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REVIEW
The superfamily of thaumatin-like proteins: its origin,evolution, and expression towards biological function
Jun-Jun Liu • Rona Sturrock •
Abul K. M. Ekramoddoullah
Received: 7 January 2010 / Revised: 26 January 2010 / Accepted: 28 January 2010 / Published online: 5 March 2010
� Her Majesty the Queen in Rights of Canada 2010
Abstract Thaumatin-like proteins (TLPs) are the prod-
ucts of a large, highly complex gene family involved in
host defence and a wide range of developmental processes
in fungi, plants, and animals. Despite their dramatic
diversification in organisms, TLPs appear to have origi-
nated in early eukaryotes and share a well-defined TLP
domain. Nonetheless, determination of the roles of indi-
vidual members of the TLP superfamily remains largely
undone. This review summarizes recent advances made in
elucidating the varied TLP activities related to host resis-
tance to pathogens and other physiological processes. Also
discussed is the current state of knowledge on the origins
and types of TLPs, regulation of gene expression, and
potential biotechnological applications for TLPs.
Keywords Antifungal activity � Defence response �Environmental stress � Phylogenetic analysis �Thaumatin-like protein
Abbreviations
AFP Anti-freeze protein
GPCRs G protein-coupled receptors
MYA Million years ago
OLP Osmotin-like protein
PR Pathogenesis-related
PR5 Family 5 of pathogenesis-related proteins
PR5K PR5-like kinase
TLP Thaumatin-like protein
Introduction
Thaumatin-like proteins (TLPs) are polypeptides of about
200 amino acid residues that share sequence similarity with
thaumatin (Velazhahan et al. 1999), a sweet-tasting protein
originally found in the fruit of the West African rain forest
shrub Thaumatococcus daniellii (Benth.) (Van der Wel and
Loewe 1972). Due to their inducible expression by stresses
like pathogen/pest attack, plant TLPs are classified as the
pathogenesis-related (PR) protein family 5 (PR5), 1 of 17
families of defence-related PR proteins (Christensen et al.
2002; van Loon et al. 2006). The most recent review of
plant TLPs by Velazhahan et al. (1999) describes occur-
rence, properties, and regulation of plant PR5 genes. Over
the past decade, TLPs have been discovered in a wide
range of organisms (Shatters et al. 2006), including nem-
atodes (Kitajima and Sato 1999), insects (Brandazza et al.
2004), fungi (Grenier et al. 2000; Sakamoto et al. 2006),
and both gymnosperms (Midoro-Horiuti et al. 2000; Pig-
gott et al. 2004; Zamani et al. 2004; Futamura et al. 2006;
O’leary et al. 2007; Liu et al. 2010) and angiosperms
(Velazhahan et al. 1999; van Loon et al. 2006). This paper
provides an overview of the knowledge gained on the TLP
superfamily over the last decade. Included are results about
the origin, structure, evolution, expression, and function of
TLPs as well as new information on their potential bio-
technological applications.
Communicated by R. Reski.
J.-J. Liu (&) � R. Sturrock � A. K. M. Ekramoddoullah
Natural Resources Canada, Canadian Forest Service,
Pacific Forestry Centre, 506 West Burnside Road,
Victoria, BC V8Z 1M5, Canada
e-mail: [email protected]
123
Plant Cell Rep (2010) 29:419–436
DOI 10.1007/s00299-010-0826-8
Origins of the TLP genes
Fungal TLPs
Grenier et al. (2000) first reported the presence of TLPs in
several basidiomycete fungi, including Irpex lacteus (Fr.),
Lentinula edodes (Berk.) and Rhizoctonia solani (J.G.
Kuhn). More recently, fungal TLP-encoding genes have
also been found in the basidiomycete yeast Cryptococcus
neoformans (Sakamoto et al. 2006). As the genome
sequences of several fungi are available, we performed
BLAST search through the available genome databank of
the Gene Index Project (at the Computational Biology and
Functional Genomics Laboratory, the Dana-Farber Cancer
Institute) and Fungal Genome Resources (at the Broad
Institute and the DOE Joint Genome Institute, JGI), and
found that fungi TLP family is small, only two or three
members in basidiomycota Coprinopsis cinerea, Crypto-
coccus neoformans, and Moniliophthora perniciosa
(Table 1).
As for ascomycete fungi, Aspergillus nidulans cet A
showed certain homology with S-type TLPs with molecular
masses of 16–17 kD from cereal plants (Osherov et al.
2002; Greenstein et al. 2006). Although a few previous
reports considered cet A and its ascomycete homologs as
TLPs (Sakamoto et al. 2006), it is worthwhile to point out
that so far no protein with typical TLP-domain features has
been found in ascomycete fungi.
Animal TLPs
Animal TLPs were first reported in the nematode Caeno-
rhabditis elegans (Maupas) (Kitajima and Sato 1999), and
later in insects Schistocerca gregaria (Forsskal) and Loc-
usta migratoria (L.) (Brandazza et al. 2004). A recent study
found that TLPs are present at least in four insect orders:
Coleoptera (Diaprepes and Biphyllus), Hemiptera (Toxop-
tera), Hymenoptera (Lysiphlebus), and Orthoptera
(Schistocerea) (Shatters et al. 2006). Our BLAST search
through the available genome databank revealed the TLP
family represented by six members each in C. elegans and
the pea aphid (Acyrthosiphon pisum), five in the red flour
beetle (Tribolium castaneum Herbst), and a total of three in
L. migratoria (Table 1). To date no TLP has been found in
Drosophila.
Plant TLPs: PR5, osmotin-like proteins, PR5-like
allergies, and PR5-like kinase receptors
TLPs are universal in plants, including angiosperms,
gymnosperms, and bryophytes like the moss Physcomit-
rella patens subsp. Patens (Table 1). Based on available
plant genomes and EST databases, the TLP superfamily
contains at least 31 genes in rice (Oryza sativa), 28 in
Arabidopsis thaliana (L.), 13 in white spruce (Picea glauca
Moench), 10 in western white pine (Pinus monticola
Dougl. ex D. Don), 6 in moss (Table 1), but no TLP gene
was retrieved from EST data of the single-celled green alga
(Chlamydomonas reinhardtii P. A. Dang.) (Liu et al. 2010).
Because environmental stresses, including pathogen/
pest invasion, drought, wounding and cold hardiness,
induce their expression, plant TLPs were assigned to form
family 5 of the PR proteins (Velazhahan et al. 1999; van
Loon et al. 2006). Some PR5 proteins, which are believed
to be capable of creating transmembrane pores (Abad et al.
1996; Anzlovar and Dermastia 2003), have also been
named permatins. Examples of permatins occurring in
particularly high concentrations in cereal seeds include
zeamatin from maize (Zea mays L.), hordomatin from
barley (Hordeum vulgare L.), and avematin from oats
(Avena sativa L.) (Roberts and Selitrennikoff 1990;
Skadsen et al. 2000).
Osmotin is a TLP that was originally regarded as a salt-
induced protein in osmotically stressed tobacco (Nicotiana
tabacum L.) cells (Singh et al. 1989). In contrast to most
other plant PR5 proteins, osmotin and osmotin-like pro-
teins (OLPs) are basic isoforms and they presumably
accumulate in the vacuoles of plant cells instead of extra-
cellularly (Yun et al. 1998; Anzlovar and Dermastia 2003).
Several members of the plant TLP superfamily have
been reported as food allergens from fruits and pollen
allergens from conifers (Hoffmann-Sommergruber 2002;
Breiteneder 2004). These food TLP allergens include
cherry (Prunus avium L.) Pru av2 (Inschlag et al. 1998),
bell pepper (Capsicum annuum L.) Cap a1 (Fuchs et al.
