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Characterisation of Stramenopile-specific mastigoneme proteins in Phytophthora parasitica

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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.

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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Truong (ANU) for assistance with the LC-MS/MS and Professor Richard W. Michelmore (UC Davis) for providing analyses of unpublished sequence data from Bremia lactucae.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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Correspondence to Wei Yih Hee.

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Handling Editor: Ulrike Mathesius

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Online Resource 1

Primer sequences used to construct the PnMas3-GFP transgene. (DOCX 12 kb)

Online Resource 2

Light micrographs of samples collected during P. parasitica flagella isolation. (a) Immunofluorescence labelling with anti-PnMas2 mAb shows that mastigonemes are still attached to the anterior flagellum (arrowheads) after the flagella isolation. (b) The posterior flagella which are not labelled by anti-PnMas2 mAb are visible using DIC optics (arrows). Scale bars = 20 μm. (PNG 476 kb)

High Resolution Image (TIF 1162 kb)

Online Resource 3

Alignment of PnMas1, PnMas2 and PnMas3 protein sequences. All three Mas proteins contain conserved cysteine residues (cyan) within EGF-like domains (yellow). (PNG 1000 kb)

High Resolution Image (TIF 4126 kb)

Online Resource 4

Statistical analysis of PnMas1, PnMas2 and PnMas3 transcript levels at six stages in the P. parasitica asexual life cycle. (DOCX 13 kb)

Online Resource 5

Details of the generation of monoclonal antibodies (mAbs) against P. parasitica PnMas1 and PnMas3 proteins. The table shows the mAb cell lines, epitopes targeted by the mAbs and the results of immunoblotting and immunocytochemical labelling. (DOCX 13 kb)

Online Resource 6

The results of immunofluorescence labelling of P. parasitica zoospores with anti-PnMas1 and anti-PnMas3 mAbs after different fixation regimes. (DOCX 12 kb)

Online Resource 7

Micrographs of P. parasitica zoospores fixed in 80% methanol and labelled with anti-PnMas1 mAbs and GAM-FITC. a-e: mAbs 2 K19, 2 K23, 2 M19, 2A21 and 2 L5 label mastigonemes on the anterior flagellum and in ER packets within the cells. f: mAb 2H18 did not show any labelling of the zoospores. Scare bars = 5 μm (PNG 1184 kb)

High Resolution Image (TIF 2854 kb)

Online Resource 8

Brightfield (a, c, e, g, i) and fluorescence (b, d, f, h, j) micrographs showing P. parasitica zoospores labelled with anti-PnMas1 mAb (2 M19) and GAM-FITC. The zoospores were treated with 80% methanol (a, b), 80% acetone (c, d), 63% ethanol/3% acetic acid (e, f), 0.05% Triton X-100 (g, h) and 0.05% Tween 20 (i, j). The anti-PnMas1 mAb labels mastigonemes on the anterior flagella (arrows) but does not label the posterior flagella (arrowheads). Methanol treated zoospores often round up and the flagella adhered to the cell surface (b). Scale bars = 5 μm. (PNG 1102 kb)

High Resolution Image (TIF 2439 kb)

Online Resource 9

Immunoblots of P. parasitica proteins separated using native PAGE with anti-PnMas1 (2 M19), anti-PnMas2 and anti-PnMas3 (2O20) mAbs. Both anti-PnMas1 and anti-PnMas2 mAbs react with a pair of bands ~400 kDa in size (arrows). Anti-PnMas3 mAb cell line 2O20 labelled a 65 kDa protein complex (arrowhead). (PNG 148 kb)

High Resolution Image (TIF 817 kb)

Online Resource 10

LC-MS/MS analysis of proteins co-precipitated by anti-PnMas2 mAb in sporulating hyphae extracts. (DOCX 14 kb)

Online Resource 11

LC-MS/MS analysis of proteins co-precipitated by anti-PnMas2 mAb in P. parasitica flagellar protein extracts. (DOCX 16 kb)

Online Resource 12

Accession number of Mas genes. (DOCX 16 kb)

Online Resource 13

MrBayes analysis of the phylogenetic relationships of concatenated Mas1, Mas2 and Mas3 protein sequences from putative homologues from the Stramenopile genomes. The EGF-like domains and N-termini of the proteins were retained for the analysis. The numbers on the branches show the posterior probabilities generated from 10,000 generations. The locations of PnMas proteins are indicated by the arrows. (PNG 748 kb)

High Resolution Image (TIF 10307 kb)

Online Resource 14

A phylogenetic tree showing neighbour joining analysis of six heat shock protein 70s from Chlamydomonas reinhardtii (Crhsp70A–F) and 12 hsp70-like proteins identified in the P. parasitica genome. The branches show bootstrap values generated from 10,000 replicates. The analysis showed that PPTG_02122 (blue arrow), which was detected in the LC-MS/MS analysis of anti-PnMas2 co-precipitated proteins and in P. parasitica isolated flagellar extracts, is closely related to Crhsp70A (red arrow), a protein found in Chlamydomonas flagella. (DOCX 1539 kb)

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Hee, W.Y., Blackman, L.M. & Hardham, A.R. Characterisation of Stramenopile-specific mastigoneme proteins in Phytophthora parasitica. Protoplasma 256, 521–535 (2019).

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