Linking Microbial Ecology with Plant Pathologyto Achieve Disease Control and IncreasePlant Productivity

Steven Lindow

University of California, BerkeleyDepartment of Plant and MicrobialBiology

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The study of epiphytes has been driven by studies of plant disease-Plant pathogens such as Pseudomonas syringae exist as epiphyteson healthy plants before inciting disease

100%

0Bacterial Populations (Log cells/leaf)

Pseudomonas syringaeIce Nucleation

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Estimated Number of Cells / Aggregate

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Cumulative fraction of living cells as a function of aggregatesize

for plants exposed to periodic low RH1

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Total Number of Cells per Aggregate

Fructose-inducible plasmid-based GFP reporter fusions

fructose fructose 1-P fructose 1,6-diP

1PfruB

2 PnptII

AAV

AAV

constitutive control

Sites of nutrient abundance suitable for formation ofbacterial aggregates are uncommon on leaves

1 hour 24 hours

Quorum sensing in Gram-negative bacteria

Low Population Densities: Low [AHL] High Population Densities : High [AHL]

HO N C3 -

O H 15

AHL = N-acyl homoserine lactone

Quorum sensing in Pseudomonas syringae

(+)

AHLsynthase

ahlI ahlR

AHLregulator

S-adenosyl methionineacyl-acyl carrier protein (+)

(-)

GacA

3-oxo-C6-HSL(+)

AefR

Expression of Density-Dependent Genes

Quorum sensing mutants ofPseudomonas syringae are hyper-swarmersand more virulent

Wet incubationDry incubation

3-oxo hexanoyl HSL production in Pseudomonas syringaeis directly related to levels of available iron in culturemedia

AHL detected in P. syringae colonies by overspray ofahlI- mutant harboring ahlI:gfp reporter gene

f ael/ufc(golgvA

B728a alone with FeCl3Inte

rnal

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(Log

cells

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Topical iron applications reduce invasion of tomatoleaves by Pseudomonas syringae B728a

4.54.03.53.02.52.01.51.00.50.0

Water alone FeCl3 (10 uM)

Reduced Incidence of infection of bean treatedtopically with various materials to alter ironavailability

Treatment Lesions/LeafGreenhouse Field

Tannic acid 30 uM 26.0 a

Control 13.9 b 6.7 a

FeCl3 2 mM 8.9 c 2.2 b

FeCl3 20 mM 0.3 c

FeNH4 Citrate 5g/L 1.1 bc

Pierce’s disease caused by Xylella fastidiosa

Xylella fastidiosa is obligately vectored by xylem sap-sucking insects

Blue-green sharpshooterGraphocephala atropunctata

www.CNR.Berkeley.EDU/xylella/

Glassy-winged sharpshooterHomalodisca coagulata

Access from vessel to vessel through bordered pits isblocked by the pit membrane

Fig. 2.9. Scanning electron micrographs of metaxylem vessels in the stem of Rhapis excelsa.Left Transversely cut stem showing intervessel pit area. Right Intervessel pits in longitudinalsection (cutting across the wall). (Zimmermann et al.1982)

X. fastidiosa cells are most commonlyfound in modest sized micro-colonieswithin xylem vessels

Most X. fastidiosa communities are relatively small,even in symptomatic leaves

AsymptomaticSymptomatic

Does X. fastidiosa really benefit from crowing into 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 infectedleaves are mostly dead

Many cells stain with PI in plugged vessels when leaf isnearly dead. When the leaf blade is still partly green, veryfew cells stain with PI.

Xanthomonas campestris pv. campestrisrpfA rpfB rpfF rpfC rpfH rpfG lysS prfB rpfD orf orf orf rpfI recJ rpfE greA

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

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

Xylella fastidiosarpfB,F Synthesis of “diffusible signal factor” (DSF)rpfG,C DSF perception and signal transduction leading to

virulence trait expression

C14-cis XfDSF1

C16-cis XfDSF2

Xcc DSF

genes

DSF-mediated gene regulation in Xylella fastidiosasuppresses virulence to plants but is nessesary forvector transmission