2002), kiwi (Actinidia chinensis Planch.) Act c2 (Gavrovic-
Jankulovic et al. 2002), apple (Malus x domestica) Mal d2
(Krebitz et al. 2003), grape (Vitis vinifera L.) TLP (Pas-
torello et al. 2003), and banana (Musa acuminate Colla)
TLP (Leone et al. 2006). Of pollen TLP allergens, there are
Jun a3 from mountain cedar (Juniperus ashei J. Buchholz)
(Midoro-Horiuti et al. 2000), Cup a3 from Arizona cypress
(Cupressus arizonica Greene) (Cortegano et al. 2004), and
Cry j3 from Japanese cedar (Cryptomeria japonica L. f.)
(Fujimura et al. 2007). Among these allergenic TLPs, Jun
a3, Mal d2, and Pru av2 each show a binding ability to Ig-E
from allergic persons by allergenic motifs in their protein
structures (Ghosh and Chakrabarti 2008).
Arabidopsis and rice both contain PR5-like receptor
kinases (PR5K) with an extracellular TLP domain and an
intracellular kinase domain (Wang et al. 1996). PR5K
genes are present in both monocots and dicots. Arabidopsis
contains three PR5K genes while rice has at least one
(Table 1).
420 Plant Cell Rep (2010) 29:419–436
123
Protein structure
Amino acid sequences for TLPs from plants, animals and
basidiomycete fungi share significant homology with
thaumatin (Fig. 1). Based on PROSITE, the database of
protein domains, families and functional sites (Hulo et al.
2008), TLPs are known to have a thaumatin family sig-
nature (PS00316): G-x-[GF]-x-C-x-T-[GA]-D-C-x(1,2)-
[GQ]-x(2,3)-C (Jami et al. 2007; Tachi et al. 2009). Most
TLPs have molecular masses ranging from 21 to 26 kD.
These large (L-) type TLPs contain 16 conserved cysteine
residues. The molecular mass of a small group of small (S-)
Table 1 Thaumatin-like protein sequences (total number in brackets) and their GenBank accession numbers or gene locus in representative
species belonging to the plant, animal, and fungal kingdoms
Plant
Arabidopsis thaliana (28)
NP_173261, NP_173365, NP_173432, NP_177182, NP_177503, NP_177640, NP_177641, NP_177642, NP_177708, NP_177893,
NP_179376, NP_180054, NP_180445, NP_192902, NP_193559, NP_194149, NP_195324, NP_195325, NP_195579, NP_195834,
NP_197850, NP_198644, NP_198818, NP_568046, NP_973870, NP_001031064, NP_001031809, NP_001078513
Oryza sativa (31)
NP_001041821, NP_001044756, NP_001049473, NP_001049531, NP_001049533, NP_001050827, NP_001050832, NP_001050833,
NP_001052130, NP_001054348, NP_001058421, NP_001059476,
NP_001062246, NP_001063785, NP_001063786, NP_001064158, NP_001064159, NP_001064589, NP_001067069, NP_001067073,
NP_001067074, NP_001067331, NP_001067332, NP_001067333, NP_001067334, NP_001067335, NP_001067336, NP_001067337,
NP_001068535, NP_001068536, NP_001068554
Picea glauca (13)
DR548648, DR550004, DR551624, DR552739, DR555245, DR566085, DR573433, DR580767, DR582629, DR584727, DV996551,
EX409023, EX409554
Pinus monticola (10)
GQ329659 to GQ329668
Physcomitrella patens subsp. Patens (6)
XP_001765997, XP_001769333, XP_001784610, AJ566726, BJ186638, FC369913
Animal
Acyrthosiphon pisum (6)
XP_001942530, XP_001942572, XP_001942718, XP_001942779, XP_001942788, XP_001951906
Tribolium castaneum (5)
XP_968724, XP_969010, XP_975156, XP_975166, XP_975175
Locusta migratoria (3)
CO821337, CO821344, CO849988
Caenorhabditis elegans (6)
NP_500747, NP_500748, NP_502360, NP_502361, NP_502362, NP_507263
Fungi
Basidiomycete
Moniliophthora perniciosa (3)
XP_002389185, XP_002395649, XP_002395825
Coprinopsis cinerea (3)
XP_001837753, XP_001837754, XP_001837765
Cryptococcus neoformans (var. grubii H99) (2)
Locus: CNAG_02497.2, CNAG_05924.2
Ascomycete
Aspergillus nidulans (2)
XP_660683, XP_680888
Aspergillus fumigatus
XP_747035, XP_754650, XP_748371
These sequences were searched out by BLAST through the available genomes at GenBank, and through the Gene Index Project data sets at Dana-
Farber Cancer Institute, and Fungal Genome Resources at the Broad Institute and the DOE Joint Genome Institute (JGI)
Plant Cell Rep (2010) 29:419–436 421
123
type TLPs mainly from conifers and cereals is around 16–
17 kD. S-type TLPs have only ten cysteines at conserved
positions because of a peptide deletion (Fig. 1). The
disulfide bridges formed by these conserved cysteines help
stabilize the molecule and allow for correct folding and
high stability under extreme thermal and pH conditions
(Fierens et al. 2009), as well as for resistance to protease
degradation (Smole et al. 2008). Both animal and plant
TLPs contain an N-terminal signal peptide targeting mature
proteins into the secretory pathway. Tobacco osmotin and
Fig. 1 Structural features of the
TLP superfamily as shown by
amino acid sequence alignment
of 15 representative TLPs from
a variety of taxa. These include
Thaumatococcus daniellii(thaumatin, AB265690), moss
Physcomitrella patens subsp.
(XP_001765997), conifer Pinusmonticola (GQ329659 and
GQ329660), Arabidopsis
(NP_173261 and NP_173365
for TLPs, and NP_198644 for
PR5K), rice (NP_001049473
and NP_001067331), nematode
(Caenorhabditis elegans,
NP_502360), pea aphid
(Acyrthosiphon pisum,
XP_001942779), basidiomycete
fungi Coprinopsis cinerea(XP_001837765) and
Moniliophthora perniciosa(XP_002389185), and
ascomycete fungi Aspergillusnidulans (XP_660683) and
Neosartorya fischeri(XP_001258659). The TLP
family signature (PS00316) in
thaumatin, G-x-[GF]-x-C-x-T-
[GA]-D-C-x(1,2)-[GQ]-x(2,3)-
C, is boxed. The conserved
cysteine residues are indicated
by a black background; and
other conserved amino acids are
indicated by a gray shadow.
Dashes in the sequences
represent single amino acid gaps
for best alignment. Conserved
positions of five amino acids are
labeled with an asterisk, and
they are responsible for
topology and surface
electrostatic potential around
the cleft proposed to determine
TLP specificity to their target
receptors or ligands
422 Plant Cell Rep (2010) 29:419–436
123
plant OLPs also have a C-terminal propeptide that is con-
sidered to be a determinant for their vacuolar targeting
(Anzlovar and Dermastia 2003).
Thus far, crystal structures have been determined for
seven plant TLPs, including thaumatin (Ogata et al. 1992),
zeamatin (Batalia et al. 1996), tobacco PR-5d (Koiwa et al.
1999) and osmotin (Min et al. 2004), plus the cherry
allergen Pru Av2 (Dall’Antonia et al. 2005), banana
allergen Ba-TLP (Leone et al. 2006) and tomato NP24-I
(Ghosh and Chakrabarti 2008). All show similar 3D
structures with three domains and a cleft structure between
domains I and II (Ghosh and Chakrabarti 2008).
In the 3D structures of these plant TLPs, domain I is a
lectin-like b-barrel that forms the compact core of a TLP
molecule. To one side of domain I is domain II made up of
several loops. On the other side of domain I is domain III,
which consists of a small loop. Each domain is stabilized
by at least one disulfide bridge linked by up to 16 cysteine
residues with a conserved spatial distribution throughout
the protein (Min et al. 2004). Very similar topological
model is predicated by homology modeling and loop
insertion for other TLPs from plants (Fig. 2) and animals
(Brandazza et al. 2004).
