Detailed studies of plant pathogens can lead to

novel means of disese control: The case of

Xylella fastidiosa in grape and citrus

Steven Lindow

Department of Plant and Microbial Biology

University of California, Berkeley

Blue-green sharpshooter

Graphocephala atropunctata

Glassy-winged sharpshooter

Homalodisca coagulata

Xylella fastidiosa is obligately vectored by various xylem-feeding insects

Xylem pits enable by-pass of water around blocked vessels

Plant Physiology 161:1529 (2013)

Many/most plants do not

excessively react to

X. fastidiosa?

SLURP!

Access from vessel to vessel through bordered pits is

blocked by the pit membrane

Xylella cell

Bordered pit opening

pit

membrane

X. fastidiosa cells are most commonly

found in modest sized micro-colonies

within xylem vessels

X. fastidiosa cells in heavily blocked regions of heavily infected

leaves are mostly dead

Xanthomonas campestris pv. campestris

Xylella fastidiosarpfB,F Synthesis of “diffusible signal factor” (DSF)

rpfG,C DSF perception and signal transduction leading to virulence trait expression

rpfA rpfB rpfF rpfC rpfH rpfG rpfDprfBlysS orf rpfIorforf recJ greArpfE

rpfA rpfB rpfF rpfC rpfG rpfE//orforf lysS prfB recJ greA

76% 70% 66% 58% 76% 55%

C14-cis XfDSF1

C16-cis XfDSF2

Xcc DSF

DSF

Activation

of response

regulatorRpfC

PeriplasmRpfC

DSF Synthesis

RpfG

rpfF

Outer

membrane

Inner

membrane

Signal sensor

+

DSF-mediated cell density-dependent gene

regulation in Xylella fastidiosa

328 genes

X. fastidiosa Decreases Virulence by Supressing

Virulence Genes in Cell Density-Dependent Fashion

Wild typerpfF mutant

More vessels

colonized &

More cells/vessel

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Temecula (wild type) KLN61 (rpfF mutant)

fre

qu

en

cy o

f tr

an

sm

issio

nrpfF mutants, which cannot signal, are severely impaired in

transmissibility by an insect vector

wild type rpfF mutant

rpfF mutants cannot form the biofilm typical of X.

fastidiosa colonies in insect foreguts

Key Virulence Genes Controlled by

RpfF-mediated cell-cell signaling

Downregulated (virulence):

Type IV pili (twitching motility)

Polyglacturonase

CellulaseMeng et al. 2005

Upregulated (anti-virulence genes):

Hemagglutinin adhesins

Extracellular polysaccharides (EPS): gum

Type I pili (anchoring)

Type IV pili

RpfF-

Fra

ction o

f cells

rele

ased

RpfF- mutants of Xylella fastidiosa which do not produce DSF

are not “sticky” in culture – and do not adhere as tightly to

plant tissue as WT strains

Inverse relationship between retention strength of X. fastidiosa

to grape xylem and their virulence to grape%

Ce

lls

Re

lea

se

d

0

Adhesive biofilms antagonize virulence of Xylella fastidiosa

X. fastidiosa appears to have a relatively promiscuous signaling system –

potentially producing and responding to various fatty acid signals –

Signal production and response dependent on environment?

XfXcc

Fo

ld c

han

ge i

n h

xfA

exp

ressio

n

XfDSF1

XfDSF2

Xcc

Xf

DSF Abundance

Gum Production

Stickiness to SurfacesExpression of adhesins

Twitching MotilityType IV pili

Pit Membrane DegradationPgl and Eng expression

Plant colonization phase Insect acquisition phase

Active vessel colonization

Low cell numbers in most vessels

Disease symptoms may not be present

Some vessels have high cell numbers

Disease symptoms may be present

Further multiplication in crowded vessels slows

Partitioning of the population of X. fastidiosa for mutually incompatible processes

by cell density signaling

Xylella fastidiosa is a very promiscuous producer of vesicles

Xylella fastidiosa

Xanthomonas

E. coli P. aeruginosa

DSF production suppresses the release of vesicles by

Xylella fastidiosa

Xylella fastidiosa does not stick to xylem vessels

in the presence of membrane vesicles

Fra

ction o

f cells

adhere

d

WT WT rpfF rpfF WT rpfF xadA xadA

Trimer

Dimer

Monomer

unknown

Filtered sap

Membrane vesicles (XadA) abundant in xylem sap

of infected grape

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, as well as vesicle release,

while increasing adhesiveness - thus also reducing

movement

How?

