phytophthora cinnamomi using rna-seq and the - · pdf file10th november 2016 root...
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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
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