The cleft between domains I and II may have an acidic,
neutral, or basic nature for binding different ligands/
receptors. In all plant PR5 proteins with known antifungal
activity this cleft is acidic because of five highly conserved
amino acids (arginine, glutamic acid, and three aspartic
acid residues) (Fig. 1). This acidic cleft is assumed to be
relevant to their specific receptor binding for an antifungal
activity (Batalia et al. 1996; Koiwa et al. 1999; Min et al.
2004). In contrast is the basic cleft region in thaumatin. A
so-called thaumatin loop that is present within the thau-
matin domain II but absent in the antifungal plant PR5
proteins may explain their differences in function (Min
et al. 2004).
Evolution
Evolutionary relationships within the TLP superfamily are
not well understood. For example, a study of phylogenetic
and structural relationships by Shatters et al. (2006) sug-
gested that plant PR5 proteins are paraphyletic in angio-
sperms and related to animal TLPs from insects and
nematodes. However, another study indicated that TLPs
are grouped into three major clades representing the ani-
mal, plant, and fungal kingdoms, respectively (Sakamoto
et al. 2006).
To better decipher the evolution of the TLP superfamily,
we conducted a comprehensive search of available genome
sequences and EST database (Table 1). Alignment analysis
was done using 118 putative TLP sequences selected from
ascomycete and basidiomycete fungi, moss, gymnosperms
and angiosperms, plus animals as represented by nema-
todes and insects. The neighbor joining algorithm was used
to construct a phylogenetic tree (Fig. 3). The topological
pattern of the resulting tree indicates that the TLP super-
family is highly divergent, with the possibility of nine
distinct groups (I–IX), each containing proteins with rela-
tively high identity.
Group I contains representative sequences from asco-
mycete fungi exclusively, and it is far away from all other
TLP groups. These ascomycete sequences shared 32–92%
identity within group but only 3–24% between group
identities. In addition to a long deletion, similar to that in the
S-type TLPs from cereals and conifers, ascomycete
sequences have other deletions at N-terminal region
(Fig. 1). Especially, they share only four of the 16 cysteines
typically occurring at conserved positions in most TLPs and
they are lack of thaumatin family signature (PS00316)
(Fig. 1). The disulfide bridges formed by the conserved
cysteines in TLPs are crucial to their characteristic 3D
structures, so it is likely that these so-called ascomycete
TLPs may have structures that are distinctly different from
other TLPs. Because of their low degree of similarity, these
ascomycete sequences may represent genes that are non-
homologous with the other eight groups of TLPs and have a
Fig. 2 Three-dimensional (3D) structure of one Pinus monticolaTLP, PmTLP-L1 (GQ329659), modulated and represented by a
ribbon diagram. The 3D structure of PmTLP-L1 was simulated using
the SWISS-MODEL software package (Swiss Institute of Bioinfor-
matics, http://swissmodel.expasy.org/). The Deep View Swiss-Pdb-
Viewer software package was used to manipulate the PmTLP-L1 3-D
model where domain I formed by a b-sandwich with 11 b-sheets,
domain II made up by three a-helical structures plus two b-sheets,
domain III composed of a junction loop and a b-sheet, and the cleft
structure localized between domains I and II
Plant Cell Rep (2010) 29:419–436 423
123
different evolutionary origin, suggesting that these asco-
mycete sequences should not be considered as TLP genes.
Group II only consists of members exclusively from
basidiomycete fungi with 55–80% within group identity
and 23–43% identity to other seven TLP groups from
plants and animals and all sequences include the 16 con-
served Cys residues (Fig. 1). Nematode TLPs constitute a
monophyletic clade as group III while all insect TLPs are
Fig. 3 Phylogenetic analysis of
118 TLPs by the neighbor-
joining (NJ) method. A
phylogenetic tree was
constructed based on amino acid
alignment of TLP sequences by
the Clustal W program. TLPs
were labeled according to
GenBank accession numbers as
shown in Table 1 and clustered
into nine clusters (I–IX). Plant
TLPs include 30 TLPs in rice
(Os-), 28 in Arabidopsis (At-),
13 in white spruce (Piceaglauca, Pig-), 10 in western
white pine (Pinus monticola,
Pm-), and six in moss
(Physcomitrella patens subsp.,
Pph-). Animal TLPs include six
TLPs in nematode
(Caenorhabditis elegans, Cae-),
six in pea aphid (Acyrthosiphonpisum, Acp-), four in red flour
beetle (Tribolium castaneum,
Trc-), and three in locust
(Locusta migratoria, Lom-).
Basidiomycete fungi TLPs
include three in both
Coprinopsis cinerea (Coc-) and
Moniliophthora perniciosa(Mop-). Ascomycete fungi
TLPs include two in Aspergillusnidulans (Asn-) and one in
Aspergillus fumigatus (Asf-),
Aspergillus oryzae (Aso-),
Neosartorya fischeri (Nef-), and
Penicillium chrysogenum (Pec-
), respectively. Arabidopsis and
rice PR5-like receptor kinases,
marked as PR5K, were grouped
here based on their TLP domain
similarities to other regular
TLPs. The scale at the bottom
indicates genetic distance
proportional to the amino acid
substitutions per site
424 Plant Cell Rep (2010) 29:419–436
123
localized in another distinct monophyletic clade for group
V. Plant TLPs are divided into five groups (IV, VI, VII,
VIII, and IX). Based on this phylogenetic pattern, we
hypothesize that one TLP gene was present in the last
common ancestor of extant plants, animals and fungi,
which existed about 1 billion years ago (Doolittle et al.
1996).
To date, plant TLP genes appear to be confined to
multicellular plants (Liu et al. 2010). In group VII, there
are two subclades (Fig. 3), one of them contains all six
moss TLPs that is proposed to have expanded from a single
precursor. The other subclade of group VII consists of
TLPs from both angiosperms and gymnosperms, suggest-
ing that group VII may represent an ancestral type of plant
TLP superfamily as compared to the other four groups (IV,
VI, VIII, and IX) of plant TLPs. Characterization of a TLP
family in moss strongly suggests the most recent common
ancestor of mosses and vascular plants, present about
425 million years ago (MYA) (Wellman et al. 2003),
already possessed at least one TLP gene with typical TLP-
domain features (Fig. 1). The fact that our phylogeny
reconstruction reveals five plant TLP clades containing
putative genes from both angiosperm and gymnosperm
lineages (Fig. 3), means that a minimum of five TLP genes
were probably present in the last common ancestor of
extant seed plants about 300 MYA. At that time, these
genes were probably already quite diverse in terms of both
sequence and function. For example, all P. monticola TLPs
(PmTLPs) except for PmTLP-L1 (GQ329659) share an
identity threshold of 48% and are grouped in one cluster
(group IX, Fig. 3). PmTLP-L1 shows only 30–37% identity
to other PmTLPs, but has 45–52% identity with angio-
sperm TLPs in the same cluster (group IV, Fig. 3). Also,
PmTLP-L1 has relatively basic surface in cleft region,
especially when compared with other PmTLPs with acidic
clefts, suggesting that its potential ligand may be different
from other PmTLPs and from Plant PR5 protein with
known anti-fungal activity (Liu et al. 2010).
The branch topology of the TLP phylogenetic tree
indicates that the nine groups may have evolved from
multiple rounds of gene duplication. Grouping of multiple
members of the same species in the same cluster or sub-
cluster suggests that gene duplication events continued to
happen throughout the evolution of plant and animal spe-
cies (Shatters et al. 2006). Clustering of multiple TLP
genes on the same chromosome and even at the same locus
has been observed in a hybrid potato (Solanaceae tubero-
sum 9 S. phureja) (Ruiz et al. 2005), apple (Gao et al.
