Robert D. Havener Seminar:
CIAT, September 14, 2017
“Innovations for
Crop Productivity”

Pieces of the phytobiome:
CIAT
September 14, 2017
Multitrophic and environmental
influences on plant health
Jan E. Leach
Colorado State University

Phytobiomes are complex
Phytobiome:
• The interaction of the
environment and
living organisms that
influence or are
influenced by plants
Plant Microbiome:
• The dynamic
community of
microbes associated
with plants
Phytobiome

It’s
bacteria!
It’s a
SYSTEM!!!
Embrace complexity!

Communication among phytobiome
members Leach et al. 2017 Cell

Achieve sustainable crop productivity
through a systems-level understanding
of diverse interacting components

INTERNATIONAL ALLIANCE
FOR PHYTOBIOMESRESEARCH
For More Information:
www.phytobiomesalliance.org
Kellye Eversole
eversole@eversoleassociates.com

Phytobiomes Alliance Goals
Identify research gaps and
help coordinate projects to
address those gaps
Establish national, international,
and multi-national public-private
projects and networks
www.phytobiomesalliance.org

Two phytobiome stories:
What is the impact of
high temperature on
plant-microbe
interactions?
Do microbes
facilitate aphid
virulence to plants?

In the phytobiome, biotic and abiotic
stresses impact plant health
Temperature Stress
Drought
Salinity
Nutrient Stress
Abiotic Stresses Biotic Stresses
Pathogen Attack
Pest Attack
Competition

…and those stresses are usually
simultaneous
Heat Stress Heat Tolerance
Pathogen Attack Disease Resistance
Disease

“Warming in the climate system is
unequivocal…..”
International Panel on Climate Change
IPCC 2007, Climate
Change Synthesis Report
Battisti and Naylor, 2009. Science

Colorado burning: 2010-2012
Colorado flooding: 2013 & 2015
“Warming in the climate system is unequivocal…..”
International Panel on Climate Change (2007)
“For the major crops (wheat, rice, and maize) in
tropical and temperate regions, climate change
without adaptation is projected to negatively impact
production ….“
Intergovernmental Panel on Climate Change, 2014
(http://www.ipcc.ch/report/ar5/)

Rice production is negatively
affected by high temperatures
PNAS 2004 101:9971-9975
PNAS 2010 107:14562-7
Nature Climate Change
2012 3:288-291
In
Rice yield ↓
CH4 ↑
Increasing
temperatures:

Rice bacterial blight disease pressure
increases with increasing temperature
• High BB disease in hot
season; little disease in
cool season
• Longer BB lesions at high
than low temperature
regimes in controlled
environments
HT
LT
HT
LT
IR24 (no R gene)
Xoo
PXO145
35°C day/29°C night = High
29°C day/21°C night = Low
What is the influence of
high temperatures on
resistance to BB disease?

Most sources of resistance to bacterial
blight of rice are single R genes
Over 30 R genes identified

Most rice BB resistance genes lose efficacy
at high temperatures
Webb et al., 2010;
Balidion et al., unpubl.
0.5
1.5
2.5
3.5
Xa3 Xa4 xa5 Xa7 Xa10
Lesionlength(cm)
35°C day/29°C night HIGH
a
b
a
b
a
b b
a a a
29°C day/21°C night LOW
Rice bacterial blight resistance gene
EXCEPT Xa7

Why are plants more susceptible to
bacterial blight at high temperatures?
Cohen et al. submitted
Why is resistance gene Xa7 more effective
at high temperatures?
RNASeq analysis
Temperature Regimes (day/night)
High = 35/29° C ; Low = 29/23° C
IRBB61 (Xa7)
3, 12 & 24 hpi

Impact of high temperatures on disease
and resistance
72 h post-inoculation
Cohen et al. submitted
9
5
8
6
7
log10CFU/leaf
Hours post-inoculation
0 48
a a
b
c
a a
a
a
XoX11-5A
(emptyvector)
XoX11-5A
(avrXa7)

Rice abscisic acid (ABA) responsive genes are
upregulated at high temperatures
log2 fold change High Temp relative to Normal Temp
kerneldensityestimate
Mock treated
ABA biosynthetic and downstream genes
Cohen et al. submitted

Abscisic acid, thermotolerance, and
disease
• ABA is important for plant adaptation to abiotic stresses
• ABA is generally antagonistic to salicylic acid in rice1,2
• ABA treatment leads to increased BB disease severity3
ABA
Abiotic
Stress
Adaptation
to stress
SAResistance
Biotroph
attack
1Jiang CJ et al. 2010. MPMI; 2Meguro & Sato 2014. Sci Rep; 3Xu J et al. 2013. PLoS ONE.
What is the relationship of the
ABA response, high
temperature, BB disease, and
Xa7-mediated resistance?

ABA responsive genes are upregulated by high
temperature during susceptible interactions
kerneldensityestimate
Mock treated
Susceptible
(no Xa7-avrXa7
interaction)
ABA responsive genes
Cohen et al. Submitted
log2 fold change High Temp relative to Normal Temp

ABA responsive genes are downregulated by
high temperature during resistant interactions
kerneldensityestimate
Mock treated
Susceptible
interaction
ABA responsive genes
Resistant
(Xa7-avrXa7
interaction)
log2 fold change High Temp relative to Normal Temp
Cohen et al. submitted

ABA responsive element (ABRE)-like motif is
enriched in promoters of genes differentially
regulated by high temperature
Cohen et al. submitted; ABRE motif from Gómez-Porras et al. 2007.
ABRE
Novel motif
Upregulated in susceptible interaction
Downregulated in resistant interaction
Enriched in promoters of genes:

Summary (part 1):
• ABA biosynthetic and responsive genes :
– Are activated by both heat and combined heat and disease stresses
– Are suppressed by Xa7- mediated resistant interactions at high
temperature
– Contain a novel motif, similar to the ABRE, in their promoters: related to
high temperature responsiveness
• H0: ABA is an important node for cross-talk between plant
response pathways to high temperature and stress and
pathogen attack
Why do we care?
• Identify/create sources of resistance that are effective and durable at high
temperatures
• Facilitate planning/breeding for sustainable crop production under
conditions of increasing global temperatures

Do microbes facilitate insect
feeding on plants?
Embracing the complexity!

