Fungal enzymes involved in grapevine trunk diseasesmanagtd.eu/images/uploads/content/172/AC ESTEVES 12 05 2015 pdf.… · Fungal enzymes involved in grapevine trunk diseases . Grapevine

Ana Cristina Esteves

Dep. Biology, University of Aveiro

Fungal enzymes involved in grapevine trunk diseases

Grapevine Trunk

Diseases

ESCA

Botryosphaeria

Dieback

(black dead arm)

Eutypa dieback

Diplodia mutila

Diplodia seriata

Neofusicoccum parvum

Lasiodiplodia theobromae

…

Phaeomoniella chlamydospora

Phaeoacremonium aleophilum

Fomitiporia mediterranea

…

Eutypa lata

The enzymes of GTD

Cell-wall-degrading enzymes produced by plant

pathogens

Achyuth

an, et al., M

ole

cule

s 2

010, 15(1

2),

8641-8

688

The enzymes of GTD


pathogens

Enzymes that degrade:

• Pectic substances

o pectin methylesterase, polygalacturonases, pectin lyase,

pectate lyase, phamnogalacturonanases, α-L-

arabinofuranosidase

The enzymes of GTD


pathogens


• Hemicelluloses

o Xylanases, Glucanases, Galactanases

o β-(1,3)-glucan is a minor component of plant tissue,

but it is important in plant disease resistance because

it occurs primarily in response to fungal penetration

o Many pathogens produce β-(1, 3)-glucanase to

degrade β-(1, 3)-glucan

The enzymes of GTD


pathogens


• Cellulose

o Complete degradation of native cellulose to glucose

requires three enzymes:

o β-1,4-glucanase; cellobiohydrolase; β-

glucosidase

• Lignin

o Laccase; lignin peroxidase; manganese-dependent

peroxidase; tyrosinase

The enzymes of GTD


pathogens


• Proteins

o proteases

The enzymes of GTD

Which enzymes?

Proteases

The enzymes of GTD

The enzymes of GTD

The enzymes of GTD

GTD

pathogens can be found in the wood but never in the leaves

of infected plants

it was hypothesized that the observed leaf and berry

symptoms are actually caused by extracellular compounds;

these compounds are most probably produced in the trunk,

which then translocate to the leaves via the transpiration

stream

M. Ramírez-Suero, et al., 2014. Protoplasma

The enzymes of GTD

GTD

pathogens can be found in the wood but never in the leaves

of infected plants

it was hypothesized that the observed leaf and berry

symptoms are actually caused by extracellular compounds;

these compounds are most probably produced in the trunk,

which then translocate to the leaves via the transpiration

stream

• Enzymes able to degrade antifungal compounds released by

the host or plant cell constituents M. Ramírez-Suero, et al., 2014. Protoplasma

The enzymes of GTD

Eutypa dieback

involvement of secondary metabolites (e.g eutypine,

eutypinol, siccayne, eutypinic acid, …)

involvement of glycosylated and non-glycosylated

polypeptide toxins that may have enzymatic activity (Octave

et al., 2008);

• Glycosylated polypeptides induced heavy plant cell

wall damage

The enzymes of GTD

Eutypa dieback

E. lata expresses (in culture) cellulases, xylanases and 1,3-β-

glucanases (Schmidt et al., 1999), chitinases and proteases (Schmidt

et al.,2011)

degradation of cell wall structure

loss of cell integrity

non-structural (mostly stored starch) and structural (hemicellulosic)

glucans are the primary targets

The enzymes of GTD

Eutypa dieback

E. lata expresses (in culture) cellulases, xylanases and 1,3-β-

glucanases (Schmidt et al., 1999), chitinases and proteases (Schmidt

et al.,2011)

degradation of cell wall structure

loss of cell integrity

non-structural (mostly stored starch) and structural (hemicellulosic)