2005), cotton (Gossypium hirsutum cv. Delta Pine 62)
(Wilkinson et al. 2005), Arabidopsis, and rice (Shatters
et al. 2006). This suggests that tandem duplication be one
of the important mechanisms for the asymmetric expansion
of the TLP superfamily during species formation.
Expression of the TLP superfamily
Velazhahan et al. (1999) reviewed the regulation of plant
PR5 gene expression by factors such as microbial infection
and pathogenic elicitors, osmotic stress, wounding and
plant hormones (e.g. abscisic acid, ethylene, salicylate, and
methyl jasmonate). In the tobacco osmotin promoter, GCC
boxes, coordinated with other types of cis-elements, are
required for full ethylene responsiveness (Raghothama
et al. 1997). A recent study of the rice TLP (Rtlp1) found
that W-box elements in the promoter region, the binding
site of transcription factor WRKY proteins, are required for
the Rtlp1 promoter to respond to fungal elicitors (Hiroyuki
and Terauchi 2008).
In addition to induced expression by various environ-
mental stresses (Velazhahan et al. 1999; Kenton et al.
2000; Jung et al. 2005; Onishi et al. 2006), some TLPs are
expressed constitutively in an organ-preferred or develop-
ment-dependent pattern (Regalado and Ricardo 1996). For
examples, in developing barley and oats seed, TLP gene
expression switches from the ovary wall to the aleurone
(Skadsen et al. 2000). An elderberry (Sambucus nigra) TLP
shows a fruit-specific expression and no antifungal and
glucanase activities during the final stages of fruit matu-
ration (Van Damme et al. 2002). A pepper TLP (PepTLP)
was also shown to have a gene expression pattern regulated
by fruit-developmental stages (Kim et al. 2002). A pro-
moter-gus fusion study found that a Japanese pear (Pyrus
serotina Burm. f.) TLP gene (PsTL1) was expressed spe-
cifically in the pistil and at low level in anthers, but not at
all in other floral organs nor in leaves (Sassa et al. 2002).
Leon-Kloosterziel et al. (2005) found that Arabidopsis
AtTLP1 is expressed specifically in the root vascular bun-
dle in response to colonization by non-pathogenic Pseu-
domonas spp. The roles of TLPs and the cascades that
regulate their expression differ among members of the
Cryptomeria japonica (L. f.) TLP family (Futamura et al.
2006). Most PmTLP members in western white pine are
responsive to rust infection, wounding, or exposure to cold
temperatures, but PmTLP-L1, PmTLP-L5 and PmTLP-S4
show constitutive expression at low levels without signif-
icant responsiveness to any tested environmental stresses
(Liu et al. 2010). These observations suggest that some
TLPs may be involved in physiological processes other
than host defence to biotic/abiotic stress.
Biological functions
Antifungal activity
While TLP antifungal activity was well documented by
Velazhahan et al. (1999), there is additional recent
Plant Cell Rep (2010) 29:419–436 425
123
evidence supporting the roles of TLPs in host defence and
other physiological processes by transgenic overexpres-
sion, in vitro antifungal activity test, or related protein
activity analyses (Table 2). For example, the TLP isolated
from chestnut (Castanea sativa Mill.) has in vitro anti-
fungal activity against Trichoderma viride and Fusarium
oxysporum and shows strong synergistic effects with
CsCh1, an abundant endochitinase from the chestnut cot-
yledon (Garcia-casado et al. 2000). The Kweilin chestnut
(Castanopsis chinensis) TLP exerts antifungal activity
against Botrytis cinerea, Fusarium oxysporum, Mycosp-
haerella arachidicola, and Physalospora piricola (Chu and
Ng 2003). A cold-induced TLP in the apoplast of winter
wheat (Triticum aestivum L.) displays antifungal activity
against the snow mold fungus (Microdochium nivale)
(Kuwabara et al. 2002). The grapevine TLP (VvTLP-1)
significantly inhibits in vitro spore germination and hyphal
growth of Elsinoe ampelina (Jayasankar et al. 2003). Two
other grape proteins, identified as TLPs by immunological
method and N-terminal sequencing, exhibits strong in vitro
antifungal activity by blocking the growth of Phomopsis
viticola and Botrytis cinerea mycelia (Monteiro et al.
2003). The recombinant Solanum nigrum TLP (SnOLP)
reveals antifungal activity toward several plant pathogenic
fungi (Fusarium solani f. sp.glycines, Colletotrichum spp.
and Macrophomina phaseolina) and an oomycete (Phy-
tophthora nicotiana var. parasitica) under in vitro condi-
tions (Campos et al. 2008).
TLPs have glucan binding and glucanase activities
Mechanisms for the antifungal activity of TLPs are believed
to be related to their ability to destroy of the cell walls of
pathogenic fungi using a variety of enzymatic activities.
Several TLPs exhibit a special binding activity to several
water-insoluble b-1,3-glucans (Trudel et al. 1998; Grenier
et al. 1999, 2000). b-1,3-glucan is a common component of
the fungal cell wall. Interaction of TLPs with b-1,3-glucan
may act in concert with PR2 enzyme to disrupt fungal cell
wall synthesis and/or prevent proper fungal wall assembly
during hyphal extension (Bormann et al. 1999; Osmond et al.
2001). A later study found that only specific barley TLP
isoforms interact tightly with b-1,3-D-glucans (Osmond et al.
2001). Among barley TLPs, Pr22-3 has glucanase activity, as
indicated by its ability to digest laminarin, a storage glucan.
Furthermore, Pr22-3 was more active in the spore bioassay
with pathogens like Rhynchosporium secalis than other
barley TLPs (Zareie et al. 2002).
Interestingly, members of some fungal TLP families have
also been reported to display b-1,3-glucanase activity
(Grenier et al. 1999, 2000). An endo-glucanase from the
edible shiitake mushroom Lentinula edodes shares sequence
homology with the antifungal TLPs (Grenier et al. 2000).
Sakamoto et al. (2006) revealed that the L. edodes TLP, tlg1,
exhibits cell wall lytic activity to degrade lentinan (an
antitumor polysaccharide) during postharvest preservation,
suggesting that the tlg1 endo-glucanase activity helps
defend against fungi that are pathogenic on other fungi. In
fact, Martin et al. (2007) show that glucanase secreted by
some pathogenic fungi and other filamentous fungi can
degrade fungal cell walls, providing further evidence to
support the suggestion that fungal TLPs are involved in
fungal–fungal interactions. Some TLPs produced by nem-
atodes and insects are likely to serve to protect them against
predation and pathogenic infection by nematophagous and
entomopathogenic fungi, respectively. Animal TLPs are
considered as potential components of the antifungal strat-
egy in animal defence (Brandazza et al. 2004).
Despite the evidence presented above, b-1,3-glucanase
activity conferred by plant TLPs is not always related to
their anti-fungal activity (van Loon et al. 2006). A banana
fruit TLP that exhibits a low but detectable in vitro endo-b-
1,3-glucanase activity is apparently lacking antifungal
activity towards pathogenic fungi (Barre et al. 2000). Also,
the apple and cherry TLPs possess a moderate endo-b-1,3-
glucanase activity but are devoid of antifungal activity too
(Menu-Bouaouichea et al. 2003). These findings suggest
that glucanase activity is not the sole mechanism for TLP
antifungal activity.
