Drechslera teres–infected barley (Hordeum vulgare L.) leaves accumulate eight isoforms of thaumatin-like proteins

Physiological and Molecular Plant Pathology (2001) 58, 183±188 doi:10.1006/pmpp.2001.0325, available online at http://www.idealibrary.com on

Drechslera teres±infected barley (Hordeum vulgare L.) leaves accumulate eight isoforms of thaumatin-like proteins E R N S T R E I S S 1* and C H R I S T I A N HO R S T M A N N 2 1

Institute for Resistance Research and Pathogen Diagnostics, Federal Centre for Breeding Research on Cultivated Plants, Theodor-Roemer-Weg, D-06449 Aschersleben, Germany and 2Institute for Plant Genetics and Cultivated Plant Research, D-06466 Gatersleben, Germany (Accepted for publication March 2001 and published electronically 16 May 2001) Eight thaumatin-like proteins of barley (Hordeum vulgare L.) (TLP1±TLP8) were detected in Drechslera teres infected leaves by two-dimensional electrophoresis followed by N-terminal microsequencing: four of them are acidic; four are basic proteins. Partial amino acid sequence data were used to generate polymerase chain reaction (PCR) clones employed for the isolation of four novel, nearly full-length cDNA sequences encoding TLP4, TLP6, TLP 7, and TLP 8. The cDNAs are characterized by sequence sites of very high GC content and they encode proteins with 171±233 amino acid residues. The N-termini of the deduced proteins were preceded by putative signal peptides of 22±25 amino acid residues. With a Clustal W based c 2001 Academic Press dendrogram similar sequences were assigned to those we obtained in this work. * Keywords: thaumatin-like protein; PR-5 protein; Hordeum vulgare; Drechslera teres; N-terminal sequencing; cDNA sequence analysis.

INTRODUCTION Induction of pathogenesis-related (PR) proteins has been associated with plant response to either biotic or abiotic stress factors. Their synthesis is considered part of an active, general defense response in the plant system. PR proteins have been grouped into families by amino acid homology, serological relationship, and biochemical properties [19, 20]. The PR-5 family comprises proteins displaying a certain degree of homology over their amino acid sequences to the sweet tasting thaumatins from Thaumatococcus daniellii [3] and includes also proteins termed osmotins [14, 15] and permatins [21]. They accumulate in plants in response to stress conditions, e.g. high salt concentrations, wounding or pathogen attack. In vitro bioassays have shown their antifungal activity [5, 21]. Also, as constitutive proteins in seeds they are thought to play a signi®cant role in protecting seeds against fungal attack during storage or germination [2, 5]. There are a few reports about PR-5 proteins of barley. The acidic protein Hv-1 was sequenced at the N-terminus and revealed a strong similarity to thaumatin-like (TL) proteins [1]. From a barley cDNA library three clones (termed pcPRHv-1a, pcPRHv-1b, and pcPRHv-1c) were isolated and sequenced [4]. They displayed a high * To whom correspondence should be addressed.

0885-5765/01/040183+06 $35.00/00

degree of homology to each other, and to Hv-1 at the protein level. In addition, in barley grains the antifungal proteins R and S were found [5]. Their N-terminal amino acid sequences also assign them to the TL proteins. Finally, from intercellular ¯uid washings of chemically stressed barley leaves three PR-5 type proteins (IFW 15, IFW 16, IFW 19) were isolated based on their capacity to bind to water-insoluble b-1,3-glucans [18]. The Nterminal sequences proved IFW 15 to be identical with PRHv-1, an IFW 19 to be almost identical to the barley seed protein R, and IFW 16 to be a 16 kDa protein with homology to TL proteins. Apart from the cDNAs encoding the acidic PRHv-1a to c, only three nucleic acid sequence entries were found in the data bank coding for TL proteins of barley: a cDNA from a library prepared of barley infected with Blumeria graminis and coding for a basic PR-5 protein (AJ001268), and two partial cDNA sequences coding for barperm1 (AF016327) and barperm2 (AF016328) which were cloned from mRNAs pooled from barley grains at various stages of development. Here, we present the results of a search in a cDNA library generated using RNA from barley primary leaves infected with Drechslera teres. This search was based on information gained by N-terminal microsequencing after 2D-electrophoretic analyses of proteins extracted from the infected leaves. c 2001 Academic Press *

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E. Reiss and C. Horstmann MATERIALS AND METHODS

