Gene Validation and Remodelling Using Proteogenomics of Phytophthora Cinnamomi, the Causal Agent of Dieback

Abstract

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draft genome sequence and annotation is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome) and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. 2,764 annotations from the draft genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome of important plant diseases and identify biologically relevant missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen.

Comparative sub-cellular proteome analyses reveals metabolic differentiation and production of effector-like molecules in the dieback phytopathogen Phytophthora cinnamomi

“…The total protein identifications for each sub-proteomes are shown in Supplementary material 1 and 2. This accounts for 17.7% of the predicted annotated genes in the P. cinnamomi genome 12 . Of these, 1070, 698 and 278 were unique to the mycelia, zoospores and secretome respectively (Figure 1).…”

“…Label-free quantification was performed using the Proteome Discoverer 2.3 using the label-free precursor quantification workflow template 14 . Spectra were matched against the P. cinnamomi MU94-48 genome 12 . Sub-proteomes were relatively quantified using the default precursor ion quantifier.…”

“…Phytophthora cinnamomi (MU94-48) cultures were obtained from the Centre of Phytophthora Science and Management (Murdoch University, Australia) and maintained by passaging through Malus domestica cv. Granny Smith fruit 11,12 . Mycelia were grown on V8 juice (Campbells) agar with growth periods of 5-7 days in the dark at room temperature.…”

“…The liquid media was passed through a 0.22 µm filter to remove mycelial fragments and obtain the secretome. Zoospores were produced as previously described 12 . Purified mycelia, secretome and zoospores were snap-frozen in liquid nitrogen and freeze-dried.…”

Proteomic analysis revealed that the oomyceticide phosphite exhibits multi-modal action in an oomycete pathosystem

“…Qualitative and label-free quantification was performed using Proteome Discoverer 2.3 37 . Mass spectra from the in vitro assay were matched to the P. cinnamomi MU94-48 genome consisting of 26,151 protein-coding sequences 38 . The proteomes between phosphite-treated MU94-48 and CPSM366 were compared to simultaneously gain insight into the effects of phosphite on sensitive P. cinnamomi and understand the differences between phosphite sensitivity and tolerance.…”