Assembly of epiphytic bacterial

communities on plants and their interactions

with the plant host

Steven Lindow

University of California, Berkeley

Department of Plant and Microbial

Biology

From: Brandl & Lindow, Appl. Environ. Microbiol. 69:1875-1883 (2003)

Fungal hypha

Glandular trichome

Vein

Bacterial aggregate20 um

Epiphytic Bacteria:

104 – 107 cells/cm2

0

100%

Bacterial Populations (Log cells/leaf)

The study of epiphytes has been driven by studies of plant disease-

Plant pathogens exist as epiphytes on healthy plants before inciting

disease

Bacterial Ice Nucleation

Considerable evidence for role of epiphytes in defense against pathogens

Composition of Bean Leaf Exudates

ug/leaf

Sucrose 5.3

Glucose 1.9

Fructose 1.6

Galactose 0.2

Amino Acids 0.7

Bacte

ria (

10

7cell

s/g

)Carbon is limiting factor on plants but is not evenly distributed

2 Hours after Inoculation

24 Hours after Inoculation

Objective [1] [2] [3] [4]Majority of bacteria live in large aggregates on leaves

0

0.2

0.4

0.6

0.8

1

1 10 100 1000 10000 100000

Estimated Number of Cells / Aggregate

Cu

mu

lativ

e P

ropo

rtio

n o

f To

tal C

ell

Ob

serv

ed

0 days

2 days

9 days

1 15 30 45

150

300

450

1500

3000

4500

5000

0

0 days

2 days

9 days1

10

100

1000

10000

Estimated Number of Cells / Aggregates

To

tal N

um

ber

of

Ag

gre

gate

s O

bserv

ed

Num

ber

of A

ggre

gate

s

Cells/Aggregate

Cells/Aggregate

Cum

ula

tive P

roport

ion o

f C

ells

Most of the cells on leaves are in aggregates

Cumulative fraction of living cells as a function of aggregate

size

for plants exposed to periodic low RH

0

0.2

0.4

0.6

0.8

1

1 10 100 1000 10000

Total Number of Cells per Aggregate

Cu

mu

lati

ve

Fra

cti

on

of

Liv

ing

Ce

lls 0 days

3 days (1c)

4 days (2c)

5 days (3c)

7 days

Immigrant bacteria survive much better in aggregates of residents

on previously colonized leaves

Morris, INRA

Quorum sensing is very common and controls behavior of plant-associated bacteria

QS repression of motility and virulence

in Pseudomonas syringae

WT AHL-

Many epiphytes either produce or inhibit

acyl homoserine lactone signal molecules

Alter behavior of neighboring

AHL-perceiving bacteria

Response of plant to AHLs:

Activation of innate defense?

12% produce

3-oxo hexanoyl HSL8% inhibit AHLs

AHL signals shared by leaf colonists can influence each

others behavior - inhibit disease caused by P. syringae

NH

O

O

O O

Le

sio

ns/L

ea

f

AHL lactonase

Ps Alone Ps + Erwinia FT1 Ps + AHL- Erwinia

QS inhibitor strains can block signaling on leaves: strain 114

increases virulence of Pseudomonas syringaeL

esio

ns/L

ea

f

Ps alone Ps + 114 Ps + 114Ant-

Frontiers Pl. Sci. 5:131 (2014)

Epiphytic

Endophytic

Functions in Pseudomonas syringae predicted to be particularly important to cells in epiphytic sites

Epiphytic

Endophytic

Fold-induction0 5 10 15 20 25

Sulfur metab & transport

Sigma factor-associated proteins

Tryptophan

Phenylalanine

Amino acid metab & transport

Chemosensing & chemotaxis

Flagellar synthesis & motilityFlagellar synthesis & motility

Chemosensing & chemotaxis

Amino acid metab & transport

Phenylalanine catabolism (phhAB)

Tryptophan synthesis (trpAB)