TLPs function as xylanase inhibitor
Xylanases (endo-b-1,4-xylanases, EC 3.2.1.8) depolymer-
ize xylan, one of the most abundant polysaccharides (next
to cellulose) in the cell wall of higher plants; both plants
and microorganisms produce xylanases. This enzyme has
been shown to be indispensable for the pathogen Botrytis
cinerea to infect plants (Brito et al. 2006). A recent study
has discovered that a novel type of xylanase inhibitor
(TLX1) from wheat (Triticum aestivum) belongs to the
TLP family (Fierens et al. 2007). The recombinant TLX1
protein displays xylanase-inhibiting activity against
Trichoderma longibrachiatum xylanase (XynI) (Fierens
et al. 2007). Wheat TLXI is among the subgroup of S-type
TLPs (15–17 kD). Based on the sequence homology
between TLX1 and other TLPs, it is proposed that xylanase
inhibitor activity is one of the mechanisms by which TLPs
function in plant defence (Fierens et al. 2007).
Do TLPs function as a-amylase and trypsin inhibitors?
Zeamatin is mainly known for its antifungal activity and
proposed as a medical agent, acting on vaginal murine
candidosis cells, or in transgenic plants, increasing their
resistance against pests and pathogens (Selitrennikoff
et al. 2000; Franco et al. 2002). Purified zeamatin was
426 Plant Cell Rep (2010) 29:419–436
123
Ta
ble
2A
sum
mar
yo
ffu
nct
ion
ally
char
acte
rize
dth
aum
atin
-lik
ep
rote
ins
(TL
Ps)
Nam
eS
ou
rces
Reg
ula
tio
n/i
nd
uct
ion
(by
/in
)F
un
ctio
n/b
iolo
gic
alro
leR
efer
ence
PR
5K
Ara
bid
op
sis
tha
lia
na
Co
nst
itu
tiv
ely
Tra
nsg
enic
anti
fun
gal
pro
tect
ion
Gu
oet
al.
(20
03
)
Cd
TL
PC
ass
iad
idym
ob
otr
yaC
ult
ure
dce
lls
Inv
itro
anti
fun
gal
acti
vit
yV
ital
iet
al.
(20
06
)
CsT
L1
Ca
sta
nea
sati
vaC
oty
led
on
Inv
itro
anti
fun
gal
acti
vit
yG
arci
a-C
asad
oet
al.
(20
00)
CaT
LP
Ca
sta
no
psi
sch
inen
sis
See
dIn
vit
roan
tifu
ng
alac
tiv
ity
Ch
uan
dN
g(2
00
3)
Dca
n1
Den
dro
ides
Ca
na
den
sis
Bee
tle
larv
aeT
her
mal
hy
ster
esis
Wan
gan
dD
um
an(2
00
6)
IFW
19
Ho
rdeu
mvu
lga
reC
hem
ical
inle
aves
En
do
-b-1
,3-g
luca
nas
eac
tiv
ity
,an
d
anti
fun
gal
acti
vit
y
Gre
nie
ret
al.
(19
99
)
Hv
PR
5c
H.
vulg
are
Ger
min
ated
gra
in(1
,3)-
b-g
luca
n-b
ind
ing
acti
vit
y,
inv
itro
anti
fun
gal
acti
vit
y,
tran
sgen
ic
anti
fun
gal
pro
tect
ion
,b
ut
no
end
o-b
-
1,3
-glu
can
ase
acti
vit
y
Osm
on
det
al.
(20
01
)
Pr2
2-3
H.
vulg
are
Lea
ves
En
do
-b-1
,3-g
luca
nas
eac
tiv
ity
,an
d
anti
fun
gal
acti
vit
y
Zar
eie
etal
.(2
00
2)
tlp
-1H
.vu
lga
reL
eav
esT
ran
sgen
ican
tifu
ng
alp
rote
ctio
nM
ack
into
shet
al.
(20
07
)
TL
PIr
pex
lact
eus
My
celi
um
En
do
-b-1
,3-g
luca
nas
eac
tiv
ity
,an
din
vit
roan
tifu
ng
alac
tiv
ity
Gre
nie
ret
al.
(20
00
)
TL
PL
enti
nu
sed
od
esF
ruit
ing
bo
die
sE
nd
o-b
-1,3
-glu
can
ase
acti
vit
yG
ren
ier
etal
.(2
00
0)
tlg
1L
.ed
od
esF
ruit
ing
bo
die
sE
nd
o-b
-1,3
-glu
can
ase
acti
vit
yS
akam
oto
etal
.(2
00
6)
AP
24
,NP
24
Lyc
op
ersi
con
escu
len
tum
Fru
its
En
do
-b-1
,3-g
luca
nas
eac
tiv
ity
,an
d
anti
fun
gal
acti
vit
y
Gre
nie
ret
al.
(19
99
)
P2
3L
.es
cule
ntu
mV
iro
id-i
nd
uce
dIn
vit
roan
tifu
ng
alac
tiv
ity
,an
dtr
ansg
enic
anti
fun
gal
pro
tect
ion
Fag
oag
aet
al.
( 20
01
)
Mal
-TL
PM
alu
sd
om
esti
cus
Fru
its
En
do
-b1
,3-g
luca
nas
eac
tiv
ity
,b
ut
no
anti
fun
gal
acti
vit
y
Men
u-B
ou
aou
ich
eaet
al.
(20
03)
Ban
-TL
PM
usa
acu
min
ate
Fru
its,
MeJ
ain
du
ced
inro
ot
En
do
-b-1
,3-g
luca
nas
eac
tiv
ity
,b
ut
no
in
vit
roan
tifu
ng
alac
tiv
ity
Bar
reet
al.
(20
00
)
Ban
-TL
PM
.a
cum
ina
taF
ruit
sIn
vit
roan
tifu
ng
alac
tiv
ity
,b
ut
no
end
o-b
-
1,3
-glu
can
ase
acti
vit
y
Men
u-B
ou
aou
ich
eaet
al.
(20
03)
TL
P(2
0k
D)
M.
ba
sjo
oF
ruit
sIn
vit
roan
tifu
ng
alac
tiv
ity
Ho
etal
.(2
00
7)
Osm
oti
nN
.ta
ba
cum
NaC
lin
cult
ure
dce
lls
(a)
Bin
dto
yea
stg
lyco
pro
tein
;Y
un
etal
.(1
99
7),
Ibea
set
al.
(20
00
,
20
01
)
(b)
Bin
dto
GP
CR
s;N
aras
imh
anet
al.
(20
05
)
(c)
An
tifu
ng
alac
tiv
ity
by
gly
can
inte
ract
ion
;
Sal
zman
etal
.(2
00
4)
(d)
Tra
nsg
enic
pro
tect
ion
toco
ld,
salt
or/
and
dro
ug
ht
Bar
thak
ur
etal
.(2
00
1),
D’A
ng
eli
and
Alt
amu
ra(2
00
7),
Hu
sain
ian
dA
bd
in
(20
08
),P
ark
hi
etal
.(2
00
9)
SE
22
,SE
39
bN
.ta
ba
cum
Sti
gm
asE
nd
o-b
-1,3
-glu
can
ase
acti
vit
yG
ren
ier
etal
.(1
99
9)
CB
P2
N.
tab
acu
mC
allu
sB
ind
tocy
tok
inin
Ko
bay
ash
iet
al.
(20
00
)
Plant Cell Rep (2010) 29:419–436 427
123
Ta
ble
2co
nti
nu
ed
Nam
eS
ou
rces
Reg
ula
tio
n/i
nd
uct
ion
(by
/in
)F
un
ctio
n/b
iolo
gic
alro
leR
efer
ence
NtT
LP
1N
.ta
ba
cum
CM
V-i
nfe
cted
leav
esIn
viv
ob
ind
toC
MV
-1a
pro
tein
Kim
etal
.(2
00
5)
TL
P-D
34
Ory
zasa
tiva
Fu
ng
us
ind
uce
dT
ran
sgen
ican
tifu
ng
alp
rote
ctio
nD
atta
etal
.(1
99
9),
Vel
azh
ahan
and
Mu
thu
kri
shn
an(2
00
3),
Fu
etal
.(2
00
5),
Kal
pan
aet
al.