glucans are the primary targets

Starch is used to

support the

pathogen’s energy

needs

The enzymes of GTD

Eutypa lapa enzymes – modulation by the host

Production of extracellular cellulase, xylanase, 1,3-beta-

glucanase, chitinase and protease was demonstrated in

Eutypa lata growing in vitro and on autoclaved wood

In grape wood extract medium, xylanase was the dominating

enzyme whereas protease activity prevailed in potato

dextrose broth

Schmidt, CS, et al., 1999. JOURNAL OF PLANT DISEASES AND PROTECTION

The enzymes of GTD

Eutypa lapa enzymes

In annual grape wood, activities of cellulase, xylanase and

protease activity were nearly equally high; low levels of 1,3-

beta-glucanase were measurable

In perennial wood, proteases were clearly the dominating

enzymes, followed by chitinases, while 1,3-beta-glucanase

activity was not detectable

The pathogen enzymes are modulated by the host

Schmidt, CS, et al., 1999. JOURNAL OF PLANT DISEASES AND PROTECTION

The enzymes of GTD

2013 Eutypa lapa (draft) genome

1,224 potentially secreted proteins

• 217 putative glycoside hydrolases

• GH61 (26 genes) breakdown of lignocellulosic

compounds

• GH43 (22 genes) hemicellulolytic activity

• GH16 (17 genes) hemicellulolytic activity

Blanco-Ulate , B, et al., Genome Announc. 2013;1(3). pii: e00228-13

The enzymes of GTD

ESCA

Several secondary metabolites have been reported (from

Phaeoacremonium aleophilum and Phaeomoniella

chlamydospora)

• scytalone, isosclerone, etc.

Polypeptides with cytotoxic activity

Phaeoacremonium aleophilum: xylanase, exo- and endo-β-

1,4-glucanase and β-glucosidase; no ligninase activity detected

Phaeomoniella chlamydospora: tannase, laccase and

peroxidase enzymes

Valtaud et al. (2009); Bruno, & Sparapano (2007) Physiological and Molecular Plant Pathology 69 : 182–194

The enzymes of GTD

ESCA - an explanation for wood pigmentation

suggests that synergism with other vascular pathogens during

plant infection may favor the effective breakdown of lignified tissues

79

212

205

Bruno, & Sparapano (2007) Physiological and Molecular Plant Pathology 69 : 182–194

The enzymes of GTD

ESCA - an explanation for wood pigmentation



79

212

205

The production of degradative enzymes such as tannase,

laccase and peroxidase probably enables Phaeomoniella

chlamydospora, and Fomitiporia mediterranea to

grow better in woody tissue

to actively degrade antimicrobial substances

to compete with each other

Bruno, & Sparapano (2007) Physiological and Molecular Plant Pathology 69 : 182–194

The enzymes of GTD

Phaeoacremonium aleophilum (draft) genome

658 potentially secreted proteins

23% consist of putative plant CW-degrading enzymes:

17 cellulases

12 hemicellulases

21 pectin-degrading enzymes

12 callose-degrading enzymes

1 cutinase

might play important roles during tissue colonization and

systemic infection

Blanco-Ulate, et al., Genome Announc. 1(3):e00390-13

The enzymes of GTD

Phaeomoniella chlamydospora (draft) genome

CW-degrading enzymes:

37 cellulases

5 cutinases

3 pectinases

10 laccases

numbers are relatively low compared to those for other wood-

degrading fungi.

Antonielli L, et al. 2014. Genome Announc. 2(2):e00098-14

The enzymes of GTD

Synergistic effects between E. lata, Pa.

chlamydospora and Pm. aleophilum

E. lata extracellular proteins cell structure damage and affect

grapevine metabolism

Pa. chlamydospora extracellular proteins reduce plant

metabolism

Pm. aleophilum extracellular proteins affects cell wall integrity

Luini et al. 2010

lead toxins in the observed pathogenic

effects.