Culture and infection of barley plants with D. teres f. teres and the extraction of leaf proteins as well as the twodimensional electrophoresis were described previously [13]. For SDS±PAGE we used 15 % (w/v) of total monomer (acrylamide and bisacrylamide) in solution. Proteins were transferred onto a PVDF membrane using the ``semidry technique'' by means of CAPS (3cyclohexylamino-propane-sulfonic acid) bu€er, pH 9.9 (NaOH). The blotting procedure was carried out for 1.5 h with a constant current of 0.8 mA cm ÿ2. The PVDF membrane-bound proteins were visualized by staining with Coomassie brilliant blue R-250. After a thorough wash with aqua dest., the membrane was airdried and the stained spots of interest were excised. The N-terminal amino acid sequences were determined by automated Edman degradation using a gas phase micro sequencer (Beckman LF 3400) equipped with an on-line Gold system microgradient HPLC. For 30 RACE, total RNA from frozen ®rst leaf material of the infected cv. Karat was puri®ed with a solution containing phenol and guanidine isothiocyanate (Trizol Reagent, Gibco BRL), and poly(A‡) RNA was isolated from total RNA using oligo-dT cellulose according to the manufacturer's instructions. Reverse transcription using SuperScript Reverse Transcriptase from the 30 RACE system (Gibco BRL) was performed at 508C for 50 min following the instructions provided by the manufacturer to avoid secondary RNA structures. Degenerate oligonucleotides, derived from the amino acid sequence of the TLP proteins, were used for the ampli®cation of a partial cDNA fragment ( ®rst primer AGGAGCTTCWSSATCACSAACCG and nested primer CGSTGCAGSTTCACSGTSTGGCCG for TLP4; ®rst primer ACIGTCGTCAACIGITGCTCITACAC and nested primer TACACIGTITGGCCIGGIGCITTICCNGG for TLP6/7; and GCCACCTTCACIGTIATHAATAAGTGYCA and nested primer TGYCARTAYACIGTITGGGCIGCNGCNGT for TLP8). PCR was carried out using programs consisting of 10 cycles of sequential steps at 948C for 30 s, 50±558C for 45 s and at 728C for 45 s, and of 30 cycles of sequential steps at 948C for 30 s, 46±508C for 45 s and at 728C for 45 s. The exact annealing temperatures depended on the expected melting temperatures of the primers. One ml of the ®rst PCR reaction sample was used as a template for the subsequent nested PCR ampli®cation. Other details agreed essentially with the protocols for the RACE system. The reaction products were electrophoresed into a 1.2 % agarose gel and the band of interest puri®ed and cloned into pNoTA/T7 (Peqlab, Biotechnologie GmbH). A cDNA library in the ZipLox phage vector was constructed from mRNA of barley leaves (cv. Karat) sampled 5 days after inoculation with D. teres f. teres. We

used the SuperScript Lambda System for cDNA synthesis and l cloning (Gibco BRL). For packaging of ligated cDNA we used the Gigapack III Gold extracts (Stratagene). After screening by hybridization, the cDNA was recovered by in vivo excision according to the protocol of Gibco BRL. Sequencing was performed using the Thermo Sequenase Fluorescent Labelled Cycle Kit (Amersham). Deduced amino acid sequences were aligned with Clustal W followed by re®nement by eye. Phylogenies were estimated with the major phylogenetic procedures maximum-parsimony and neighbor joining methods. The con®dence of tree topology was estimated with the bootstrap method. In each case, 1000 resamplings of the data were carried out. Thaumatin was added as outgroup.

RESULTS AND DISCUSSION It was shown previously, that a broad range of infectionrelated proteins is induced in leaves of susceptible and resistant barley cultivars in response to infection with various pathogens or after treatment with a toxic fraction from the infecting fungus D. teres [13]. D. teres infection resulted in very similar 2D protein patterns in resistant and susceptible cultivars and Fig. 1 gives a representative example of the proteins extracted from the primary leaves of the susceptible cultivar Karat. After blotting of the two-dimensional PAGE gel we con®rmed now among the analysed, infection-related spots the accumulation of eight TL proteins (TLP1±TLP8) by N-terminal microsequencing (Table 1). The spots are arranged in a broad pH range, from acidic (TLP1±TLP3) via weakly acidic (TLP4) to basic (TLP5±TLP8). The apparent molecular masses range roughly from 15 to 24 kDa. The proteins were classi®ed as thaumatin-like based on their strong similarities to known TL proteins as revealed in a data bank search. For the time being, they were assigned TLP1±TLP8 in order of increasing isoelectric points ( pI). These TLPs belong to the family 5 of the PR proteins (PR-5) [19, 20]. As TLP4 is detectable also in the healthy barley leaves (data not shown), it should be classi®ed rather as a PR-5-like protein. The proteins TLP1, 2 and 3 show N-terminal sequences identical to that of the acidic Hv-1 protein [1] as well as to those of the proteins deduced from the three cDNA clones, which were found with a heterologous probe in a cDNA library produced from barley inoculated with Rhynchosporium secalis [4]. The TLP4 sequence is identical to that of IFW 16 isolated from stressed barley leaves [18]. The N-terminal region of TLP5 closely resembles that of avematin, a TL protein isolated as an antifungal protein from oat seeds (Avena sativa) [21]. N-terminal sequencing did not reveal any di€erence between TLP6 and TLP7. The sequenced

Isoforms of thaumatin-like protein

185

pH 3

pH 10

66–

45–

36– 29–

24–

5 6

20.1–

7

8

3

1,2

4 14.2–

kDa F I G . 1. Two-dimensional resolution of the proteins isolated from the susceptible cv. Karat inoculated with D. teres f teres. First dimension: IEF ( pH 3±10). Second dimension: SDS±PAGE. Spots which were proved to represent thaumatin-like proteins were designated with 1±8 beginning in the acidic part of the gel.

