F) Localization of point-mutated MoGga1^I208N-GFP and truncated MoGga1^GAT-GFP in conidia. The averaged GFP punctate numbers determined for 50 conidia were counted and analyzed. Asterisks indicate significant differences. Images were observed using confocal fluorescence microscopy (Zeiss LSM710 laser scanning microscope; 63× oil). Bar, 5 μm. (G) Rice spraying assays for the ΔMogga1-related mutants. (H) Detached barley assays for the ΔMogga1-related mutants.