Clean caterpillars and grubby beetles: the role of oral secretions in

Clean caterpillars and grubby beetles: the role of oral secretions in

Cristina Rosa,
Plant Pathology and Environmental Microbiology Department,
Pennsylvania State University
Plant Phytobiome

The case of Ourmia melon virus: plant
interaction
Rosa Lab
2

Single amino acid
substitutions in the
movement protein
of Ourmia melon
virus can change
the symptoms
induced in
Nicotiana
benthamiana and
can modulate viral
movement. (Rosa
lab).
3
OuMV
MP
4

Protoplasts infected
with Ourmia melon
virus are
surrounded by
tubular protrusion
that can be
abolished by single
aa substitution in
the viral movement
protein (Rosa lab).
5


Can we ignore the interplay between plants
and everything else?
Note: the majority of plant viruses are
vectored by arthropods!
environment
vector
disease
host
pathogen
host
virus
environment
Real world?
6
What are the evolutionary implications of the Plant
Phytobiome?
7
Gary W. Felton
Department of Entomology
Pennsylvania State University
People working on this project: Seung Ho Chung, Jie Wang, Michelle Peiffer, Erin Scully, Kelli
Hoover, Cristina Rosa, Dawn Luthe.
8
9
MAMPs= microbe associated
molecular patterns
HAMPs= herbivore associated
molecular patterns
DAMPs= damage associated
molecular patterns
From Bruinsma (2014)
10
• Plants use hormone cross-talk to fine tune their
responses to their attackers
• Jasmonate and salicylate pathways are frequently
antagonistic
Harman et al. 2004; Nature Reviews Microbiology
11
Leptinotarsa decemlineata
Colorado potato beetle ‘CPB’
Helicoverpa zea
12
500
450
Identify component(s)
responsible for suppression
CysPI
Relative Expression
400
350
 Heat labile
300
 MW exclusion suggests
*
250
200
150
100
50
0
control
JA-responsive CysPI
wound wound +
only
OS
peptide
 Fractionation was
inconsistent
 Unable to identify an
insect-derived component
CHUNG ET AL. J CHEM ECOL 2011
13
Felton Lab
14
Felton Lab
15
Pretreat CPB
with antibiotic
for 48 h
CPB feeds 12 to
24 h on plant
Assay defense
gene expression
by qRT-PCR
AB(+)
AB(-)
16
Untreated larvae suppress anti-herbivore defenses
250
CysPI
50000
40000
30000
PR-1(P4)
a
b
20000
10000
Relative expression
200
150
100
c
0
1500
1200
b
900
600
300
500
Con
AB(-) AB(+)
PPOB
300
b
100
b
60
30
c
c
c
0
0
Con
a
a
400
200
90
Chung et al., 2013 PNAS
AB(-) AB(+)
a
Relative expression
Con
PPOF
Relative expression
b
50
c
0
1800
Con, undamaged plants;
AB(-), Plants damaged by untreated larvae
AB(+), Plants damaged by AB-treated larvae
a
PPO activity
mOD/min/mg tissue
Relative expression
60000
AB(-) AB(+)
0
Con
AB(-) AB(+)
Con
AB(-) AB(+)
JA-responsive CysPI and PPOF/B; SA-responsive pathogenesis-related protein 1 (P4), PR-1(P4)
17
Five day growth on treated plants
Chung et al., 2013 PNAS
18
Chung et al., 2013 PNAS

Collect regurgitant

Isolate 22 individual
colonies from regurgitant on
solid media

Test colonies on defense
expression

Identify bacteria by 16S
rRNA sequencing
19
(mOD/min/mg tissue)
PPO activity
100
80
60
*
**
**
***
40
20
0
CON
YT
I
CON= undamaged plants
YT= Wounding + media
S
A
R
L
D
I, S, A, R, L, D = bacteria from OS
• Screened 22 isolates followed by16-S r-RNA sequencing
JA-responsive PPO
Chung et al. (2013) PNAS. 20



Pseudomonas sp.
Enterobacter sp.
Stenotrophomonas sp.

