10th November 2016

Root transcriptome analysis of resistance against

Phytophthora cinnamomi using RNA-seq and the

association of β-cinnamomin elicitin with virulence

Md Tohidul Islam

PhD student

Deakin University

Part-1: Root transcriptome analysis of resistance

against Phytophthora cinnamomi using RNA-seq

Part-2: Association of β-cinnamomin elicitin with

virulence of Phytophthora cinnamomi

Background to this study

Phytophthora cinnamomi is a soil borne pathogen

This oomycete causes die-back or root rot disease in plants

P. cinnamomi is known to infect numerous plant species across a broad range of plant families,

many of them native to Australia

Very few plant species are resistant

P. cinnamomi infected Xanthorrhoea

australis plant

Phytophthora root rot on avocado Phytophthora root rot on pineapplePhytophthora root rot on chestnut

Protecting susceptible species against P. cinnamomi

Phosphite is a systemic fungicide and is used to control P. cinnamomi

Treatment with phosphite is not a long term control method and it sometimes causes phytotoxicity and the

pathogen may develop resistance

An improved understanding of key mechanisms employed by resistant species is necessary to design durable

control methods for P. cinnamomi

Application of phosphite by light aircraft to infested vegetation in the Anglesea

heathlands Ref: Cahill et al. 2008

Some plant species are highly resistant to P. cinnamomi

Some native species have been identified as field resistant species to P. cinnamomi

Very little information is available about resistance responses

No information available about resistance-related genes in these species

Lomandra longifolia L. filiformis Gahnia radula G. sieberiana Corymbia calophylla

Islam et al., 2017. Active defence by an Australian native host, Lomandra longifolia, provides resistance

against Phytophthora cinnamomi. Functional Plant Biology, 44: 386-399.

Lomandra longifolia showed highly resistant to P. cinnamomi

Callose, lignin and hydrogen peroxide are associated with resistance

Resistance-related genes (GST, PAL, CHS, CSs) were induced in L. longifolia roots

Resistance-related genes in plants to P. cinnamomi

Microarray analysis of Zea mays infected with P. cinnamomi (Allardyce et al. 2013)

Next Generation sequencing is a powerful tool for transcriptomic research

To date only two studies published using next generation sequencing for interactions with P. cinnamomi

Castanea crenata candidate genes were associated

with a number of functional groups

Representative putative defence-related genes

present in avocado dataset

No one has analysed transcriptome of native species using RNA-Seq analysis

Avocado root (highly

tolerant variety DUSA)