Who’s in the Phytobiome?
Who’s in
Whoville?

Tripartite interactions:
Insects-microbes-plants
• Bacteria associated with the
Colorado potato beetle
manipulate plant defenses
to facilitate beetle feeding
• Microbiome composition
differs if larvae are raised on
different plant hosts
• Different microbiome
composition affects ability
of larvae to manipulate
plant defense
S-H Chung et al. 2013 PNAS; 2017 Sci Reps

Aphids use stylets to probe/feed:
https://vimeo.com/64269766
By Kimberly Falk

Russian wheat aphid salivary proteome
contains no known effectors
Luna, Van Eck, Campillo et al., in prep

Russian wheat aphid salivary proteome
99% bacterial proteins!
Luna, Van Eck, Campillo et al., in prep

Aphid stylets harbor bacteria
Luna et al. unpubl.
http://www.plantphysiol.org/co
ntent/158/4/F1.medium.gif

Diets
Aphids
Bacteria isolated from aphids, saliva
(diets) and infested leaves
Leaves
Enterobacteriacae
Moraxellaceae

0.05
Bacteria isolated from aphids, saliva and infested
leaves match proteome predictions
Predicted bacterial genera
in salivary proteome

• Do bacteria associated
with Russian wheat aphid
enhance aphid virulence
to wheat?
Aphids-Bacteria-Plants
Luna et al., in review

Abducted aphid babies:
Fewer bacteria than mom
Clean Dirty DirtyClean
Clean aphids = less bacteria
Log10(CFU/ml)
Luna et al. in prep

Dirty aphid microbiome: more diverse and
more Enterobacteriacea and Moraxellaceae
Bifidobacteriaceae
Prevotellaceae
Weeksellaceae
ChitinophagaceaeRuminococcaceae
Caulobacteraceae
Rhodobacteraceae
Acetobacteraceae
Sphingomonadaceae
Aeromonadaceae
Enterobacteriaceae
(Buchnera)_
Enterobacteriaceae
(non-Buchnera)
Moraxellaceae
Pseudomonadaceae
Brevibacteriaceae
Weeksellaceae
Streptococcaceae
Lachnospiraceae
Ruminococcaceae
Veillonellaceae
Caulobacteraceae
Methylobacteriace
ae
Moraxellaceae
Enterobacteriaceae
(Buchnera)
Other
Dirty aphids
Clean aphids
• Buchnera dominates
• Dirty aphid microbiome
more diverse & enriched
in Enterobacteriaceae
and Moraxellaceae

Clean Dirty
DirtyClean Clean
Aphid num
Chloroticleafarea(%)
Totalaphidnumber
Clean aphids = less chlorosis
*
‘Clean’ aphids cause less damage to
wheat than ‘Dirty’ aphids!
Luna et al. in prep

Are RWA bacterial associates virulent
to wheat?
• Bacteria do not cause disease symptoms
(water-soaking, chlorosis, etc) or HR when
infiltrated into wheat leaves
Erwinia sp.
Pantoea sp.
Arthrobacter sp.
Water
Enterobacter sp.
E. coli

How do bacteria facilitate aphid virulence?
Transcriptome analysis
Salicylic acid (SA)Jasmonic acid (JA)
log2 fold change
-8 80 4-4
Top: 5 h
Bottom: 48 h
Luna et al. in prep
By 5 h, both JA and SA
genes are induced
48 h, JA down and SA
genes still induced
• Consistent with the SA pathway being
antagonistic to the JA pathway

Dirty aphidsClean aphidsUntreated plants
ng/gFWSA
ng/gFWJA
Trend for lower JA at 48 h
Bacteria contribute to induction of SA
How do bacteria facilitate aphid
feeding/virulence?
Luna et al., in prep
Clean aphids induce less SA than dirty

• H0 : Bacteria associated with
Russian wheat aphid
manipulate plant defenses to
enhance aphid virulence
Managing the phytobiome to control
plant pests
Luna et al. in prep
Phytobiome knowledge can guide
management strategies
• Should breeding programs/treatment regimes target
the bacteria rather than the insect?
• Can the wheat leaf microbiome be altered to protect
plants from aphid feeding?
• Have Russian wheat aphids evolved to ‘rely’ on
bacterial effectors for virulence?

Embrace the complexity!

Pieces of the phytobiome:
Leach Lab members
Emily Luna
Tony Campillo
Leon van Eck
Stephen Cohen
Rene Corral
Hongxia Liu
CSU Insectary
Jeff Rudolph
Frank Peairs
Collaborators
Cris Argueso
Paul Ode
Anna-Maria Botha
Michelle Cilia
Nora Lapitan
Ned Tisserat
Valerie Verdier
Casiana Vera Cruz
Electron Microscopy
Kim Vanderpool

INTERNATIONAL ALLIANCE
FOR PHYTOBIOMESRESEARCH
For More Information:
www.phytobiomesalliance.org
Kellye Eversole
eversole@eversoleassociates.com