The enzymes of GTD

Lignin degrading enzymes



79

212

205

49

Pm. aleophilum

N. parvum

E. lata

Pa. chlamydospora



The enzymes of GTD

Botryosphaeria dieback – N. parvum (draft) genome

1,097 potentially secreted proteins

• 167 consist of putative plant GH

• 22 polysaccharide lyases

• 8 cutinases

• 212 lignin degrading enzymes (similar to E. lata)

Blanco-Ulate B, Genome Announc. 2013 Jun 13;1(3). pii: e00339-13

The enzymes of GTD

Botryosphaeria dieback (N. parvum and D. seriata)

phytotoxic metabolites

• lipophilic compounds, exopolysaccharides

extracellular proteins

proteins produced by N. parvum appeared to be more

aggressive than those from D. seriata

denatured proteins had no effect the 3D

structure/activity of these proteins is crucial for toxicity

M. Bénard-Gellon, et al., 2015. Protoplasma (2015) 252:679–687

interaction between extracellular proteins and

secondary metabolites could be responsible for the

toxicity of the extracellular

compounds produced by Botryosphaeriaceae

The experience at Microlab/Fungal and Plant Biology lab

Botryosphaeriales enzymes

Neofusicoccum, Diplodia, etc.

D. corticola

L. theobromae

The experience at Microlab/Fungal and Plant Biology lab – Univ. Aveiro

amylases laccases xylanases proteases lipases cellulases





Species

Neofusicoccum luteum

Neofusicoccum parvum

Neofusicoccum australe

Neofusicoccum mediterraneum

Neofusicoccum vitifusiforme

Neofusicoccum ribis

Dothiorella iberica

Dothiorella sarmentorum

Dothiorella prunicola

Dothiorella iberica

Dothiorella parva

Spencermartinsia plurivora

Spencermartinsia viticola

Diplodia seriata

Diplodia intermedia

Diplodia pinea

Diplodia cupressi

Diplodia subglobosa

Diplodia mutila

Neodeightonia phoenicum

Diplodia scrobiculata

Botryosphaeria dothidea

Melanops tulasnei

Botryosphaeria corticis


Lasiodiplodia gonubiensis

Lasiodiplodia parva

Lasiodiplodia pseudotheobromae



70%

30% 9 out of 9

7 out of 9

All strains expressed cellulases and laccases

while the least detected enzymes were lipases

Esteves, et al., 2014. Can. J. Microbiol. 60(5):332-42

Endoglucanases



Neofusicoccum Dothiorella Diplodia

Botryosphaeria Lasiodiplodia

N. luteum

N. parvum

B. dothidea

Proteases





Proteases





The enzymatic activities could not be associated with any particular

species nor with any particular phylogenetic group, host or niche

Some strains concomitantly expressed low extracellular cellulolytic

and endoglucanolytic activities and high proteolytic and lipolytic

activities (e.g. Melanops tulasnei) indicative of a symbiotic lifestyle

The relative lack of secreted polysaccharide-degrading enzymes

may reflect a strategy to avoid eliciting host defence responses.



Temperature modulates the activity of these enzymes

Some are active at 70ºC

Such high temperature activity profiles had never been described for

cellulases from the Botryosphaeriales order, which were thought to be

active at temperatures below 40ºC



Diplodia corticola

Causes infections in Portugal, Spain, Morocco, Italy, Greece,

Hungry, and the USA

Endophyte / pathogen

Hosts:

Quercus sp.

Vitis vinifera

Eucalytus sp.

