Abstract
Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.
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Acknowledgments
This work was supported by Chinese Universities Scientific Fund (Grant No. 2013XJ012) and National Fundamental Basic Research program (Grant No. 2012CB114002) of the Ministry of Sciences and Technology, China.
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We declared that no conflict of interest exists.
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Communicated by S. Hohmann.
Nucleotide sequence data are available in the GenBank database under the accession number KF201869 (MoSPA2).
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294_2014_431_MOESM1_ESM.tif
Fig. S1 Phylogenetic tree of fungal Spa2 proteins. Protein sequences were aligned using the programs ClustalX and Mega 4.0 to create the dendrogram. A. fumigates, Aspergillus fumigatus Spa2 (XM744410.1); A. gossypii, Ashbya gossypii Spa2 (NM209428.1); A. nidulans, Aspergillus nidulans SpaA (XM656327.1); A. niger, Aspergillus niger SpaA (EF474481); C. albicans, Candida albicans Spa2 (XM706557.1); G. zeae, Gibberella zeae Spa2 (XP_381108.1); M. oryzae, Magnaporthe oryzae Spa2 (XP_003716178.1); N. crassa, Neurospora crassa Spa2 (XM959881.1); S. cerevisiae, Saccharomyces cerevisiae Spa2 (NM209428.1); U. maydis, Ustilago maydis Spa2 (XP_760615.1). The numbers at nodes represent the percentage of branch support of 1,000 bootstrap replicates, and the scale bar indicates the number of amino acid differences per site (TIFF 156 kb)
294_2014_431_MOESM2_ESM.tif
Fig. S2 Alignment of the SHD-I of fungal Spa2 homologs. Alignment of the conserved domain of Spa2 from M. oryzae with those from other organisms (shown in Fig. S1) was performed using ClustalX and ESPript programs. The region of highest homology corresponds to the SPA2 direct repeat (SDR, Roemer et al. 1998) and is indicated by arrows (TIFF 1041 kb)
294_2014_431_MOESM3_ESM.tif
Fig. S3 Nuclei and septa in vegetative hyphae of the wild-type P131, the ∆Mospa2 null mutant MH1, and one complementation strain CM20 were co-stained by Hoechst 33258 and Calcofluor White. The white arrows and triangles point to the septa and nuclei, respectively. Bar = 20 μm (TIFF 1217 kb)
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Li, C., Yang, J., Zhou, W. et al. A spindle pole antigen gene MoSPA2 is important for polar cell growth of vegetative hyphae and conidia, but is dispensable for pathogenicity in Magnaporthe oryzae . Curr Genet 60, 255–263 (2014). https://doi.org/10.1007/s00294-014-0431-4
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DOI: https://doi.org/10.1007/s00294-014-0431-4