(20
06
),M
aru
thas
alam
etal
.(2
00
7)
CH
TL
Pru
nu
sa
viu
mF
ruit
sE
nd
o-b
-1,3
-glu
can
ase
acti
vit
y,
bu
tn
oan
tifu
ng
alac
tiv
ity
Gre
nie
ret
al.
(19
99
)
Pru
-TL
PP
.a
viu
mF
ruit
sE
nd
o-b
1,3
-glu
can
ase
acti
vit
y,
bu
tn
o
anti
fun
gal
acti
vit
y
Men
u-B
ou
aou
ich
eaet
al.
(20
03)
TL
PR
hiz
oct
on
iaso
lan
iM
yce
liu
mE
nd
o-b
-1,3
-glu
can
ase
acti
vit
y,
and
anti
fun
gal
acti
vit
y
Gre
nie
ret
al.
(20
00
)
TL
P(1
6,2
5k
D)
Sec
ale
cere
ale
Co
ld-i
nd
uce
din
leav
esA
nti
free
zeac
tiv
ity
Yu
and
Gri
ffith
(19
99
)
Sn
iOL
PS
ola
nu
mn
igru
mO
smo
tic
stre
ssan
do
ther
sIn
vit
roan
tifu
ng
alac
tiv
ity
,b
ut
no
end
o-b
-
1,3
-glu
can
ase
acti
vit
y
Jam
iet
al.
(20
07)
Sn
OL
PS
.n
igru
m–
Inv
itro
anti
fun
gal
acti
vit
yC
amp
os
etal
.(2
00
8)
OL
PS
.d
ulc
am
ara
Ste
ms
Cry
op
rote
ctio
nN
ewto
nan
dD
um
an(2
00
0)
CB
P2
S.
tub
ero
sum
xd
emis
sum
Cu
ltu
red
cell
sB
ind
toac
tin
Tak
emo
toet
al.
(19
97
)
Th
aum
atin
IIT
ha
um
ato
cocc
us
da
nie
llii
Fru
its
Tra
nsg
enic
anti
fun
gal
pro
tect
ion
Mar
iaet
al.
(20
02
),S
ches
tib
rato
van
d
Do
lgo
v(2
00
5)
Th
aum
atin
T.
da
nie
llii
Fru
its
(a)
Bin
dto
GP
CR
s;L
iet
al.
(20
02
)
(b)
Tra
nsg
enic
anti
fun
gal
,sa
lin
ity
and
dro
ug
ht
pro
tect
ion
Raj
amet
al.
(20
07)
WA
S-3
aT
riti
cum
aes
tivu
mC
old
,A
BA
and
elic
ito
rsIn
vit
roan
tifu
ng
alac
tiv
ity
Ku
wab
ara
etal
.(2
00
2)
TL
X1
T.
aes
tivu
mG
rain
sX
yla
nas
ein
hib
ito
rF
iere
ns
etal
.(2
00
7)
VV
TL
-1V
itis
vin
ifer
aE
mb
ryo
gen
iccu
ltu
res
Inv
itro
anti
fun
gal
acti
vit
yJa
yas
ank
aret
al.
(20
03
)
TL
P,O
LP
V.
vin
ifer
aF
un
gi-
infe
cted
leaf
and
ber
ryIn
vit
roan
tifu
ng
alac
tiv
ity
Mo
nte
iro
etal
.(2
00
3)
Zea
mat
inZ
eam
ays
Gra
ins
a-am
yla
se/t
ryp
sin
inh
ibit
or?
Ric
har
dso
net
al.
(19
87
),G
om
ez-L
eyv
aa
and
Bla
nco
-Lab
raa
(20
01
)
428 Plant Cell Rep (2010) 29:419–436
123
able to inhibit porcine pancreatic trypsin and digestive a-
amylases of insect (Tribolium castaneum) and bacteria
(Bacillus sp.) (Richardson et al. 1987; Schimoler-
O’Rourke et al. 2001). However, zeamatin did not inhibit
fungal a-amylase and fungi do not contain trypsin, sug-
gesting that zeamatin’s antifungal activity is not the result
of inhibition of these enzymes (Schimoler-O’Rourke et al.
2001). Gomez-Leyvaa and Blanco-Labraa (2001) found
that purified zeamatin actually contains five different
fractions: four of them correspond to different 22 kD
isoforms showing antifungal activity but no inhibitory
activity on a-amylase/trypsin; the other small band cor-
responds to a contaminant 14 kD protein that functions as
the a-amylase/trypsin inhibitor. These authors thus sug-
gest that the protease and a-amylase inhibitory activity
should not be assigned to the PR5 proteins (Gomez-
Leyvaa and Blanco-Labraa 2001). On the other hand,
Tsukuda et al. (2006) demonstrated that the rough lemon
(Citrus jambhiri Lush.) miraculin-like protein
(RlemMLP2) contains a thaumatin motif and shows both
antifungal activity against Alternaria citri and trypsin
inhibitor activity. A full understanding of the antifungal
mode of action of TLPs is clearly incomplete and still
under debate (Franco et al. 2002).
TLPs mediate signaling by interaction with their
receptors or ligands
Most TLPs have specific wide-spectrum antifungal
activities, suggesting that target recognition may be
determined by their interaction with pathogen cell surface
components. Ibeas et al. (2000) found that tobacco
osmotin binds to phosphomannan, a cell wall glycoprotein
of yeast (S. cerevisiae). The binding of osmatin with
phosphomannan is required for the maximal TLP toxicity
to walled cells but not spheroplasts, suggesting that
phosphomannan promotes osmotin uptake across the cell
wall. Phosphomannans are conjugated to several cell wall
proteins and several cell wall mannoproteins could bind to
immobilized osmotin in vitro (Ibeas et al. 2000). Salzman
et al. (2004) found that Ca2? facilitates binding of os-
motin to the fungal cell surface, but K? blocks this
interaction by competing for binding to mannosephos-
phate groups. Several other TLPs have been shown to
bind glucans in vitro (Trudel et al. 1998), suggesting that
carbohydrate binding is a common feature of TLPs that
determines target specificity.
The microbial cell wall contains resistance determinants
to TLPs such as osmotin. The fungal cell surface compo-
nents enhance or suppress TLP antifungal activity (Vero-
nese et al. 2003). Resistance of the model fungus S.
cerevisiae to osmotin is strongly dependent on the natural
polymorphism of its SSD1 gene. The yeast mutant (ssd1) is
sensitive to osmotin due to its deficiency in cell wall gly-
coproteins of the PIR (proteins with internal repeats)
family, alkali-insoluble glucans, and other unidentified cell
wall components (Yun et al. 1997; Ibeas et al. 2001).
Susceptibility of Aspergillus nidulans to osmotin is nega-
tively correlated with cell wall chitin content (Coca et al.
2000). The toxic activity of osmatin on A. nidulans and S.
cerevisiae involves in a heterotrimeric G protein mediated
signal pathway (Yun et al. 1998; Coca et al. 2000). Over-
expression of a heterologous cell wall glycoprotein in
Fusarium oxysporum leads to an increased virulence and
resistance to osmotin, which indicates that fungal cell wall
components can affect resistance to plant defense proteins
and pathogenic virulence (Narasimhan et al. 2003). These
resistance determinants probably form ‘‘barriers’’ that
prevent TLP transportation across the cell wall.
