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Transcriptome of the foliar forest pathogenDothistroma septosporum.
APPS, Fremantle WA, September 2015Rosie Bradshaw, Yanan Guo, Andre Sim, Shahjahan Kabir, Pranav Chettri,
Kutay Ozturk, Lukas Hunziker, Rebecca Ganley* and Murray CoxBio-Protection Research Centre, Institute of Fundamental Sciences, Massey University, New Zealand
*Scion (forest research), Rotorua, New Zealand
Dothistroma needle blight,a global problem.
Drenkan et al (Dothistroma COST Action FP1102; manuscript in prep.) Emergence of an epidemic:Dothistroma needle blight in Europe and globally. A review of pathogen spread and host susceptibility.Map prepared by Petr Vahalík, Department of Forest Management and Applied Geoinformatics,Mendel University in Brno, Czech Republic (http://arcgis.mendelu.cz/monitoring/ )
Dothistroma septosporum – a hemibiotrophic Dothideomycete1-4 asymptomatic
1Spore
germination
2Epiphytic2-6 weeks
3Penetration
4Stomatalchamber
colonisation
5Mesophyll
colonisation& early lesions
5-6 symptomatic (lesions)
6Mature
sporulatinglesions
(8-12 weeks)
Kabir, M.S., Ganley R.J., and Bradshaw, R.E. (2015) The hemibiotrophic lifestyle ofthe fungal pine pathogen Dothistroma septosporum. Forest Pathology 45: 190-202
Why a transcriptome?
• To gain knowledge of fungalpathogen – gymnosperm interactionsat the molecular level.
• To identify key genes expressed inplanta that will help in developmentof new methods of disease control.
Prolific sporulation ofDothistroma septosporum
on radiata pine. [Photo:Kabir and Bradshaw]
Aim: To develop a time-series transcriptomics resource for D. septosporumby RNA sequencing of a semi-synchronized infection of P. radiata needles.
(8 w) (12 w)
Stages of dothistroma needle blight used for transcriptome samples.Early
(3 wpi)Mid
(8 wpi)Late
(12 wpi)epiphytic and penetration early lesions sporulating lesions
in vitrofungus(FM)
myceliumonly
ground wholeneedle samples
150lesions
150lesions
Bradshaw, R.E., Guo, Y., Sim, A., Kabir, M.S., Chettri, P., Ozturk, I.K., Hunziker, L., Ganley, R.L., Cox, M.P.,2015. Genome-wide gene expression dynamics of the fungal pathogen Dothistroma septosporumthroughout its infection cycle of the gymnosperm host Pinus radiata. Mol. Plant Pathol. In Press.doi:10.1111/mpp.12273
Dothistroma septosporum transcriptome statistics
Early(3 wpi)
Mid(8 wpi)
Late(12 wpi)
epiphytic and penetration early lesions sporulating lesions
Total reads
(plant and fungal)
Mapped fungal
readsa
% of total
readsb
Early 683,221,605 512,742 0.1%
Mid 1,033,815,851 871,668 0.5%
Late 17,146,358 1,883,653 17.1%
Total 1,734,183,814 3,268,063aIllumina reads mapped to D. septosporum JGI frozen gene catalogue(12,548 genes). Total from two biological replicates for each stage.bNormalised by comparison to fungal in vitro mapping rate
D. septosporum genes significantly up-regulated in planta
FM FM FM
E M L
Predicted protein typea FM-Early FM-Mid FM-Late
secreted proteins (all) 112 (19.2%) 94 (14.8%) 222 (17.7%)
oxidoreductase 88 (15.1%) 195 (30.7%) 258 (20.6%)
secondary metabolism 4 (0.7%) 29 (4.6%) 25 (2.0%)
CAZy 35 (6.0%) 30 (4.7%) 79 (6.3%)
SSCP 6 (1.0%) 10 (1.6%) 22 (1.8%)
Total genes up-reg in plantab 583 635 1253
Total DE genes c 1357 1592 2814aCAZy (carbohydrate active enzymes)SSCP (short secreted cysteine-rich proteins)
bNumber of genes up-regulated in planta compared to in vitro (FM)cNumber of differentially expressed (DE) genes (up- or down-regulated)
Only a few genecategories areshown here.