Diplodia corticola

Diplodia corticola

Affects plants of different ages:

dieback;

cankers;

vascular necrosis;

…

Little information regarding the exact mechanism of infection

toxins involved

host stress/ host wounds

Diplodia corticola

Diplodia corticola

1) Quantify the production of extracellular enzymes

2) Determine the proteome of the fungus

Ensaios de infeção

Quercus suber; stem; canker; Aveiro PT

• CAA003

• CAA004

• CAA007-1

• CAA008

Quercus suber, stem; canker; Samora Correia PT

• CAA009-1

• CAA009-2

• CAA009-3

• CAA010

Eucalyptus globulus; stem; canker; Anadia PT

• CAA499

• CAA500

Diplodia corticola

D. corticola interaction with Q. suber

Gelatinases

D. corticola + PDB

D. corticola + PDB +

cork oak stem

data not published


Gelatinases ↑↑

Caseinases ↑↑

Endoglucanases ↔ Amylases ↑

Total extracellular enzymatic activity increased on the presence of host tissue data not published


Proteome of Diplodia corticola secretome

PROTEOMIC WORKFLOW

method? genome?

Diplodia corticola

Protein extraction methods

I Fernandes, et al. 2014. Fungal Biology. 118: 516-523.

Diplodia corticola

Basal secretome of Diplodia corticola


Basal secretome of Diplodia corticola

Spot Protein Accession

number Organism Theoretical pI1 Subcellular localization2

1 Glucoamylase K2S7L9 Macrophomina phaseolina (strain MS6) 5.37 Extracellular

2 Glycoside hydrolase family 71 K2R498 Macrophomina phaseolina (strain MS6) 4.84 Extracellular

3 Putative carboxypeptidase S1 R1GF60 Neofusicoccum parvum UCRNP2 4.45 Extracellular

4 Neuraminidase K2SSW0 Macrophomina phaseolina (strain MS6) 4.27 Extracellular

5 Putative serine protease R1GM11 Neofusicoccum parvum UCRNP2 6.07 Extracellular

6,7 Peptidase M35 deuterolysin K2SDQ0 Macrophomina phaseolina (strain MS6) 5.34 Extracellular

8 Uncharacterized protein K2RZ98 Macrophomina phaseolina (strain MS6) 5.59 Extracellular

9 Putative ferulic acid esterase R1EDH3 Neofusicoccum parvum UCRNP2 4.79 Extracellular

10 Putative glucan-β-glucosidase R1GIC9 Neofusicoccum parvum UCRNP2 4.73 Extracellular

11, 12 Spherulation-specific family 4 K2RK67 Macrophomina phaseolina (strain MS6) 4.04 Extracellular

Involved in the virulence of the phytopathogenic

fungus Alternaria alternata

Important virulence

factor Involved in the

virulence of some microorganisms (p.e.

HIV)

Ferulic acid esterase: may be involved in

pathogenisis compromising suberin

integrity

Glicoside hydrolases

Proteases

Diplodia corticola


Which proteins are induced (or repressed) by the presence of host?

BASAL INFECTION-INDUCED

Diplodia corticola

data not published

Diplodia corticola

37 spots differentially expressed

59 spots common to basal and infection-induced conditions. data not published

Diplodia corticola

Protein Homology FASTS Evalue BaCelLo(subcellular

localization predictor)

Peptidase M35 deuterolysin Macrophomina phaseolina (strain MS6) 8,70E-26 Secretory

Putative leucyl aminopeptidase Botryosphaeria parva (strain UCR-NP2) 7,20E-24 Secretory

Peptidase A1 Macrophomina phaseolina (strain MS6) 2,00E-06 Secretory

Uncharacterized protein Macrophomina phaseolina (strain MS6) 7,50E-02 Secretory

Putative 5 3-nucleotidase protein Botryosphaeria parva (strain UCR-NP2) 1,90E-13 Secretory

Glucoamylase Penicillium marneffei (strain ATCC 18224) 3,50E-13 Secretory

Putative lipase protein Botryosphaeria parva (strain UCR-NP2) 5,70E-10 Secretory

Peptidase M35 deuterolysin Macrophomina phaseolina (strain MS6) 6,20E-12 Secretory

Spherulation-specific family 4 Macrophomina phaseolina (strain MS6) 3,50E-19 Secretory

Phosphoesterase Macrophomina phaseolina (strain MS6) 5,50E-08 Secretory

Putative extracellular guanyl-specific ribonuclease protein Botryosphaeria parva (strain UCR-NP2) 2,70E-20 Secretory