All are gram negative bacteria
21
2h
Genomic DNA from leaves
Quantification of rpoD
using qPCR
Relative Pseudomonas Abundance
AB-treated/untreated larvae
500
rpoD
400
300
200
100
*
0
AB(-)
AB(+)
Pseudomonas rpoD (sigma
factor subunit of RNA
polymerase)
• rpoD gene was not detectable in undamaged plants.
AB treatment decreased abundance of Pseudomonas sp. in CPB.
Chung et al., 2013 PNAS
22
Pretreat CPB
with antibiotic
for 48 h
Re-inoculate
bacteria with
feeding for 6 h
Transfer CPB to
the plant for
assay
AB(+)
AB(-)
Chung et al., 2013 PNAS
23
PPO activity
PPO activity
PPO activity
JA-responsive PPO
Chung et al., 2013 PNAS
24
What is is the mechanism of suppression?
25
Chung et al., 2013 PNAS
2h feeding
0
2
4
24
48
Harvest
400
250
cis-JA (ng/g FW)
A
150
100
B
50
0
A
B
C
C
A
A
N.S.
A
SA (ng/g FW)
Con
CPB AB(-)
CPB AB(+)
200
300
200
N.S.
A
B
B
B
A
B
B
B
Con
CPB AB(-)
CPB AB(+)
100
B
0
24
24
Time (h) after placing insects
48
24
24
Time (h) after placing insects
48
26
Moneymaker
NahG
60
c
1000
a
500
b
c
6000
4000
2000
0
1000
Con AB(-) AB(+)
a
100
80
b
60
40
20
c
800
8
a
6
4
a
2
0
120
Con AB(-) AB(+)
PR-1(P4) a
Con AB(-) AB(+)
a
PPOB
a
a
600
400
200
100
a
80
60
40
20
b
b
0
Con AB(-) AB(+)
b
PPOF
b
0
Relative expression
10
Relative expression
20
8000
10
0
Con AB(-) AB(+)
0
Con AB(-) AB(+)
Con AB(-) AB(+)
JA-responsive CysPI and PPOF/B; SA-responsive pathogenesis-related protein 1 (P4), PR-1(P4)
Chung et al., 2013 PNAS
1500
30
a
10000
12
a
CysPI
12000
PPOB
a
2000
40
0
120
Con AB(-) AB(+)
PPOF
50
Relative expression
b
20000
0
2500
Relative expression
Relative expression
40000
Relative expression
Relative expression
60000
14000
PR-1(P4) a
Relative expression
a
80000 CysPI
27
Recognition of Biotic Signals:
DAMPs (damage-associated
molecular patters)
HAMPs (oral secretions or
herbivore-associated molecular
patterns)
MAMPs (microbe-associated
molecular patterns)
MAMPs include elongation factor
Tu (EF-Tu), lipopolysaccharide (LPS),
flagellin, etc.
28
The tomato
flagellin receptor
recognizes a 15
amino acid motif
SA-responsive PR-1
is induced
From Henry et al. 2013 New Phytologist
29
30
Pseudomonas flagellin at different conc.
*Pseudomonas flagellin contains the 15 amino acid motif
recognized by the tomato flagellin receptor
JA-responsive PPO
Chung et al., 2013 PNAS
31
Does bacterial suppression occur in other Solanum host
plants of the Colorado potato beetle?
32
Subgenus Solanum sensu stricto
Section Lycopersicon
Tomato group
Currant Tomato
Wild Tomato
S. pimpinnelifolium S. lycopersicum var. cerasiforme
Tomato
S. lycopersicum
Subgenus Leptostemonum
Section Melongena
Eggplant group
Buffalobur
S. rostratum
Horsenettle
S. carolinense
Eggplant
S. melongena
Subgenus Solanum sensu stricto
Section Petota
Potato
Felton Lab
Potato
S. tuberosum
33
Bacterial suppression in other host plants
Exp. 1: Reared on tomato, transferred to other hosts
34
Defense suppression by symbiotic bacteria from CPB
2000
1500
c
500
0
AB(-)
3000
2000
b
c
1000
AB(+)
AB(-)
b
80
60
C
40
20
0
6000
b
4000
C
AB(-)
AB(+)
600
JA-responsive PPO
AB(+)
a
Eggplant
500
400
300
b
b
200
100
Con
AB(-)
Con
AB(+)
PPO activity
mOD/min/mg protein
1200
AB treated/untreated
larvae
AB(-)
0
0
Con
500
700
8000
2000
c
1000
Con
a
Horsenettle
PPO activity
mOD/min/mg protein
100
b
1500
Felton Lab
Buffalobur
2000
AB(+)
10000
a
a
Tomato
0
Con
140
PPO activity
mOD/min/mg protein
4000
0
Con
120
a
PPO activity
mOD/min/mg protein
b
2500
1000
Wild T
PPO activity
mOD/min/mg protein
3000
2500
5000
a
Currant T
PPO activity
mOD/min/mg protein
PPO activity
mOD/min/mg protein
3500
AB(-)
AB(+)
a
Potato
b
1000
800
600
c
400
200
0
Con
AB(-)
AB(+)
35
Bacterial suppression in other host plants
Exp. 2: Reared on respective hosts throughout experiment
36
Defense suppression occurred only in cultivated hosts”
Fold change to control
Cultivated hosts
Wild hosts
AB(-)
3
2
AB(+)
*
*
1
0
Tomato
Potato
Eggplant
Buffalobur Horsenettle Nightshade
PPO activity of control was set to 1 in each Solanum plant.
* , Significant difference between AB(-) and AB(+)
JA-responsive PPO
Felton Lab
37
• When larvae were reared on wild hosts, the larvae could not
manipulate defense response.
 the larvae secreted gut bacteria.
 the larvae secreted similar amounts of OS.
• What caused the specific responses of defense manipulation?
 Host plants change microbial communities.
 Wild host-fed larvae could have less abundant bacteria
that are involved in defense suppression.
Gut DNA extraction
16S rRNA V3-V4 region
Miseq
RDP classifier
10-12 days
Felton Lab
38
Relative adundance of each family
Host plants affect microbial communities of CPB
100%
80%
Burkholderiaceae
60%
Bradyrhizobiaceae
Enterobacteriaceae
40%
Lactobacillaceae
Spiroplasmataceae
Sphingomonadaceae
20%
Pseudomonadaceae
Streptococcaceae
0%
Xanthomonadaceae
Felton Lab
39
Tomato (T), Potato (P), Eggplant (E), Horsenettle (HN), S. rostratum (SR), S. dulcamara (SD)
HN
HN
HN
E
P
E
SR
SR SR
SD
E
P
P
SD T
SD
T
T
Non-Metric
Multidimensional Scaling
Felton Lab
40
A.'
regurgitant'
midgut'
flagellin'
B.'Beetles rely on subterfuge using beneficial oral bacteria to

suppress plant defenses
regurgitant'

Host plant plays an important
midgut'role in determining gut bacterial
FACs'
communities

saliva'
Microbial mediation of inducedsignaling'
plant defenses to herbivores
has
GOX'
been overlooked
ATPases'
Phosphatase'
41
42
43

Can we integrate climate and phytobiome in a
microbial envelope?
44
Assistant Professors of Plant Pathology and
Environmental Microbiology
(75% Research, 25% Teaching)
Phytobiomes
Microbial Ecology
Contact: David Geiser; pick up a flyer!
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