Transgenic DSF-producing plants

Direct application of DSF

Endophytes that produce DSF?

Disease control

Sharpshooter

Inoculation

Non-mobile

Mobile

Early - Inoculum becomes mobile and starts to grow and spread

Sharpshooter

Acquisition

Non-mobile

Mobile

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

Sharpshooter

Inoculation

In transgenic DSF-producing plant signal would be high at all times, restricting

growth and movement

Non-mobile

Grape transformed with rpfF gene from Xylella fastidiosa

that produce DSF are much more resistant to Pierce’s Disease

compared to wild-type grape

Field trials of transgenic grapes conducted at UC Davis and Riverside

Severity of PD much lower on RpfF-expressing Freedom – Solano County August, 2012

RpfF

WT

Large reduction in disease in DSF-producing plants - Riverside County October, 2012

Normal grape

DSF-producing

Expression of Xylella fastidiosa rpfF in Citrus reduces growth and movement

of pathogen and Citrus Variegated Chlorosis symptoms

Hamlin WT Hamlin rpfF

Palmitoleic acid is attractive as a commercially available signal for X. fastidiosa

XfDSF2

Some disease control can be conferred by topically applied signaling molecules

7

0

1

2

3

4

5

6

Bu

rkh

old

eri

aP

sJN

(L

og

(cell

s/g

)

0 20 40 60 80 100

Distance from POI (cm)

Paraburkholderia phytofirmans colonizes grape xylem, unlike other bacteria tested

Biological Control of Disease

Inoculation of Paraburkholderia phytofirmans itself with Xylella fastidiosa greatly

reduces disease severity

Co-inoculation of Xf and Paraburkholderia results in HUGE reductions in the

population size of the pathogen in plants

Inoculation of Paraburkholderia at time of X. fastidiosa inoculation reduces

disease in all grape varieties tested

Bacteria can be introduced into plant tissue with penetrating surfactants

Large populations of Xylella fastidiosa within leaves immediately after spraying

No

surfactant

Paraburkholderia phytofirmans mediates disease control even after infection

of grape with Xylella fastidiosa when applied in different ways

Priming of innate immune system of grape by Paraburkholderia phytofirmans

B=

B = Paraburkholderia alone

BX = Paraburkholderia + Xylella

X = Xylella alone

0

2

4

6

8

10

12

14

PsJ

N n

ee

dle

+X

f

PsJ

N &

Xf

PsJ

N S

pra

y +

Xf

PsJ

N n

ee

dle

bef

ore

Xf

PsJ

N s

pra

y b

efo

re X

f

Co

ntr

ol X

f

PsJ

N n

ed

dle

aft

er

PsJ

N s

pra

y af

ter

Bre

aktr

+X

f

PsJ

N n

ee

dle

(X

3)

+X

f

PsJ

N s

pra

y (X

3)

+Xf

PsJ

N n

ee

dle

( X

3)

+X

f (

X3

)

PsJ

N s

pra

y (

X3

) +

Xf

( X

3)

Co

ntr

ol X

f( X

3)

PSJ

N t

run

k +

Xf

Soap

+X

f

AU

DP

C

b

Disease severity in field trials - 2018

Control

PsJN & Xf mix

PsJN needle before Xf

Nu

mb

er

of

sh

oo

ts

Fraction of symptomatic leaves

Disease reduction conferred by PsJN was associated with complete control on most

shoots rather than reduced severity on a given shoot – All or none response

Nian Wang

Nian Wang Subhadeep Chatterjee Clelia Baccari

Karyn Newman Ellen Beaulieu Miki Ionescu

Rodrigo AlmeidaAline da Silva

Kenji Yakota

Nian Wang

Elena Antonova