Sulfur metab & transport

Sigma factors & Anti-sigma factors

Functions predicted to be particularly important to cells in endophytic sites

Fold-induction0 5 10 15 20 25

HCN

Syringofactin

Non-ribosomal peptide synthases

Trehalose

NAGGN

Syringolin

Syringolide

Syringomycin & Syringopeptin

Phytotoxin metab & transport

Syringomycin & syringopeptin

Syringolide

Syringolin

Compatible solute synthesis

NAGGN

Trehalose

Secondary metabolite synthesis

Non-ribosomal peptide synthases

Syringafactin

HCN

Epiphytic

Endophytic

Gene expression very context-dependent in and on plants

IAA production by epiphytic bacteria : a story of resource conversion

and plant modification

Most bacteria on plants produce

3-indole acetic acid (IAA)

Epiphytic IAA producers can alter auxin-mediated responses in plants

Fruit RussetFlower and fruit abscission

But – Bacteria also use their own and plant IAA as signal molecules

Agrobacterium tumefaciens

Cell. Micro. 10:2339 (2008)

Fruit russet severity varies between years but is predicted by early-season

populations of epiphytic IAA-producing bacteria

Climatic and agro-ecological impacts on epiphytic bacterial populations

Reduction of fruit russet of pear by application of non-IAA producing

Pseudomonas fluorescens A506 to trees at flowering

Treatment Fruit Russet (% of surface)

Site1 Site2 Site3 Site4

Control 4.46a 3.29a 3.7a 3.4a

P. fluorescens A506 2.78b 1.48b 1.17b 2.2b

Ratio 2

99R

:299R

XY

LE

Hours after Inoculation

0 10 20 30 40

IAA Production Contributes to Fitness of Erwinia herbicola on Bean Leaves

Do epiphytes produce IAA to increase nutrient leakage

from plants?

From: Lindow & Brandl, AEM 69:1875-1883 (2003)

-10

-8

-6

-4

-2

0

0 3 7 24

Lo

g (

ice n

ucle

i/cell)

Hours After Inoculation0 3 7 24

Hours after Inoculation

Log (

ice n

ucle

i/cell)

0 NAA

100 uM NAA

10 uM Sucrose

Pscr InaZ

Biosensor cells sense LESS sucrose in presence of auxin

0

100

200

300

0 3 7

Me

an

rela

tive G

FP

Time after inoculation (hrs)

0

20

40

60

80

100

0 1 3 5

% G

FP

Po

sit

ive

HpiHours after Inoculation

0 1 3 5

0 3 7

PfruB gfpR

ela

tive

GF

P F

luo

resce

nce

% G

FP

po

sitiv

e c

ells

100 uM NAA

0 NAA

0 NAA

100 uM NAA 10 uM Fructose

Cells sense MORE fructose & more quickly in presence of auxin

5

6

7

0 Hr. 0 0.05 0.1 0.5 1

Lo

g (

cells/m

L)

Sugar Concentration (mg/L)

Sugar Concentration (mg/L)

0 0.05 0.1 0.5 1.00 hr

Ma

x.

Popula

tion s

ize (

Log (

cells

/ml)

)

Fructose

Glucose

Sucrose

Cells can grow at lower substrate concentrations

of mono-saccharides than di-saccharides

6 hr

Cycloserine sensitivity is low in cells grown in low concentrations of sucrose

GlucoseFructose

Sucrose

Cells inactive in presence of low concentrations of sucrose

Leaf Cuticle

SUT

Bacterial IAA in the phyllosphere

Sucrose

Glucose

Fructose

cw

Inv

Phloem cell

Mesophyll cell

ApoplastSUT

Glucose

Fructose

Sucrose

Diffu

sio

n/

Leakage

Glucose

Fructose

SucroseEpiphyte

Diffu

sio

n/

Le

aka

ge

Key features of the model:

• Auxin-dependent changes in epiphytic

sugars

• Increased fructose availability

• Decreased sucrose availability

• Resource conversion makes adaptive

sense

1. Microbial IAA promotes plant

invertase activity

2. Invertase cleaves apoplastic

sucrose, yielding hexose sugars that

diffuse to leaf surface and apoplast

3. Preferential use of glucose and

fructose at the low concentrations

found on leaves and apoplast

Biosurfactants: Common good products prominent on leaves

Syringafactin

Syringafactin contributes to nutrient acquisition:

alters cuticular permeablility

Effects on Plant Fitness?

