Chapter 8

Plant Defense Proteins –

Pathogenesis-Related (PR) Proteins
Plant defense against pathogens
(A-)

(R-)

(SAR)
PR proteins
 Pathogenesis-related (PR) proteins

 15 ~ 35 kD proteins
 Induced in HR tissues
 Localized predominantly in the intercellular
spaces
 Also accumulated during certain compatible host-
parasite interactions
 Tobacco PR proteins were originally grouped into
five families (Van Loon et al. 1987).
PR-1, -2, -3, -4, -5
 Currently, 14 families of PR proteins have been
recognized (Van Loon and Van Strien, 1999)
 Pathogenesis-related (PR) proteins
PR-1 Acidic unknown protein
PR-2 Basic b-1,3-glucanase (I), acidic/basic b-1,3-
glucanase (II, III)
PR-3 Basic chitinase (I), Acidic chitinase (II)
PR-4 Type I, II chitinase
PR-5 Acidic/basic thaumatin-like (TL), osmotin proteins
PR-6 Basic protease inhibitor
PR-7 Endoprotease
PR-8 Acidic chitinase (III), acidic/basic chitinase (IV)
PR-9 Lignin-forming peroxidase
PR-10 Ribonuclease-like proteins
PR-11 Basic Chitinase (V)
PR-12 Plant defensin
PR-13 Thionin
P. 152-154
PR-14 Lipid-transfer protein
 Elicitation of PR proteins
 Biotic elicitor
Viruses, fungi, bacteria, nematodes, viroids, and
insects
Exogenous elicitors
Fungal cell walls
Certain fungal metabolites
Elicitin
Endogenous elicitors
Salicylic acid (SA)
 Abiotic elicitor
UV, air pollutants, heavy metals, chemicals
(polyacrylic acid), and 2,6-dichloroisonicotinic acid
 Elicitin

 A class of 10 kD proteins (98 amino acid residues) secreted

only by various Phytophthora spp. and Pythium spp.
 The N-terminal 20 amino acids is a signal peptide.
 Some elicitins have no glycosylation.
 Some elicitins possess C-terminal domains of variable length.
Many of which have a high threonine, serine, or proline
content, suggesting an extensive O-linked glycosylation and
association with the cell wall.
 Grouped into 2 classes
 a elicitin : pI of 3.5 ~ 4.5
 b elicitin : pI of 9.0 ~ 9.5
 It induces HR, phytoalexin production, and PR proteins in
tobacco.
 ELI : elicitin
ELL : elicitin-like protein

GPI : glycosylphosphatidyl-
inositol-anchored proteins

Mol. Biol. Evol. 23:338–351. 2006

 PR-1

 168 amino acid residues

 With antifungal activity
 Biological function unknown
 Secreted out of cells
 Transgenic plants overexpressing PR-1 showed
resistance to pathogens
 PR-2 (b-1,3-glucanase)

 b-1,3-glucanase is abundant in higher plants.

 Has antifungal activity – degrade fungal cell wall
components
 Groups into 3 classes – Class I, II, III
Class I – basic isoform, localized in vacuole
Class II – acidic isoform, localized extracellularly
Class III – acidic isoform, localized
extracellularly, 54-59% similarity to class II
 Chitinase (PR-3, -4, -8, -11)

 Chitin is a linear homopolymer of b-1,4-linked N-

acetyl-D-glucosamine.
 Chitin is a structural component of fungi and
insects but not higher plants.
 Chitinases are grouped into 5 classes – Class I, II,
III, IV, V.
Class I, IV
N–SP-chitin-binding domain – variable hinge
region – catalytic domain-C
Class II, III, V – SP-catalytic domain
 Chitinase (PR-3, -4, -8, -11)

 Chitinase I, II – PR-3
 Chitinase I, II - PR-4
 Chitinase III IV – PR-8
 Chitinase V – PR-11
Polysaccharides
of Bacterial Cell
Walls and Insect
Exoskeletons

 Bacterial cell wall :

pepditoglycan
(GlcNAc-
MurNAc)

 Fungal cell walls

and insect
exoskeletons
 Chitin
GlcNAc
b(1-4) linkage
 Notes

 Not all isoforms of chitinase and b-1,3-glucanase

have antifungal activities.
 The class I (vacuolar) chitinase and tobacco b-1,3-
glucanase are the most active against F. solani
germlings, resulting in lysis of hyphal tips and
growth inhibition.
 The class II isoforms of these hydrolases exhibit no
antifungal activity.
 Synergistic antifungal activity of transgenic
plants

 Enhanced protection against fungal attacks has

been obtained by transforming more than one PR
gene into crops.
 Zhu and Lamb (1994) overexpressing chitinase
and glucanase genes in tobacco.
 PR-4

 Acidic intracellular type I, II chitinase

 20 kD
 Causes lysis of the germ tubes of Trichoderma
viride and Fusarium solani
 Thaumatin-like proteins (PR-5)
 Thaumatin is a sweet protein present in the fruit of the
monocot Thaumatococcus danielli, a West African shrub.
 Thaumatin-like (TL) proteins
 Homologous to permatins that permeabilize fungal
membranes
 Osmotin -Induced in response to osmotic stress (25g NaCl/L)
 Zeamatin – a maize seed protein
Causes the rapid release of cytoplasmic materials from
Candida albicans and Neurospora crassa.
Probably cause permeability or disruption of the fungal
plasma membrane
 P23 (tomato infected with citrus exocortis viroids)
accumulated in vacuoles and has antifungal activity.
50mg/ml of purified P23 protein inhibits 75% growth of
F. oxysporum f.sp. lycopersici and 60% of Phytophthora
citrophthora.
Sweet-tasting proteins
The infection of oat stem rust fungi on oat leaf
Appressorium
The infection process of stem rust
The development of oat stem rust fungal hyphae
 Nucleotide
sequence
alignment of the
thaumatin-like
gene family
Amino acid
sequence
alignment of
various TL
proteins
 Multiple copies of tlp genes

TLPs are encoded by a gene family.