Putative cell surface spherulin 4-like protein Botryosphaeria parva (strain UCR-NP2) 5,40E-04 Secretory

Putative acid phosphatase protein Botryosphaeria parva (strain UCR-NP2) 6,70E-05 Secretory

Uncharacterized protein (α-amylase) Macrophomina phaseolina (strain MS6) 2,30E-28 Secretory

Putative carboxypeptidase s1 protein Botryosphaeria parva (strain UCR-NP2) 1,00E-17 Secretory

Cerato-platanin Colletotrichum graminicola (strain M1.001) 1,50E-09 Secretory

Putative extracellular guanyl-specific ribonuclease protein Botryosphaeria parva (strain UCR-NP2) 2,20E-12 Secretory

Putative cell surface spherulin 4-like protein Botryosphaeria parva (strain UCR-NP2) 3,40E-03 Secretory

Uncharacterized protein (ferulic acid esterase) Macrophomina phaseolina (strain MS6) 3,40E-14 Secretory

Cerato-platanin proteins are small,

secreted proteins that are abundantly

produced by filamentous fungi with all types

of lifestyles

These proteins appear to be readily

recognized by other organisms and are

therefore important factors in interactions of

fungi with other organisms

Cerato-platanin family protein abundantly

secreted by Botrytis cinerea, is required

for full virulence and elicits the

hypersensitive response in the host, that

leads to host death



≥20 different species in the family Botryosphaeriaceae have

been associated with Botryosphaeria dieback symptoms

But, L. theobromae was the only species isolated from

diseased grapevines in Piura, Peru

Rodríguez-Gálvez, et al. Eur J Plant Pathol (2015) 141:477–489


Rodríguez-Gálvez, et al. Eur J Plant Pathol (2015) 141:477–489


Phenotypic characterisation

Strains from several hosts (grapevine, mango tree, …,

human)

Effect of temperature, pH and enzyme production


temperature

24h

48h

72h

96h

0

2

4

6

85ºC15ºC

20ºC25ºC

30ºC35ºC

37ºC40ºC

gro

wth

(c

m)

24h

48h

72h

96h

0

2

4

6

85ºC15ºC

20ºC25ºC

30ºC35ºC

37ºC40ºC

gro

wth

(c

m)

24h

48h

72h

96h

0

2

4

6

85ºC15ºC

20ºC25ºC

30ºC35ºC

37ºC40ºC

gro

wth

(c

m)

24h

48h

72h

96h

0

2

4

6

85ºC15ºC

20ºC25ºC

30ºC35ºC

37ºC40ºC

gro

wth

(c

m)

data not published


pH

24h

48h

72h

96h

0

2

4

6

84

5

5,6

7

8

9

10 A

gro

wth

(c

m)

24h

48h

72h

96h

0

2

4

6

845

5,67

89

10

gro

wth

(c

m)

data not published


Enzyme production

amylases

laccases

xylanases

proteases

pectinases and pectin lyases

lipases

cellulases

at 25ºC, 30 and 37ºC

data not published


Enzyme production

data not published


Enzyme production

Amylases

Xylanases

Proteases

Cellulases

25ºC 25ºC 37ºC 37ºC

data not published


Enzyme production - the broad picture


PCA biplot of effect of temperature (25ºC)

on the enzymatic profile of L.theobromae

30ºC

37ºC

data not published


Concluding

There seems to be a host-specific expression of enzymes

But environmental temperature has major effect on the

modulation of extracellular enzymes

How will these pathogens behave under a climate

change scenario?

Thank you

Pacheca Farm, Douro

http://www.malhadinhanova.pt/pt/herdade/vinha/

Fungal enzymes involved in grapevine trunk diseasesmanagtd.eu/images/uploads/content/172/AC ESTEVES 12 05 2015 pdf.… · Fungal enzymes involved in grapevine trunk diseases . Grapevine

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