DSF

RpfC SignPael rsiepnlassomrActivationofRrpesfGponse

Outermembrane

rpfF

RprefCgulator

+

Innermembrane

DSF SynthesisAdhesion Plantvirulencegenes

X. fastidiosa Decreases Virulence by CoordinatingVirulence Genes in Cell Density-Dependent Fashion

Wild type rpfF mutant

Frac

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RpfF- mutants of Xylella fastidiosa that do not produce DSFare not “sticky” in culture – and do not adhere as tightly toplant tissue as WT strainsVirulence inversely related withadhesiveness

RpfF-

frequ

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rpfF mutants, which cannot signal, are severely impaired intransmissibility by an insect vector

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0Temecula (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 Controlledby RpfF in culture

Type IV pili

Downregulated:Type IV pili (twitching motility)PolyglacturonaseCellulase

Upregulated:Hemagglutinin adhesins

Meng et al. 2005

Extracellular polysaccharides (EPS): gumType I pili (anchoring)

Partitioning of the population of X. fastidiosa for mutually incompatible processesby cell density signaling

Plant colonization phase Insect acquisition phaseActive vessel colonizationLow cell numbers in most vesselsDisease symptoms may not be present

Some vessels have high cell numbersDisease symptoms may be presentFurther multiplication in crowded vessels slows

DSF Abundance

Type IV piliTwitching Motility

Expression of adhesinsStickiness to Surfaces

Pgl and Eng expressionPit Membrane Degradation

Gum Production

CFU

1000/V

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Species comparisonVe

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Xylella fastidiosa is a particularly prominentproducer of outer membrane vesicles

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Xf PD3

Xac PD3

Ec LB

Ec PD3

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Xylella fastidiosa does not stick to xylem vesselsin the presence of membrane vesicles

Quorum sensing suppresses release of vesicles by X. fastidiosa – reduces virulence

OMV adhesinDSF

DSF

Low cell density High celldensity

The goal: Confuse pathogen by exposing it toexcessive amounts of signal molecule even when itis in low population sizes

The expectation: The premature presence of DSF in vesselswill suppress extracellular enzyme production and thus bothmovement and multiplication, while increasing adhesivenessand thus also reducing movement

How?Endophytes that produce DSFDirect application of DSFTransgenic DSF-producing plants

Early - Inoculum becomes mobile and starts to grow and spread

Non-mobile

Mobile

SharpshooterInoculation

Later- Extensive growth and spread of X. fastidiosa – some vessels become crowdedand accumulate DSF – suppression of further growth and movement in that vessel –acquired by insects

Non-mobile

Mobile

SharpshooterAcquisition

In transgenic DSF-producing plant signal would be high at all times, restrictinggrowth and movement

Non-mobile

SharpshooterInoculation

Direct Application of Signal MoleculesPalmitoleic acid is attractive as a commercially available signal for X. fastidiosa

-2

wild typeTRA1TRA1TRA3TRA4TRA5TRA6TRA7TRA8TRA9

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Grape transformed with rpfF gene from Xylella fastidiosathat produce DSF are much more resistant to Pierce’s Diseasecompared to wild-type grape

10Wild type

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6DSF-producing lines

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Plant Genotype

Large reduction in disease in DSF-producing plants - 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

Phytobiomes Initiative Goal:• By 2025, build a foundation for the

comprehensive understanding ofphytobiomes to improve crop productivity

– how organisms associated with plantsinfluence or are influenced by theplant or the plant environment

– how that information can be used toimprove crop productivity, quality,nutrition, safety, and security

www.phytobiomes.org

Texasdroughtproject.org

d plants

Phytobiomes include: All organisms living in, on and aroun microbes (the plant & soil microbiomes) animals (insects, nematodes, amoeba, etc) other plants

the Environment

Phytobiomes are systems

pproductionroductionmmustusti•increasencrease

70%70%

2050Total aggriculturalricultural

2011 pproductivityroductivityX 1.7mmustust ddoubleouble

Why a Phytobiomes Initiative Now?

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To Understand Phytobiomes:

Interdisciplinary,systems levelapproach

Consider interactionsin context

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s,

Technological & Scientific Advances• Advances in systems-level approaches

– High-throughput sequencing– Metagenomics & other ‘-omics technologies– Data sciences (data mining, predictive analytics, …)– Computational biology and modeling– Computer science (machine learning, image processi

and graphics, …)

• Human microbiome discoveries– Conserved patterns despite variation– Strength of longitudinal vs cross-sectional studies– Unexpected impacts on the host– Successful translation to treatments (fecal transplant

probiotics)

• Management strategies in agriculture– Precision Ag– Decision support systems

A strong scientificfoundation….

