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