Identification of Plasmodiophora brassicae effectors — A challenging goal

Figures & data

Figure 1. Brassica plant root affected by Plasmodiophora brassicae. A. Canola root with typical galls after 1 month of inoculation with P. brassicae resting spores. B. Life cycle of P. brassicae showing the steps involved in infection through to the production of secondary plasmodia in the host plant cortical cells. Scheme based on that of Kageyama and Asano [7], representing spindle-shaped resting spores, biflagellate primary zoospores, zoospores, and primary and secondary plasmodia (oval black figure in root hairs and cortical cells, respectively). Further steps in P. brassicae´s life cycle, such as the formation of resting spores in cortical cells and its ejection to the soil, are not shown in this scheme.

Table 1. Steps to identify putative effectors within the secretome of P. brassicae in the European strain Pbe3 and the Canadian strain Pb3.

	Europe strain (Pbe3)	Canadian strain (Pb3)
Signal Peptide/No trans-membrane domain^a	533*	590*
D score > 0.7	NA	431
Size (Small secreted proteins) ^b	416	221
Over-expression ^c	300	NA
Plant-free library ^d	92	NA

NA, Not applied

^a In the Canadian strain, other subcellular localization signals were also used to remove putative proteins from further analysis.

^b For the European strain the cutoff was < 450 aa, while for the Canadian protein proteins < 300 aa were selected.

^c At least 10 expected fragments per kilobase of transcript per million fragments (FPKM)

^d FPKM log2 fold change > 5 in plant-free library

* Putative secreted proteins.

Table 2. Bioinformatics tools used in a coherent pipeline to identify putative effectors of P. brassicae.

Bioinformatics tool	Description	Website	Reference
Trimmomatic	A fast, multithreaded command line tool that can be used to trim and crop Illumina data as well as to remove adapters.	http://www.usadellab.org/trimmomatic	^[Citation80]
STAR	An algorithm for aligning high-throughput long and short RNA-seq data to a reference genome.	http://code.google.com/p/rna-star	^[Citation81]
SignalP	Software that predicts signal peptide cleavage sites in proteins from eukaryotes and prokaryotes.	http://www.cbs.dtu.dk/services/SignalP	^[Citation82]
WoLF PSORT	An algorithm that predicts the subcellular localization sites of proteins based on their amino acid sequences. It makes predictions based on both known sorting signal motifs and some correlative sequence features such as amino acid content.	www.wolfpsort.org	^[Citation83]
TargetP	Tool that predicts subcellular location of eukaryotic proteins.	http://www.cbs.dtu.dk/services/TargetP	^[Citation84]

Figure 2. Coherent pipeline to identify putative effector proteins of Plasmodiophora brassicae. The pipeline assumes: (1) Researchers are starting with RNA-Seq reads from a host plant infected with P. brassicae; (2) The draft genomes available for P. brassicae, other plasmodiophorids, oomycetes, and other plant pathogens are used; (3) Motifs mentioned in the review or structural similarities with previously described effector proteins were identified.

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