Bradshaw et al (2015) MPP In Press
E M
Differential expression of geneontology (GO) groups in planta
up- or down-regulated:early to mid stage, 1172 genesmid to late stage, 977 genes
BUT first transition
M L (biotrophic/epiphytic tonecrotrophic) entails muchbroader range of GO terms(biological process)
Bradshaw et al (2015) MPP In Press
REVIGO (Supek et al 2011; PLoS One 6: e21800) circlesize indicates frequency; shading indicates probability
Fig. 3: Differentially expressed gene ontology (GO) groups in planta (REVIGO maps)
Heat maps of gene expressionbetween stages in planta
Waves of gene expressionsimilar to other hemibiotrophs,i.e. stage specificity
Valuable resource to search forvirulence mechanisms at eachdisease stage
Focus on:1) Secondary metabolites (SM)2) Carbohydrate active enzymes (CAZy)3) Effectors (SSCP) Log2 fold increase
or decreasecompared to mean
Bradshaw et al (2015) MPP In Press E M L
1) Secondary Metabolites.Dothistromin toxin is a virulence factor
OH
HO O O
OH O OH
O OH
Dothistromaseptosporum
wild-type
Late stage lesions onPinus radiata needles
D. septosporumdothistromin KO
mutant(6-fold
reduction insporulation)
Pinus radiata cross-section withgfp-labelled D. septosporum
Kabir, M.S., Ganley R.J., and Bradshaw, R.E. (2015) Dothistromin toxin is a virulence factor indothistroma needle blight of pines. Plant Pathology 64: 225-234. Doi: 10.1111/ppa.12229
Read
sper
milli
onpe
rkilo
base
Expression of core secondary metabolite genes in planta
1000
100
10
Early
Mid
Late
1PksA Pks1 Pks2 Pks3 Nps1 Nps2 Nps3 Hps1 Hps2 Tub1
Dothistromin core geneexpression coincides with
production
Pks: polyketide synthase (PKS)Nps: non-ribosomal peptide synthase (NRPS)Hps: Hybrid PKS-NRPSTub: beta tubulin
E-M and M-L differences all significant except Pks3 E-M and Hps1 E-M,M-L
Read
sper
milli
onpe
rkilo
base
Expression of core secondary metabolite genes in planta
1000
100
10
Early
Mid
Late
1PksA Pks1 Pks2 Pks3 Nps1 Nps2 Nps3 Hps1 Hps2 Tub1
Dothistromincore gene
Nps3(cyclosporin
synthase family)
PksA(dothistromin)
Pks1 (melanin-like)Pks2 (fumonisin-like)
Kutay Ozturkposter
Early Mid Late
2) Carbohydrate-active enzyme (CAZy) genes
Predicted numbers of CAZy genes:
CAZy gene Dothistroma Cladosporiumfamily* septosporum fulvum
(hemibiotroph) (biotroph)
GH (glycosyl hydrolases) 201 274PL (polysaccharide lyases) 4 9CE (carbohydrate esterases) 23 35
Different types of GH enzymes have different substrates:fungal cell wall or plant cell wall components, or 'energy'
*www.cazy.orgTable adapted from de Wit et al (2012) PLoS Genetics 8(11): e1003088
Expression of some classes of CAZy genes in planta.
GH11 xylanase Ds137959 (4.5-fold up E to M)65% aa identity to Zymoseptoria tritici Xyl1
hemicellulose
Fewer PCW-modifying enzymesthan other Dothideomycetes
Early (E) Mid (M) Late (L)
E M L each line represents one gene
Expression of some classes of CAZy genes in planta.
fungal cell wall
6th & 19th most highly expressedof all genes at early stage(beta glucanases GH17, GH64)
- cell wall re-modeling?- competition with other fungi?