The antifungal activity of TLPs also involves in sig-
naling by specific plasma membrane components of the
fungal targets. For example, osmatin induces programmed
cell death in yeast (S. cerevisiae) by signaling suppression
of stress-responsive genes via the RAS2/cAMP pathway
(Narasimhan et al. 2001). A receptor-like polypeptide with
seven transmembrane domains and structural features of G
protein-coupled receptors (GPCRs) is the osmotin binding
plasma membrane protein in S. cerevisiae that regulates
lipid and phosphate metabolism and is required for full
sensitivity to osmotin (Narasimhan et al. 2005). Interest-
ingly, human GPCRs are the thaumatin sweet taste
receptors (Li et al. 2002). These results suggest that the
GPCR-binding activity may be a conserved characteristic
of TLPs (Veronese et al. 2003). Selectivity against target
microorganisms is probably due to a variety of defense and
susceptibility capabilities of the host and target micro-
organism, respectively (Veronese et al. 2003; Narasimhan
et al. 2003).
TLPs do not just interact with fungal cell wall compo-
nents, as evidence by a study that found a N. tabacum TLP
(NtTLP1) interacting with cucumber mosaic virus (CMV)
1a protein in vitro and in planta (Kim et al. 2005). This
specific in planta interaction with a virus replication-asso-
ciated protein suggests that NtTLP1 may affect CMV
multiplication as well the rate and extent of CMV
spreading (Kim et al. 2005). Also, genes encoding putative
receptor kinases with an exocellular TLP domain (PR5K)
have been identified in Arabidopsis (Wang et al. 1996; Shiu
and Bleecker 2001). A 22 kD potato OLP and a 26 kD
tobacco OLP were found with binding activities to actin
(Takemoto et al. 1997) and cytokinin (Kobayashi et al.
2000), respectively. Despite having a high structural sim-
ilarity, thaumatin, zeamatin, and Cassia didymobotrya TLP
(CaTLP) showed very different folding characteristics,
Plant Cell Rep (2010) 29:419–436 429
123
which was considered to affect their roles significantly
(Perri et al. 2008). Such findings indicate that the domains
of plant PR5 proteins can recognize various self and
environmental signal molecules and may be involved in
host defence as well as other mechanisms.
Role of TLP genes in response to abiotic stresses (cold,
drought, and salinity)
Freeze-tolerant organisms survive subzero temperatures
by the accumulation of anti-freeze proteins (AFPs) that
have the ability to adsorb onto the surface of ice crystals
and retard ice crystal growth (Griffith and Yaish 2004). In
addition to their roles in pathogen resistance, plant TLPs
accumulate in the apoplasts to enable plant adaptation to
cold climates. Two apoplastic TLPs (16- and 25-kD) were
revealed to function as AFPs in cold acclimated winter
rye (Yu and Griffith 1999). Winter rye AFPs, including
TLPs and other PR proteins (glucanases and chitinases),
do not act as cryoprotectants; rather, they bind to ice
crystal directly via hydrophobic interaction and modify
the freezing process by inhibiting intercellular ice growth
and recrystallization in planta (Griffith et al. 2005). The
PR proteins that accumulate in winter rye leave apoplasts
in response to abscisic acid (Yu and Griffith 2001) or
salicylic acid (Yu et al. 2001) do not have antifreeze
activity. Instead, AFPs accumulate in winter rye leaves in
response to cold, drought or ethylene (Yu et al. 2001),
indicating that PR proteins with and without antifreeze
activity are regulated by different signal transduction
pathways. It is hypothesized that genes encoding PR
proteins arose from gene duplication followed by muta-
tion, resulting in their different forms and functions (Yeh
et al. 2000).
TLPs from both plants and animals also show a cryo-
protection role. Such a plant OLP was purified from
Solanum dulcamara stems, suggesting a functional role in
freezing tolerance (Newton and Duman 2000). Recently, a
beetle (Dendroides Canadensis) TLP (TLP-Dcan1) was
found to significantly enhance the thermal hysteresis of
AFPs by interaction with these proteins, even though TLP-
Dcan1 itself lacks AFP activity (Wang and Duman 2006).
The mechanism for TLPs’ role as cryoprotectant appears
complex. It was found that potato OLP binds to actin
directly (Takemoto et al. 1997). Osmotin overexpression
shows multiple effects on programmed cell death, cyto-
skeleton organization and Ca2? signaling, which contribute
to cold protection of the transgenic olive tree (D’Angeli
and Altamura 2007).
In addition to enhancing cold tolerance, overexpression
of the tobacco osmotin gene also improves plant tolerance
to salinity and drought stresses in transgenic tobacco
(Barthakur et al. 2001), salt tolerance in transgenic
strawberry (Husaini and Abdin 2008) and drought toler-
ance in transgenic cotton (Parkhi et al. 2009).
How have the TLPs adapted diverse biological
functions?
TLPs appear to have different functions in host–pathogen
interaction, stress tolerance, and cell signaling. A major
unanswered question regarding the molecular mechanism
underlying TLP biological functions is how the conserved
TLPs have adapted functional diversification. Resolution of
this question depends on understanding the interaction of
distinct TLPs with a variety of their binding ligands/target
proteins, which include actin (Takemoto et al. 1997), b-
1,3-glucan (Trudel et al. 1998), ice crystal (Yu and Griffith
1999), cytokinin (Kobayashi et al. 2000), fungal cell wall
mannoproteins (Ibeas et al. 2000), human or fungal GPCRs
(Li et al. 2002; Narasimhan et al. 2005), viral proteins
(Kim et al. 2005), AFPs (Wang and Duman 2006), xy-
lanase (Fierens et al. 2007), and human Ig-E (Ghosh and
Chakrabarti 2008). Because these interactions are usually
family member specific, even isoform specific, we
hypothesize that substitution, insertion or deletion of some
key amino acid residues during evolution have resulted in
the family diversification for potential multiple functions.
Ancestral gene duplication followed by short sequence
insertions or deletions, domain juxtapositions, and/or gene
recombination have probably contributed to the origin of
novel TLP genes for adaptively important new functions
(Wang et al. 1996; Rep et al. 2002; Brandazza et al. 2004;
Liu et al. 2010). Natural selection of genes with new
functions under environmental pressure has very likely
played an important role in the evolution of the genetic
diversity of this complex gene family.
Advance of genomics study through the availability of
many genome and EST databases (Table 1) has allowed
classification of TLPs based on their origins, structures, and
expression profiles, while crystallographic studies have
provided powerful insights into their structural biology.
TLP molecules differ from each other in the distribution of
charges on their surface. The electronegative character of
the interdomain cleft in TLP 3D structure makes an
important contribution towards their b-1,3-glucan binding
and b-1,3-glucanase activities. For example, located in the
central cleft region of the tobacco NP24-I protein are
Glu84, Asp97, Asp102, which constitute a catalytic Glu–Asp
pair required for the b-1,3-glucanase activity, while a
longer distance ([5 A´
) between this catalytic Glu–Asp pair
may account for a lower level of its activity as compared to
authentic b-1,3-glucanase for the hydrolytic cleavage of
glycan polymers (Ghosh and Chakrabarti 2008). 3D mod-
eling showed a similar catalytic Glu–Asp pair in the acidic
430 Plant Cell Rep (2010) 29:419–436
123
cleft of other TLPs with b-1,3-glucanase activity (Menu-
Bouaouichea et al. 2003). On the other hand, thaumatin has
a basic cleft structure. Chemical modification found that
five lysine residues (Lys78, Lys97, Lys106, Lys137 and
Lys187) of thaumatin I contribute markedly to its sweetness
(Kaneko and Kitabatake 2001). These five lysine residues,
specific for thaumatin and not existent in non-sweet TLPs,
are separate and spread over a broad surface region around
the cleft, which implies that the positive charges and
positions of these lysine residues play an important role in
a multipoint interaction of thaumatin I with human GCPRs.
