SMRT sequencing reveals a transferrable pathogenicity chromosome in Fusarium oxysporum f.sp. radicis-cucumerinum

Abstract

The genome of Fusarium oxysporum (Fo) is typically divided into a set of eleven ‘core’ chromosomes, shared by most strains and responsible for housekeeping, and one or several accessory chromosomes. In Fo f.sp. lycopersici, one of these chromosomes was shown to be horizontally transferrable and responsible for pathogenicity towards tomato. In de novo Illumina assemblies, these accessory chromosomes are dispersed over many contigs, making it impossible to identify how many accessory chromosomes are present in a strain. We sequenced a strain of Fo f.sp. radicis-cucumerinum (Forc) using PacBio SMRT sequencing. The majority of the core chromosomes were assembled into a single contig, and a chromosome that shows all the hallmarks of a pathogenicity chromosome comprised two contigs. Part of this chromosome is very rich in repeats and transposons and contains all identified candidate effector genes, including homologs of SIX6, SIX9, SIX11 and SIX13. Through individual knockout of several SIX homologs as well as Horizontal Chromosome Transfer (HCT) towards a non-pathogenic strain (Fo47), we were able to show that (a) SIX6 is an important effector for this root rot pathogen and (b) that this chromosome is indeed a pathogenicity chromosome for cucurbit infection. Strains that received this chromosome into a Fo47 background are fully pathogenic, essentially changing Fo47 into a virulent root rot pathogen infecting multiple cucurbits. One strain obtained by means of HCT showed a duplication of the entire pathogenicity chromosome and is still pathogenic, while another shows partial duplications and is reduced in virulence towards cucumber and watermelon but not towards muskmelon. These observations allow us to conclude that also non-wilt-inducing Fo pathogens like Forc rely on effector proteins for successful infection and that the Forc pathogenicity chromosome contains all the information necessary for causing root rot of cucurbits. Moreover, the fact that three out of nine HCT strains have undergone large-scale chromosome alterations shows the enormous plasticity of Fo genomes.