Early (E) Mid (M) Late (L)E M L
Log 2
RPM
Kenergy Expression of CAZy
genes in planta
GH13 alpha amylase genesDs75147 (16-fold up M to L)Ds70643 (29-fold up M to L)
Early (E) Mid (M) Late (L)
dothistromindeficientmutant
D. septosporumwild-type
Utilisation of starchin green islands?
3) Effectors, eg. Cladosporium fulvum Avr4- 'assists' in disease process (virulence function)
[by protecting fungal cell wall from plant chitinases]- triggers resistance if recognised by host (avirulence function):
Host immune receptor(Resistance) gene eg. Cf-4
Cf-4 no Cf-4
Fungalpathogen
avirulence geneeg. Avr4
Avr4R
noAvr4
Avr4 51.7%Ecp2-1 59.8%Ecp2-3 52.7%Ecp6 68.6%Ecp4/5 pseudogenes in D. septosporum
D. septosporum effectors are recognised by tomato Cf receptors
Orthologues ofC. fulvum effectors
% amino acid identity of D. septosporumand C. fulvum effector candidates
de Wit et al (2012) PLoS Genetics 8(11): e1003088
Agrobacterium-mediatedtransient assay with
D. septosporum DsAvr4shows a resistance responsein Cf-4 transgenic tobacco.
RPM
K
Low expression of most D. septosporum orthologs of Cf effectors
10000
D. septosporumAvr4 down-regulated to
evade detectionin pines??
1000
100
10
In vitro
Early
Mid
Late
1Avr4 Ecp2-1 Ecp2-3 Ecp6 Act1
C. fulvum effectors expressed athigher levels than actin in planta onlyde Wit et al (2012) PLoS Genetics 8(11): e1003088
Other D. septosporum effector candidates in the genome
Analysed SSCPs (short secretedcysteine-rich proteins)
Generally higher expression at laterather than early stage compared toother studied hemibiotrophs
Early: biotrophic functions?
Mid and late: necrotrophic functions?
Numbers of D. septosporum effectorcandidate genes up-regulated in planta.
Gene expression in planta helps to determine best effector candidates
Gene in vitro Early Mid Late Predicted function/notesDs69335
Ds70057
426
311
1448 1012 554 SCP/TAPS superfamily
467
491
2569 3312 not known
C-type lectin / most highlyexpressed gene at late stagecerato-platanin / defence-related
Ds72737 41 245 8995
Ds70155 257 71
Ds131885 1 9
507
369
1303
203 not known
Gene expression reads per million per kb (RPMK), mean of two biological replicates
Key challenge for the future: effector delivery to pine tissue.
Development of methods to test effector protein recognition in pines
In progress
• vacuum infiltration
• needle injection
• pine tissue culture
• agroinfiltration (of tobacco)
Conclusions
• Gene expression dynamics of a fungal gymnosperm pathogenwere revealed.
• Deep sequencing was necessary due to low fungal reads inearly and mid stages.
• Stage-specific expression was shown for many genes,including potential virulence and avirulence factors.
• Development of a system to test protein function on pinetissue will assist functional analysis of these genes.
Acknowledgements New Zealand collaborators:Murray Cox, Carla Eaton, Kee Sohn,IFS, Massey University, Palmerston NorthCarl Mesarich, Plant and Food, AucklandBeccy Ganley & Rebecca McDougal, Scion, Rotorua
Lab Dothistromateam:Shahjahan KabirPranav ChettriMelissa GuoKutay OzturkLukas Hunziker
Financial support:NZ Bio-Protection Research CentreMassey UniversityWillie Commelin Scholten FoundationDutch Experimental Plant Sciences FoundationCOST Action FP1102 (Dothistroma)
Overseas collaborators:Pierre de Wit, Wageningen UniversityDothideomycete Comparative Genomics Consortium
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