Steven Lindow - Biocontrol 2016

The many cell density-dependent behaviors of
Xylella fastidiosa: achieving disease control
via pathogen confusion
Steven Lindow
Department of Plant and Microbial Biology
University of California, Berkeley
Blue-green sharpshooter
Graphocephala atropunctata
www.CNR.Berkeley.EDU/xylella/
Glassy-winged sharpshooter
Homalodisca coagulata
Citrus Variegated Chlorosis
From Katajima
Plant Physiology 161:1529 (2013)
Many/most plants do not
excessively react to
X. fastidiosa?
SLURP!
Pit
Pit
Access from vessel to vessel through bordered pits is
blocked by the pit membrane
X. fastidiosa cells are most commonly
found in modest sized micro-colonies
within xylem vessels
Large colonies probably block flow
- plant can shuttle sap to
neighboring vessels
- bacteria will become starved for
nutrients/ O2
- insects avoid feeding from tissue
with extensive blockage
X. fastidiosa cells in heavily blocked regions of heavily infected
leaves are mostly dead
Does Xylella fastidiosa sense its numbers and prevent overgrowth in vessels?
Xanthomonas campestris pv. campestris
rpfA
rpfB
rpfF
76%
rpfC
70%
rpfA
rpfH
66%
orf orf rpfB
rpfG
58%
//
rpfF
lysS prfB
rpfD orf orf orf
rpfI
recJ
76%
rpfC
rpfE
55%
rpfG
lysS prfB recJ
rpfE
greA
Xylella fastidiosa
rpfB,F
rpfG,C
Synthesis of “diffusible signal factor” (DSF)
DSF perception and signal transduction leading to
virulence trait expression
C14-cis XfDSF1
C16-cis XfDSF2
Xcc DSF
greA
DSF-mediated gene regulation in Xylella fastidiosa
DSF
Signal
sensor
Periplasm
Activation
ofRpfG
response
regulator
RpfC
RpfC
+
rpfF
DSF Synthesis
328 genes
Outer
membrane
Inner
membrane
X. fastidiosa Decreases Virulence by Coordinating
Virulence Genes in Cell Density-Dependent Fashion
Wild type
rpfF mutant
rpfF mutants, which cannot signal, are severely impaired in
transmissibility by an insect vector
0.9
frequency of transmission
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Temecula (wild type)
KLN61 (rpfF mutant)
rpfF mutants cannot form the biofilm typical of X.
fastidiosa colonies in insect foreguts
wild type
rpfF mutant
Key Virulence Genes Controlled
by RpfF in culture
Type IV pili
Downregulated (virulence):
Type IV pili (twitching motility)
Polyglacturonase
Cellulase
Meng et al. 2005
Upregulated (anti-virulence genes):
Hemagglutinin adhesins
Extracellular polysaccharides (EPS): gum
Type I pili (anchoring)
Fraction of cells released
RpfF- mutants of Xylella fastidiosa that do not produce DSF
are not “sticky” in culture – and do not adhere as tightly to
plant tissue as WT strains
RpfF-
Inverse relationship between retention strength of X. fastidiosa
to grape xylem and their virulence to grape
%%Cells
Released
CellsReleased
4
3
0 2
1
0
Partitioning of the population of X. fastidiosa for mutually incompatible processes
by cell density signaling
Plant colonization phase
Active vessel colonization
Low cell numbers in most vessels
Disease symptoms may not be present
Insect acquisition phase
Some vessels have high cell numbers
Disease symptoms may be present
Further multiplication in crowded vessels slows
DSF Abundance
Expression of adhesins
Stickiness to Surfaces
Type IV pili
Twitching Motility
Pgl and Eng expression
Pit Membrane Degradation
Gum Production
Disease control
The goal: Confuse pathogen by exposing it to
excessive amounts of signal molecule even when it
is in low population sizes
The expectation: The premature presence of DSF in vessels
will suppress extracellular enzyme production and thus both
movement and multiplication, while increasing adhesiveness
and thus also reducing movement
How?
Endophytes that produce DSF
Direct application of DSF
Transgenic DSF-producing plants
Early - Inoculum becomes mobile and starts to grow and spread
Non-mobile
Mobile
Sharpshooter
Inoculation
Later- Extensive growth and spread of X. fastidiosa – some vessels become crowded
and accumulate DSF – suppression of further growth and movement in that vessel –
acquired by insects
Non-mobile
Mobile
Sharpshooter
Acquisition
In transgenic DSF-producing plant signal would be high at all times, restricting
growth and movement
Non-mobile
Sharpshooter
Inoculation
Symptomatic leaves/plant
Grape transformed with rpfF gene from Xylella fastidiosa
that produce DSF are much more resistant to Pierce’s Disease
compared to wild-type grape
10
8
Wild type
6
DSF-producing lines
4
2
0
-2
Plant Genotype
wild type
TRA1
TRA1
TRA3
TRA4
TRA5
TRA6
TRA7
TRA8
TRA9
Proportion of Vessels Colonized with X. fastidiosa
The movement of Xylella fastidiosa
in DSF-producing transgenic grape is reduced
WT Freedom
RpfF
Freedom
10
60
120
Stem Sampling Location (cm from POI)
Fraction of Cells Released
Cells of Xylella fastidiosa adhere much more tightly to xylem vessels of
RpfF-expressing Freedom grape
Wild Type
RpfF-expressing
Field trials of transgenic grapes established in Solano and Riverside counties
Incidence of infected shoots after inoculation much lower on RpfF-expressing Freedom
– Solano County 2012
WT
RpfF
Severity of PD much lower on RpfF-expressing Freedom – Solano County August, 2012
WT
RpfF
Large reduction in disease in DSF-producing plants: natural insect inoculation
- Riverside County October, 2012
DSF-producing
Normal grape
Incidence and severity of PD on RpfF Freedom much lower than wild type Freedom
– Riverside County, October 2012
WT
RpfF
WT
RpfF
Xylella fastidiosa has a promiscuous signaling system –
responds to several different unsaturated fatty acids
Palmitoleic acid is attractive as a commercially available signal for X. fastidiosa
XfDSF2
Topical applications of Palmitoleic Acid to alter X. fastidiosa behavior in grape:
Penetrating surfactants promising method to introduce the material
Injection
Biological Control:
Burkholderia phytofirmans colonizes grape xylem, unlike other bacteria tested
Inoculation of Burkholderia phytofirmans with Xylella fastidiosa greatly reduces
disease severity
Topical application of Burkholderia phytofirmans with penetrating surfactants
established populations within petioles
+ 0.2% Breakthru
Buffer alone
Biological control of Pierce’s disease by Burkholderia phytofirmans can be
conferred by various inoculation strategies
Even small amounts of culture supernatant of Burkholderia phytofirmans
induces a tenacious biofilm by Xylella fastidiosa
Note subterranean biofilm
Burkholderia phytofirmans apparently produces compounds related to DSF
Biofilm production in Xylella fastidiosa induced by non-polar compound
produced by Burkholderia phytofirmans
Cannot yet rule out that control is conferred by effect on plant
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