Lineage specific regions in the onion basal rot
pathogen Fusarium oxypsporum f.sp. cepae
A. Armitage1 , J.P. Clarkson, A. Taylor, A. Jackson, S. Ott, L. Baxter, G. Teakle, R. de
Heer, T. Ferber and R. Harrison
Andrew.Armitage@EMR.ac.uk 1East Malling Research, East Malling, Kent ME19 6BJ, UK
b
a
The onion pathogen Fusarium oxysporum ‘formae speciales’ cepae (FoC) causes basal rot of
onion bulbs and leads to annual losses of £11M the UK alone. Lack of effective control measures
means there is pressure to identify natural disease resistance in onion and breed new cultivars
with robust disease resistance. This requires an understanding of the basis of pathogenicity.
Isolates show host adaptation, allowing the separation formae speciales on the basis of hostselective pathogenicity. The tomato pathogen F. oxysporum f.sp. lycopersisi (FoL) has been
shown to possess lineage specific regions containing pathogenicty genes and effectors1.
Fig.1: Control onion bulbs (a) and bulbs
grown in F. oxysporum fsp. cepae infected
compost (b).
Sequencing
Library prep. (Truseq)
GA2 (2x70bp) & MiSeq (2x300bp)
Assembly
QC (fastqc & fastq-mcf)
Assembly (Velvet)
Gene prediction (Augustus)
Functional annotation (Interproscan)
Identifying putative effectors
Motif searching in predicted genes
(SignalP, RxLR)
Pathogenicity gene regulators
Presence of promotor motif in
assembled contigs (MIMP)
Homologs to known & novel
effectors
Homology searching (tBLASTx)
Gene expression during infection
QC (fastqc & fastq-mcf)
Align reads to genome (bowtie2)
Idenitfy expressed genes (bedtools)
Aims:
Identitfy putative effectors in FoC genomes
Determine core effectors and genes in lineage specifc regions
Identify the role of lineage specific effectors in pathogenicity
Methodology
1. F. oxysporum isolates were characterised for differential virulence (Fig.1).
2. NGS data was generated for 3 pathogenic, 1 intermediate and 3 non-pathogenic FoC isolates
(Tab.1).
3. A bioinformatic pipeline was developed to identify lineage putative effectors in genomes (Tab.1).
4. Preliminary RNAseq data was generated for a pathogenic FoC isolate infecting onion to identify
genes involved in pathogenicity (Tab.1).
Tab.2: Results from NGS effector identification
pipeline for the tomato pathogen reference genome
(4287), along with isolates identified as pathogenic,
intermediate and non-pathogenic on onion.
Genome assembly stats (A) are shown along with
identification of novel effector candidates through
presence of RxLR motifs (B) genes downstream of
promotors associated with pathogenicity (C) and
presence of homologs to previously characterised
effectors (D). RxLR and MIMP associated proteins
were characterised as putative core (E) or lineage
specific (F) on the basis of BLAST homology.
Tab1: NGS pipeline for identification of pathogenicity
genes & effectors in oomycete and fungal pathogens.
Results
 Assemblies of F.oxysporum f.sp. cepae are comparable to the reference
sequence 4287 before scaffolding (Tab.2A).
 Putative RxLR effectors were identified using motif searches within
predicted proteins (Tab.2B). This motif, common in Oomycyete
pathogens, has not previously been described in Fusarium spp..
 Pathogenicity genes In FoL are often under regulation by a promotor
containing a MIMP motif. This also appears to be true in FoC, as
pathogenic isolates contain greater numbers of MIMPs and MIMPassociated genes than non-pathogenic isolates (Tab.2C).
 Previously characterised, publically available, FoL effectors (SIX genes) had
homologs in FoC pathogens (Tab.2D).
 Putative core gneome effectors were identified from genes containing RxLRs and
MIMP-associated genes (Tab.2E).
 Putative lineage specific effectors were identified from genes containing RxLRs
and MIMP-associated genes (Tab.2F).
 The genes of FoC pathogenic isolate Fus2 that showed greatest expression
when infecting onion included SIX gene homologs along with other genes
showing pathogenicity functions (Tab.3).
Future work
 RNAseq to investigate expression of core and lineage specific effectors.
Tab.3: Top 18 most expressed genes in FoC isolate Fus2 when infecting
onion roots at 96hrs post inoculation. Homologs to previously
characterised FoL pathogenicity genes (SIX genes) show high expression.
Functional annotation of other highly expressed genes indicate roles in
pathogenicity such as proteases, cellulases, secondary metabolite
synthesis and chitin binding.
et al. Nature (2010) 464, 367-373
2. Schmidt et al. BMC Genomics (2013) 14, 119
1. Ma
 Determine gene-for-gene interactions between effectors and resistance genes.
 Perform PacBio sequencing and scaffold genomic contigs.
 Investigate structure of lineage specific contigs.