Infection types
Accumulation of tlp gene transcripts in Rodney
(Pg-2) infected with Puccinia graminis

Compatible

Incompatible

Incompatible
Accumulation
of tlp-1, -2, -3,
and -4 gene
transcripts in
response to
infection
Accumulation of tlp-1, -2, -3, and -4 gene
transcripts in response to wounding
 Pga-6A Pga-1H

Development of
16 h
compatible and
incompatible isolates
of Puccinia graminis
f.sp. avenae in oat
30 h
Rodney (Pg-2)

AP : appressorium 42 h
SV : substomatal vesicle
IH : infection hyphae
HMC : haustorial
mother cell
60 h
 Appearance of HR

42 h

60 h

Oat Rodney (Pg-2) infected with incompatible isolates of

Puccinia graminis f.sp. avenae Pga-1H
Overexpression of
TLP-1 in barley
shows resistance
to barley powdery
mildew fungi

Without TLP-1 gene

With TLP-1 gene

 PR-6 (Protease inhibitors, PI)

 During plant growth and development, the protein

turnover rate is high since certain proteins needed
to be eliminated at one stages of life cycle. The
process is catalyzed by protease.
 However, this process can be regulated by
protease inhibitor.
 Some PI inhibit serine proteinase while others
inhibit cysteine proteinase.
 In tomato, two classes of PI – protease inhibitor I
and II.
 Plant defense against herbivore damage
 Protease inhibitors
Found in legumes, tomatoes, and other plants
Block the protein digestion by binding tightly
and specifically to the active site of protein-
hydrolyzing enzymes such as trypsin and
chymotrypsin.
 a-amylase inhibitor
Found in some legumes
Block the action of the starch-digestive enzyme
 Lectins
Bind to the epithelial cells lining the digestive
tract and interfere with nutrient absorption
 Sporamin (甘藷儲蛋白)

Isolated from sweet potato (甘藷)

A type of trypsin inhibitor
Wound-inducible
Transgenic tobacco expressing
sporamin
 Cystatin

Isolated from taro (芋頭)

A type of cysteine protease inhibitor
With antinematode activity
Antifungal activity, such as Sclerotium
rolfsii
 Plant
defense
against
herbivore
damage
含Protease inhibitor gene的煙草

非轉殖煙草 轉殖煙草
 PR-7

 Endoproteinase (P69) from tomato infected with

citrus exocortis viroid
 MW. 69kD, pI of 9.0
 With hydrolytic enzyme activity against bacteria,
oomycetes, and viruses.
 P69 selectively degrade the large subunit of
Rubisco.
 P69 is located in vacuoles and in intercellular
spaces.
 PR-9 (Peroxidase)

 Anionic peroxidases catalyze a number of

reactions that fortify plant cell walls.
 These reactions include the incorporation of
phenolics into cell wall and lignification
(polymerization of cinnamyl alcohols into lignin)
and suberization of plant cell walls.
 PR-10 (Ribonuclease-like protein)

 The betv1 gene family of birch (樺樹, Betula

verrucosa) encodes a group of 17 kD polypeptides,
including constitutively expressed pollen allergens
and PR proteins.
 The Betv1 proteins have 70-88% identity to the
pollen allergens and are homologous to ginseng
ribonuclease.
 Enzyme assays confirm that Betv1 possess
nuclease activity specific for RNA but not for
single- or double-stranded DNA.
 PR-12 (Defensin)

 A type of thionins (g-thionins)

 Has structural and functional similarity to those of insect and
mammalian defensin.
 Most of plant defensins are constitutively expressed, but can be
induced in response to pathogen attack.
 Defensins have antifungal activities against a broad spectrum
of filamentous fungi by causing membrane permeabilization.
 They may bind to membrane phospholipids, and then insert
into membrane and cause pores.
 e.g. In seeds of radish, the defensins Rs-AFP1 and Rs-AFP2
are located in the cell wall and released during seed
germination. They accumulate systemically at high levels
after infection by Alternaria brassicicola.
 PR-13 (Thionin)

 Thionins are low MW proteins (~5 kD) occurring in

seeds, stems, roots, and leaves of certain monocot
and dicot plants.
 Thionins are rich in cysteine (6 or 8 residues) and
are positively charged. They may interact with
negatively charged membrane phospholipids,
causing membrane disruption by forming pores.
 Thionins can be triggered by pathogen and exhibit
toxicity for plant pathogens.
 Thionin may be divided into two major groups
based on their structure: thionins and g-thionins
(defensins).
 PR-14 (Lipid-transfer protein, LTP)
 LTP are basic, 9 kD proteins with the ability to bind
and transfer a variety of lipids between membranes
in vitro.
 Several roles of LTPs in vivo have been proposed:
Participation in cutin formation
Defense reactions against plant pathogens
Symbiosis
Adaptation of plants to various environmental
conditions
 With antibiotic activity against bacterial and fungal
pathogens
 The sugar beet LTPs exhibit a strong in vitro
antifungal activity against Cercospora beticola.
END