Phytobiomes: include individual organisms that function as commensals,

pathogens/pests and beneficialsMost studies of one/few organismsWhat about communities?Most studies focus on prokaryotesWhat about eukaryotes?Most studies of cultured organisms Roles for non-cultured?

are influenced by many biotic and abiotic stressesMost studies of one or two plant/microbe/insect/environmental stresses at a time Integrate knowledge of the SYSTEM?

Phytobiomes Visions:

• Management strategies thatcreate disease-suppressivecommunities

• Plants that select for andmaintain beneficialphytobiomes

Jorge Greuel/Getty Images

Biota

Crop Soil

Technology Water

Data Weather

Raj Khosla, CSU

Phytobiomes will build a foundation to:– Move from today’s detect & respond to

tomorrow’s predict, act, manipulate, andprevent

– Assess phytobiome-mediated attenuation ofclimatic impacts on crops

– Understand inter-relationships with nutrientuptake and their utilization

– Relate the phytobiome to its impacts on animaland human health and safety

– Safely and sustainably intensify production offood, feed and fiber

– Change the discovery paradigms for plantdisease/pest control, crop improvement,etc.…..

Science 332:1099 (2011)

Analyses of soil microbes revealed that disease occurrence in thepresence of a soil-borne fungal pathogen was reduced in soils withhigher proportions of Proteobacteria and Actinobacteria, thussuggesting that “healthy” plant microbiomes can be described anddeveloped

The rhizosphere microbiome: significance of plant beneflclal,plant pathogenic.. and human pathogenic microorganismsRodrigo Mendes 1, Paolina Garbeva2 & Jos M. Raaijmakers3 FEMS Microbiol. Rev. 37:634 (2013)

Analysis of bacterial communities in soils suppressive of plant disease (“Healthy Soil”)revealed that opportunistic human pathogens were also usually much less abundant

a-Proteobacteria Sphmgomonas paucunob1/tsOchrobactrum enlhrop1

Ochrobacrrvmtntic,fj-Proteobactena Achromobecter xylosoxldans

AJcal,genesfaeca/JsA/csllg6f19sxyfosoxKJans

Burkholder,acepac,eJ911thinobactenum/Jvidum

Chromobsctenumvussceumy-Proteobactena Aeromonas veron11

Enrerooacter cloacaePanroeaegglomerans

Proteus vv{gansCnterobacter emr,,genusEnterobacter intermedius

Klebsrells pneumon,aeSalmonella typh,murwm

Serretra gr1mes11Serralls /,que(ac,ensSerrat,a msrcescens

Serretia proteamacutansAc1Mtob8cler b81.m1,mn11

Acmetooocter celcoacetJcusPseudomonas aerugmosa

Ste11011op/>01nw,as111aJtopt111taF1TTT11Cutes Bacillus cereus

Staphy1ococcusaureusBactefOldeles Chfyseobactenwn 1ndologenes

Sphlngobecterwm spmuvorum

(a)

• Suppressive soil- Conducive soil

- Suppressive soil- Conducive soil

(b)

0 ,o 20 30 40 50 60Number of OTlJ$

70 0 2 3 4 5 6Relative abundance

Nature Reviews Microbiology 9:99 (2011)

Not all viruses are pathogenic: many have symbiotic interactions with plants – and canengage in complex interactions with other organisms such as fungi to confer stresstolerance to plants

Complex interactions between leaf endophytic fungi and poplar genotype influenced disease.Endophytes often increased disease severity

Both stimulatory and inhibitory interaction observed between other resident bacteriaon lettuce and the plant pathogen Xanthomonas campestris pv. vitians usingculture-independent community analyses

Not only can the distinctive bacterial communities on rice leaves and roots be comparedwith those in the water in which the plants live, but the preferential expression of traits thatenable colonization of these sites can be deciphered by proteome analysis

16th International Symposium on Microbial Ecology

21-26 August, 2016