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

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 host-selective pathogenicity. The tomato pathogen F. oxysporum f.sp. lycopersisi (FoL) has been shown to possess lineage specific regions containing pathogenicty genes and effectors1.

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

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 MIMP-associated 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).

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)

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.

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. Determine gene-for-gene interactions between effectors and resistance genes. Perform PacBio sequencing and scaffold genomic contigs. Investigate structure of lineage specific contigs.

Tab1: NGS pipeline for identification of pathogenicity genes & effectors in oomycete and fungal pathogens.

1. Ma et al. Nature (2010) 464, 367-373 2. Schmidt et al. BMC Genomics (2013) 14, 119

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.

Fig.1: Control onion bulbs (a) and bulbs grown in F. oxysporum fsp. cepae infected compost (b).

a b

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).