Full text

Illumina reads were mapped against Forc016’s assem-
bly. Mapping densities relative to total # mapped reads
are plotted for HCT strains #2, #4, #8, #9. Since the core
genomic sequences in these strains are derived from a
Fo47 background, mapping eciency is lower in these
regions, but remain high for chrRC.
Strain #8 underwent a chromosome duplication event.
All four tested strains (#2, #4, #8, #9) caused abundant symp-
tom development in each of the three plant species, compa-
rable to the control strain, Forc016∆SIX6#46 (at 20°C).
5.7-
4.6-
3.5-
Mb
-2.2
-1.6
-1.1
Mb
HCT#1
HCT#2
HCT#3
HCT#4
HCT#5
HCT#6
HCT#7
HCT#8
HCT#9
Fo47pGRB
Forc016
∆SIX6#46
*
Forc016 chrRC (Mb)
Fom001 contig 127 (Mb)
AB
SIX9
SIX13 SIX6
SIX11
% similarity
70 90
858075 95 100
mimps
effectors
1.5
1.0
0.5
0
0.5 1.0 2.01.5
01.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7
0.9
1.0
1.1
1.2
1.3
SMP1
A
B
chrRC
Fusarium oxysporum f.sp. radicis-cucumerinum (Forc), the causal agent of root rot in multiple cucurbit species, was sequenced
using PacBio SMRT sequencing. All but one of the core chromosomes were assembled into single contigs, and a chromosome
that shows all the hallmarks of a pathogenicity chromosome was identied. e aims of this study were to (i) determine the ge-
nome structure of Forc, (ii) investigate whether Forc, like wilt-causing strains of F. oxysporum, relies on eector proteins for suc-
cessful colonization and (iii) identify which part(s) of the Forc genome are necessary for the root- and shoot-rot phenotype as
well as the extended host range of Forc.
chr01
0
1
2
3
4
5
6
chr02
0
1
2
3
4
5
chr04
0
1
2
3
4
5
chr05
0
1
2
3
4
5
chr07
0
1
2
3
4
chr08
0
1
2
3
4
chr09
0
1
2
3
chr10
0
1
2
3
chr11
0
1
2
3
4
chr12
0
1
2
3
chr13
0
1
2
chrRC
0
1
2
c53
0
c3
0
c6
0
c7
0
chr15
0
1
2
chr14
0
1
chr13
0
1
chr12
0
1
2
chr11
0
1
2
chr10
0
1
2
chr09
0
1
2
3
chr08
0
1
2
3
chr07
0
1
2
3
4
chr06
0
1
2
3
4
chr05
0
1
2
3
4
chr04
0
1
2
3
4
5
chr03
0
1
2
3
4
5
.
0
chr02
0
1
2
3
4
.
0
chr01
0
1
2
3
4
5
A.) karyotype
B.) gene density (50kb windows)
C.) repeat density (50kb windows)
D.) eector candidates
E.) nucmer comparison to Fol4287 reference genome.
- Forc016 possesses 11 core, 1 pathogenicity and 2 accessory chromosomes
- Central region of chrRC is packed with eectors and TEs
- Six6 is a secreted eector protein that promotes virulence in Forc
- ChrRC can be horizontally transferred, conferring virulence to the recipient strain
- ChrRC is responsible for root and shoot rot in cucumber, melon, watermelon
- ChrRC (and with it, virulence) can be lost in Forc
[1] van Dam P, Fokkens L, et al. 2016. Eector proles distinguish formae speciales of Fusarium oxysporum. Environ Microbiol 18:4087–4102.
Forc016ΔSIX9 strains grown in the presence of benomyl
were selected for Hyg-sensitivity, yielding 5 strains that
lost the chromosome containing the HygR locus (chrRC).
ese turned out to have lost virulence (at 20°C).
cucumber melon watermelon cucumber melon watermelon
Fol4287
Forc016
A
B
C
D
E
SMRT sequencing reveals a transferrable pathogenicity
chromosome in Fusarium oxysporum f.sp. radicis-cucumerinum
Peter van Dam1, L. Fokkens1, Y. Ayukawa2, M. van der Gragt1, A. ter Horst1, B. Brankovicz3, P. M. Houterman1, T. Arie2, M. Rep1
1. Molecular Plant Pathology, University of Amsterdam, e Netherlands; 2. Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology (TUAT), Japan;
3. Westerdijk Fungal Biodiversity Institute, Utrecht, e Netherlands
Watch: Forc016-GFP
growing in planta
https://youtu.be/
cutT-ZC3w
300 μm
WT ΔSIX6#30 ΔSIX6#40 ΔSIX6#46 ectopic mock
Forc’s pathogenicity chromosome
is similar to Fom’s
A central part of this chromosome (named ‘chrRC’) con-
tains most of the identied candidate eector genes, in-
cluding homologs of SIX6, SIX9, SIX11 and SIX13.
Six6 is a virulence factor in Forc
ree individual SIX6 knockout strains show reduced
symptom development in cucumber plants (at 25°C).
Horizontal transfer to Fo47
Coincubation of Forc016ΔSIX6 (HygR on chrRC) with
Fo47 (PhleoR on core) resulted in nine strains that un-
derwent horizontal chromosome transfer (HCT) of chr-
RC (marked *). ree strains show large-scale chromo-
somal rearrangements (white arrowheads)
Read mapping shows specic
transfer of chrRC to Fo47
e SMRT genome assembly of Forc016 includes
eleven core chromosomes (light blue), sev-
eral repeat-rich, gene-poor accessory
regions (dark blue) and one chro-
mosome (red) enriched in can-
didate eector genes iden-
tied in [1]. Sequences
on this chromosome
show similarity to
Fol4287’s accessory
Forc016’s SMRT genome assembly
Chromosome transfer: pathogenicity Chromosome loss: avirulence
Conclusions
p.vandam@uva.nl