infected with P. cinnamomi

Engelbrecht and van den

Berg 2013Santos et al. 2014

Castanea hybrid infected

with P. cinnamomi

Primary response of root to P. cinnamomi

Transcriptome analysis of the native species, L. longifoliaL

. lo

ng

ifo

lia

Lu

pin

us

an

gu

sti

foli

us

Control Inoculated

Resis

tan

t p

lan

t

Su

scep

tib

le p

lan

t

120 h

pi

120 h

pi

Illumina HiSeq data

Harvested root

samples at 0

hpi, 6 hpi and 24

hpi

Raw reads

Summary of RNAseq data of control and P. cinnamomi

inoculated Lomandra longifolia root

Average raw reads - 31,097,665

Read length - 126 bp

% GC - 46.5

De novo assembly statistics of L. longifolia transcriptome

Average contig number - 66,143

Mean contig length - 633 bp

Maximum contig length - 11,148 bp

Minimum contig length - 200 bp

GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway

analysis of selected L. longifolia transcripts at 6 hpi

GO analysis of L. longifolia contigs generated 3 main

categories

1. Biological process

-Biosynthetic process

-Cellular protein modification process

2. Molecular function

-Nucleotide binding

-Hydrolase activity

3. Cellular components

-Plastid

-Membrane

Biosynthesis of antibiotics

Purine metabolism

Starch and sucrose

metabolism

Phenylpropanoid biosynthesis

Phenylalanine metabolism

Flavonoid biosynthesis

KEGG analysis of L. longifolia contigs generated 25

categories

Contig

number Predicted function/Gene Fold change at 6 hpi Reported function, notes

254 Glutathione s-transferase 274

GSTs have multiple function in plant pathway including

Antioxidative activity

2002 Subtilisin-like proteases 13.00

Act as a receptor to activate downstream immune signalling

process

13320 2-alkenal reductase 10.53 Antioxidative activity

11336 Callose synthase-7 9.1 Callose biosynthesis

Representative defence-related transcripts identified through Blast analysis

A. Plant defence network associated transcripts

B. Plant hormone biosynthesis and signalling transcripts

Contig

number Predicted function/Gene Fold change at 6 hpi Reported function, notes

25514 Cytochrome 450 like protein 37.75

Involved in biosynthetic reaction of plant hormones and

defensive compounds

13026 RNA binding KH domain containing protein 23 Jasmonic acid (JA) signalling pathway

15766 F-box protein 23

Important receptor and signalling components in plant

hormone signalling pathway

11284 Putative 12-oxophytodienoate reductase 5 11.4 Jasmonic acid biosynthetic pathway

30984 Lipoxygenase 5 5.04 Jasmonic acid biosynthetic pathway

o Induction of these genes suggested the involvement of callose and H2O2 in L. longifolia resistance

o Induction of these genes suggested the involvement of JA in L. longifolia resistance

Conclusions for Part-1

L. longifolia transcripts is involved in important gene ontology category

L. longifolia induced different layer of resistance in response to P. cinnamomi

A range of transcripts involved in antibiotic biosynthesis in L. longifolia root

The induction of jasmonic acid pathway genes indicate the involvement of JA in L. longifolia resistance

Islam MT, Hussain HI, Rookes JE, Cahill DM. 2017.Transcriptome analysis, using

RNA-seq, of Lomandra longifolia roots infected with Phytophthora cinnamomi

reveals the complexity of the resistance response. Accepted to publish in Plant

Biology

Part-2: Association of β-cinnamomin elicitin with

virulence of Phytophthora cinnamomi

Elicitins of Phytophthora can act as both avirulence and virulence factor

Elicitins are 10 kDa protein

P. cinnamomi secretes elicitin family protein cinnamomins whose biological role remain unclear

P. cinnamomi has gene cluster consisting of four elicitin genes

β-cinnamomin silenced (FATSS) strain of P. cinnamomi scored as weakly virulent pathogen

β-Cinnamomin structure

Rodrigues et al. 2006Cell-wall and membrane associated proteins

Cyt

op

lasm

Released into the medium

Released into the medium

Released into the medium

Horta et al. 2008

Analysis of the specificity of the β-

cinnamomin antiserum using western-blotβ-cinnamomin production in Lupinus

angustifolius infected with P. cinnamomi

Tris-tricine gel and western blot

Cell wall extract protein

and M1 medium extract

protein showed band at 10

kDa level

M1 extract protein (elicitin)

reacts only with β-

cinnamomin antiserum

Red channel Merged Blue channel Merged

Transverse root sections harvested at 72 hpi and labelled

with β-cinnamomin and Phytophthora antiserum. Red colour

indicates the presence of β-cinnamomin and blue colour

indicates the presence of Phytophthora.

o β-cinnamomin is produced through out the inoculated root

tissue

Effect of β-cinnamomin antiserum on lesion formation on L. angustifolius infected

with P. cinnamomi

Inoculation with pre-treated zoosporeswith the β-cinnamomin antiserum inLupinus angustifolius roots revealed apartial loss of virulence

A. Water B. Zoospores

C. β-cinnamomin antiserum (1:100 dilution)

D. β-cinnamomin antiserum (1:300 dilution)

E. β-cinnamomin antiserum (1:1000 dilution)

F. Pre-immune serum (1:100 dilution)

Zoospores incubatedwith antiserum for 1hour

Inoculate roots andmeasure lesionlength until 120 hpi

Conclusions for Part-2 (Elicitins)

• β-cinnamomin antiserum is specific to react with only β-cinnamomin

• P. cinnamomi produces β-cinnamomin which is involved in susceptibility in Lupinus angustifolius

• The mechanism of β-cinnamomin that is involved in induced susceptibility is not known.

Acknowledgements

Prof. David Cahill

Dr. James Rookes

Dr. Hashmath Hussain

DUPRS scholarship

Thanks for your attention

Overview of methodology

Lomandra longifolia root

Inoculated with P.

cinnamomi

Inoculated with water

Harvested root samples at 0

hpi, 6 hpi and 24 hpi

Isolated RNA from root

samples and quality check

Sent RNA samples to

Marcogen, South Korea for

sequencing

Quality control of sequencer

generated reads using CLC

Genomics workbench

Assembled read into contigs

Differentially expressed gene

(DEG) analysis using contigs

Functional description of

contigs using Blast2go

program

Quantitative PCR to validate

RNA-seq DEG data

Assigned contigs in GO terms