Differences in the topology and surface electrostatic
potential around the cleft are considered to determine the
specificity of TLPs to their target proteins and ligands (Min
et al. 2004). Recently, a site mutagenesis revealed that the
mutant TLX1 protein (His22 to Ala22) was unable to form a
complex with xylanases, resulting in a protein lacking
xylanase-inhibition capacity (Rombouts et al. 2009). This
His22 is situated on a loop of the protein surface which
distinguishes TLX1 from other, non-inhibiting TLPs, sug-
gesting that the positive charge of this basic amino acid
residue (His22) is crucial for TLX1-xylanase interaction.
It is common that different members may have been co-
opted to additional functions during evolution of a multiple
PR gene family. In the PR3 family of winter rye chitinases,
it has been found that two cold-induced members possess
antifreeze activity while other pathogen-induced members
have been shown only enzymatic activity (Yeh et al. 2000).
A similar case has been observed in the PR14 family of
plant lipid transfer proteins (LTPs). Despite a high identity
between two LTP amino acid sequences, an algorithm
revealed a clear ice-binding face on one (LTP1), but not on
the other (LTP2), and experiment confirmed this predica-
tion as LTP1 had clear ice-binding activity and LTP2 did
not (Doxey et al. 2006). Site-directed mutations in the AFP
ice-binding face caused the loss of up to 90% of their
antifreeze activity, but did not disrupt the fold of the ice-
binding face (Middleton et al. 2009). Such findings suggest
that amino acid substitutions followed by natural selection
may have resulted in adaptive functional alterations for
some plant PR proteins.
The TLP family thus presents a functional genomics
conundrum to answer how it is that a complex protein
family can diversify to fulfill multiple functions while
conserving their structure of the TLP domain. The
challenge remains to identify and explain fully what the
functions of these proteins are, why they are so numer-
ous and subject to such complex expression profiles
regulated by environmental factors and developmental
stages, and how they interact with their specific ligands?
Continued analysis of this protein superfamily will no
doubt reveal many other examples of their functional
diversification.
Potential application
Application of thaumatin as sweetener
and flavor enhancer
Thaumatin has wide applied usage in food and pharma-
ceutical industries because it is 1,600 times sweeter than
sucrose on a weight to weight basis. Since 1983, thaumatin
has been approved and commercialized as a safe sweetener
and flavor enhancer in food (Zemanek and Wasserman
1995; Faus 2000). For taste modification of vegetables, the
thaumatin gene was transformed into potato (Witty and
Harvey 1990) and tomato (Bartoszewski et al. 2003).
Transgenic cucumber (Cucumis sativus L.) fruits with
thaumatin II accumulation exhibited a sweet phenotype and
showed a positive correlation between thaumatin accumu-
lation levels and intensity of sweetness (Maria et al. 2002).
A recent study showed that transgenic expression of the
thaumatin II gene resulted not only in a sweeter taste of
cucumber fruits in comparison with the control, but also
higher aroma acceptability (Zawirska-Wojtasiak et al.
2009). Thus, it appears that transgenic expression of
thaumatin could be useful for modifying the taste of a
variety of foods and plants bearing edible fruits (Masudaa
and Kitabatake 2006).
Application of TLP genes in engineered
plant disease resistance
In the past decade several transgenic plants have been
developed to express a variety of TLP genes for enhanced
resistance against pathogens. Transgenic plants constitu-
tively overexpressing TLPs often show an enhanced fungal
resistance (Datta et al. 1999; Fagoaga et al. 2001; Kalpana
et al. 2006). For example, transgenic strawberry plants
expressing the thaumatin II gene showed a significantly
higher level of resistance to gray mold (Botrytis cinerea)
(Schestibratov and Dolgov 2005). Overexpression of barley
TLP-1 in transgenic wheat lines resulted in significant
reductions in Fusarium head blight severity in greenhouse
evaluations (Mackintosh et al. 2007).Transgenic tobacco
plants with the thaumatin gene exhibited delayed disease
symptoms caused by Pythium aphanidermatum and Rhi-
zoctonia solani, higher germination percentage, and seed-
ling survival under salinity and PEG-mediated drought
stress (Rajam et al. 2007). The rice TLP (D34) showed a
similar effect in transgenic tobacco: at high D34 expression
levels, transgenic tobacco plants showed enhanced toler-
ance to necrotization caused by the pathogen Alternaria
alternata (Velazhahan and Muthukrishnan 2003). A field
test of creeping bentgrass lines with rice D34 demonstrated
that the transgenic plants displayed an improved resistance
to the causal agent of dollar spot (Sclerotinia
Plant Cell Rep (2010) 29:419–436 431
123
homoeocarpa), but no improvement against brown patch
(Rhizoctonia solani) under greenhouse conditions (Fu et al.
2005). In a field test with inoculation of S. homoeocarpa,
some transgenic lines of creeping bentgrass with AtPR5K
overexpression also showed delays in disease development
of 29–45 days as relative to the control plants (Guo et al.
2003).
Plants transformed with TLPs do not always show
enhanced pathogen resistance. Transgenic cucumbers with
an accumulation of the thaumatin II protein and a sweeter
phenotype did not exhibit tolerance to the pathogenic
fungus Pseudoperonospora cubensis (Maria et al. 2002).
Coexpression of TLP and chitinase in the progenies of a
transgenic rice line revealed an enhanced resistance to the
sheath blight pathogen (Rhizoctonia solani) as compared
to transgenic lines expressing the individual genes
(Maruthasalam et al. 2007), suggesting disease resistance
may be the effect of multiple defence-responsive proteins
(Lorito et al. 1996). However, transgenic wheat plants
stably expressing genes for TLP, chitinase, and glucanase
did not show resistance to Fusarium-caused scab under
field conditions (Anand et al. 2003). AtTLP1 knockout
mutant plants showed normal levels of rhizobacteria-
induced systemic resistance (ISR) against the bacterial leaf
pathogen, suggesting that expression of AtTLP1 in the
roots is not required for systemic expression of ISR in the
leaves (Leon-Kloosterziel et al. 2005). These results indi-
cate the application of TLP genes to engineering disease
resistance in plants needs to consider many factors, such as
the specialization of TLPs on pathogen range, the syner-
gistic interaction of TLPs with other defence-related pro-
teins, and the complexity of ecosystems.
Application of TLPs as mammalian therapeutic agents
The antifungal capability of various TLPs suggests the
existence of a signaling or recognition role mediated by
their specific interaction with targets at the fungal plasma
membrane (Veronese et al. 2003). Osmatin induces apop-
tosis in the yeast S. cerevisiae and activates AMP kinase in
C2C12 myocytes via a receptor that has homology to
mammalian receptors for the hormone adiponectin (Nara-
simhan et al. 2005). Similar to related TLPs, osmatin has a
structurally homologous lectin-like b-barrel domain (Min
et al. 2004). This b-barrel domain in the TLP structure,
overlapped with that of adiponectin, could be used as a
surrogate for adiponectin in the development of new ther-
apeutic agents for the treatment of a wide variety of
mammalian disorders involved in the adiponectin receptor-
mediated pathway. Such disorders include diabetes, arte-
riosclerosis, heart disease, and others (Narasimhan et al.
2005).
Conclusion
Additional research and data generated in the past decade
have led to a better understanding of the TLP family in
variety of organisms, especially the biological functions
of TLPs relative to disease resistance and adaptation to
stressful environments. This emerging knowledge indi-
cates great potential for the application of TLP genes in
gene engineering technologies for crop improvement and
for use in medicine. Unfortunately, information on fun-
damental aspects of the TLP family remains quite lim-
ited, particularly on the precise mechanisms of protein
regulation and function. This means that challenging
fundamental and applied studies need to be conducted to
characterize TLP receptors and their interacting compo-
nents in the many life processes affected by this complex
protein superfamily.
Acknowledgments This work is supported in part by the Canadian
Forest Service and the CFS-Genome R&D Initiative.
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