Characterisation of Stramenopile-specific mastigoneme proteins in Phytophthora parasitica
Mastigonemes, tripartite tubular hairs on the anterior flagellum of Phytophthora zoospores, are instrumental for disease dissemination to new host plants. A previous study showed that PnMas2 was part of the tubular shaft of Phytophthora parasitica mastigonemes. In the current study, genes encoding two related proteins, PnMas1 and PnMas3, were identified in the genome of P. parasitica. PnMas1 interacts with PnMas2 and also occurs along the mastigoneme shaft. RNA-Seq analyses indicate that PnMas1 and PnMas2 genes have similar expression profiles both in vitro and in planta but that PnMas3 is expressed temporally prior to PnMas1 and PnMas2 during asexual development and plant infection. Immunocytochemistry and GFP-tagging document the occurrence of all three PnMas proteins within the specialised compartments of the ER during mastigoneme formation, but only PnMas1 and PnMas2 occur in mature mastigonemes on the flagellar surface. Anti-PnMas1 and anti-PnMas2 antibodies co-labelled two high-molecular-weight (~400 kDa) protein complexes in native gels but anti-PnMas3 antibodies labelled a 65 kDa protein complex. Liquid chromatography-mass spectrometry analysis identified PnMas1 and PnMas2 but not PnMas3 in flagellar extracts. These results suggest that PnMas3 associates with mastigonemes during their assembly within the ER but is not part of mature mastigonemes on the anterior flagellum. Phylogenetic analyses using homologues of Mas genes from the genomes of 28 species of Stramenopiles give evidence of three Mas sub-families, namely Mas1, Mas2 and Mas3. BLAST analyses showed that Mas genes only occur in flagellate species within the Stramenopile taxon.
KeywordsCo-immunoprecipitation GFP-tagging Immunolocalisation Liquid chromatography-mass spectrometry (LCMS) Mastigonemes Phytophthora Stramenopiles Zoospores
The authors would like to thank Dr. Thy Truong (ANU) for assistance with the LC-MS/MS and Professor Richard W. Michelmore (UC Davis) for providing analyses of unpublished sequence data from Bremia lactucae. We would also like to thank Simon Michnowicz and Eugene Kapp from Walter and Eliza Hall Institute of Medical Research for incorporating genomic data of P. parasitica into the Mascot database. We also thank Dr. Megan Mcdonald for advice on phylogenetics analysis. The work was supported by grants from The Hermon Slade Foundation and the Australian Research Council.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Australian Government Department of the Environment (2014) Threat abatement plan for disease in natural ecosystem caused by Phyotophthora cinnamomi. Australian Government Department of the Environment. http://www.environment.gov.au/resource/threat-abatement-plan-disease-natural-ecosystems-caused-phytophthora-cinnamomi Accessed March 2015
- Balci, Y, Bienapfl, JC & Lamour, K (2013) Phytophthora in US forests. In: Lamour, K (ed.) Phytophthora: a global perspective. CABI, Croydon, pp 135–145Google Scholar
- Blackman LM, Cullerne DP, Torreña P, Taylor J, Hardham AR (2015) RNA-Seq analysis of the expression of genes encoding cell wall degrading enzymes during infection of lupin (Lupinus angustifolius) by Phytophthora parasitica. PLoS One 10:e0136899. https://doi.org/10.1371/journal.pone.0136899 CrossRefPubMedPubMedCentralGoogle Scholar
- Bottin A, Larche L, Villalba F, Gaulin E, Esquerré-Tugayé M-T, Rickauer M (1999) Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phytophthora parasitica var. nicotianae. FEMS Microbiol Lett 176:51–56. https://doi.org/10.1016/S0378-1097(99)00218-9 CrossRefPubMedGoogle Scholar
- Erwin DC & Ribeiro OK (1996) Phytophthora disease worldwide. The American Phytopathological Society Press, St. Paul, Minnesota, USAGoogle Scholar
- Hardham A, Gubler F, Duniec J, Elliott J (1991) A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens. J Microsc 162:305–318. https://doi.org/10.1111/j.1365-2818.1991.tb03142.x CrossRefGoogle Scholar
- Hee W, Torreña P, Blackman L, Hardham A & Lamour K (2013) Phytophthora cinnamomi in Australia. In: Lamour, K (ed.) Phytophthora: a global perspective. CABI, Croydon, pp 124–134Google Scholar
- Jahn, TL, Lanoman, MD & Fonseca, JR (1964) The mechanism of locomotion of flagellates. II. Function of the mastigonemes of Ochromonas*. 11, 291–296Google Scholar
- Kamoun S, Furzer O, Jones JD, Judelson HS, Ali GS, Dalio RJ, Roy SG, Schena L, Zambounis A, Panabieres F, Cahill D, Ruocco M, Figueiredo A, Chen XR, Hulvey J, Stam R, Lamour K, Gijzen M, Tyler BM, Grunwald NJ, Mukhtar MS, Tome DF, Tor M, Van Den Ackerveken G, McDowell J, Daayf F, Fry WE, Lindqvist-Kreuze H, Meijer HJ, Petre B, Ristaino J, Yoshida K, Birch PR, Govers F (2015) The top 10 oomycete pathogens in molecular plant pathology. Mol Plant Pathol 16:413–434. https://doi.org/10.1111/mpp.12190 CrossRefGoogle Scholar
- Milne I, Lindner D, Bayer M, Husmeier D, McGuire G, Marshall DF, Wright F (2009) TOPALi v2: a rich graphical interface for evolutionary analyses of multiple alignments on HPC clusters and multi-core desktops. Bioinformatics 25:126–127. https://doi.org/10.1093/bioinformatics/btn575 CrossRefPubMedGoogle Scholar
- Morris BM, Reid B, Gow NAR (1992) Electrotaxis of zoospores of Phytophthora palmivora at physiologically relevant field strengths. Plant Cell Environ 15:645–653. https://doi.org/10.1111/j.1365-3040.1992.tb01006.x CrossRefGoogle Scholar
- Perkins DN, Pappin DJC, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567. https://doi.org/10.1002/(sici)1522-2683(19991201)20:18<3551::aid-elps3551>3.0.co;2-2 CrossRefPubMedGoogle Scholar