Diffusion across waxy cuticle

Syringafactin

added

3H2O

Isolated

cuticle

Measure movement of 3H2O

Apply surfactant

Method: Schreiber et. al., 2005

-10

0

10

20

30

40

50

5 7 9 11 13 15

minutes

inte

nsit

y

Syringafactin extract

WT in wash

WT in waxes

Wax control

Majority of syringafactin adsorbs to leaf waxes

Syringafactin is hygroscopic and can bind a lot of water at

high relative humidity

Hygroscopic wetting of Teflon by syringafactin

and its retention during washing

Washed Dried Humid

WT

ΔsyfA

Syringafactin-producing cells of Pseudomonas syringae experience

less water stress on dry leaves

GFP fluorescence intensity

Fra

cti

on

of

ce

lls

Benefit of syringafactin production pronounced after multiple daily

dry/humid cycles

WT

ΔsyfA

Ba

cte

ria r

eco

vere

d (

Lo

g(c

ells/g

))

Syringafactin is hygroscopic and locally alters wetness of leaf surface habitat

Bean Soybean

Pumpkin Tomato

Bacterial numbers are usually very low on young plants:Lack of suitable colonists since young plants can support high numbers?

Soil is poor source of immigrants to leaves?

Assembly of epiphytic communities

Leaf isolates Soil isolates Rhizosphere isolates

Lo

g (

dry

-we

t)

UV

Bacteria isolated from leaves are much more fit on leaves exposed to stresses than

those from soil or rhizosphere: Source of inoculum for new plants?

Soil-borne bacteria are not good epiphytes!

uninoculated

flowers

Pseudomonas fluorescens

inoculated flowers

Limitation of immigration of suitable colonists restricts bacterial

population sizes on young plant tissue such as flowers

Aerial plant surfaces are an “open” microbiological habitat

Immigration plays an important role in structuring epiphytic communities

How do immigrant arrive and where do they come from?

Water or vegetation-free

Wind direction

Determining contribution of vegetation to local airborne microbial communities

Various uniform plant species

30 meters

Lymperopoulou et al. AEM 82:3822 (2016)

The abundance and composition of airborne bacteria is strongly influenced by

nearby plants

Prominent bacterial taxa on plants were much more common in downwind air than upwind air

Upwind air

Plants

Downwind air

Bacteria

Prominent bacterial taxa on plants were much more common in downwind air than upwind air

Upwind air

Plants

Downwind air

Bacteria

Large influence of neighboring plant species on bacterial communities of tall fescue

within a local area

Selection of epiphytic communities apparently occurring from different local

metacommunities

While distinct communities

on given plant species:

Communities on fescue influenced by neighboring plants

PNAS 108:14288 (2011)

Bacterial species assemblages on algae distinct from seawater but also differed greatly between algal samples

High similarity in functional genes on different algal samples

Competitive lottery model of community assemblyCombination of niche- or guild-based selection and random components

“Species with similar trophic or other ecological properties are able to occupy the same niche within an ecosystem and the particular species that occupies a particular space is then determined by stochastic recruitment”

Continuous immigration and subsequent selection and growth

determine eventual communities on mature leaves:

Context dependent?

Plants will sample from different metacommunities in an agro-ecological context

in which crops are grown – driven by amount and type of nearby vegetation

Management practices can influence process of establishing the plant microbiome

Large (10,000 m2) replicated blocks of vegetation management

established in 80 ha pear orchard to test for under-tree vegetation

effects on pear epiphytes

Differences in airborne microflora and subsequent

bacterial-induced fruit russeting in a replicated pear field

plot having different cover crop plant species

Cover crop species Bacteria Deposited Fruit Russet

(cells/petri dish/hr) (% of surface)

Mixed weeds 28.0 ab 9.5 ab

Annual Ryegrass 23.3 ab 13.0 a

Mixed Grasses 15.0 bcd 7.9 b

Perennial Clovers 18.0 cd 7.8 b

Berseem Clover 12.7 cd 7.8 b

Bare soil 11.0 cd 6.3 b

Pea+Vetch 7.8 d 5.7 b

Burr Clover 6.3 d 6.3 b

Hig

h leaf

popula

tions

Intercellular signaling

& habitat modification

Signaling to plant

X

Emigration

Airborne epiphytes

Colonization of plants involves many processes occurring at different scales

X

Tracy Powell

Adrien Burch

Despoina Lymperopoulou

Pseudomonas syringae transcriptome team

Monica Hernandez