T A B L E 1. N-terminal sequences of eight PR-5 proteins of barley compared with similar sequences (italic) from the data base 1 TLP1 TLP2 TLP3 PRHv-1 (Hordeum vulgare) TLP4 Hv-IFW 16 (H. vulgare) TLP5 Avematin (Avena sativa) TLP6, 7 Protein R (H. vulgare) TLP8 Protein S (H. vulgare)

A A A A R R T T A A A A

5 T T T T S S T T T T T T

F F F F F F I I I I F F

N N N N S S T T T T T T

I I I I I I V V V V V V

10 K K K K T T V V V V I I

N N N N N N N N N N N N

N N N N R R R K R R K K

C* C* C* C C* C C* C C* C C* C

G G G G S S S S S S Q Q

15 S S S S F F Y Y Y Y Y Y

T T T T T T T T T T T T

I I I I V V I V V V V V

W W W W W W W W W W W W

P P P P P P P P P P A A

20

A

G

T

P

V

A X G G G

A G A A A

T L L L L

P X P P P

V G G G G

A

A

V

P

A

*Designates a presumed cysteine residue. Completely conserved amino acids are in bold face type.

parts of the molecules were identical to protein R, one of two TL proteins found in barley seed [5]. Similarly, the N-terminus of TLP8 has the same sequence as protein S, the second TL protein isolated from barley seeds. However, thus far the IFW 16 protein as well as proteins R and S have only been sequenced N-terminally. For the TL proteins TLP4±8, the information obtained about the partial amino acid sequences was used to elucidate the corresponding mRNA/cDNA sequences. A 30 RACE procedure (rapid ampli®cation of cDNA ends) resulted in PCR-fragments for TLP4, 6, 7 and 8. In contrast, no fragment corresponding to the cDNA of TLP5 could be ampli®ed. A cDNA library, constructed using mRNA from infected barley leaves, was probed with the cloned PCR-fragments to isolate the respective cDNAs. Subsequently, the largest clones were completely sequenced in both directions to obtain the nearly full length cDNA sequences. The GenBank accession numbers for the four nucleotide sequences are:

AF355455 (TLP4), AF355456 (TLP6), AF355457 (TLP7), and AF355458 (TLP8). The discovered nucleotide sequences show a high degree of identity to the cDNAs encoding thaumatin and known TL proteins (data not shown). All clones comprise open reading frames de®ned by translation start and stop codons. The protein coding regions of the cDNAs reveal a high (G ‡ C) content, which is partly attributable to a marked preference for the use of G or C in the degenerate third base of codons. This is comparable to the b-1,3-glucanases [6] and PR-1 proteins [8] of barley. Therefore, in reverse transcription and DNA ampli®cation it was very important to apply conditions that restrict the formation of secondary structures in barley RNA/DNA encoding TL proteins. The nucleotide sequences allowed to de®ne primary structures of barley TL precursor proteins TLP4, 6, 7 and 8. Complete identity was found between part of the deduced amino acid sequences and the N-terminal amino

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F I G . 2. Alignment of the deduced sequences of ®ve PR-5 proteins of barley (H. vulgare) including the acidic protein PRHv-1a [4] for comparison. Arrow indicates the 50 ends of the mature proteins. Highly conserved amino acids are in boldface type. Character to show that a position in the alignment is perfectly conserved: (*); character to show that a position is well conserved: (.).

acids sequences of the blotted proteins. The deduced amino acid sequences of the proteins together with that of the acidic barley PRHv-1a [4] have been aligned (Fig. 2) according to the method of Myers and Miller of pairwise similarity computations and ®nal alignments [9].

Furthermore, a dendrogram was generated (Fig. 3) based on the alignment of the new amino acid sequences and other already known deduced amino acid sequences of PR-5 proteins including two sequences found in the data bank and that of PR-Hv1a representing the three

Isoforms of thaumatin-like protein

76 88 100 100

100 99

66 71 96 64

Barperm1 TLP7 BasPR5 TLP6 TLP8

100 100

TLP4 PR–Hv1a Thaumatin

F I G . 3. Phylogenetic relationships of amino acid sequences of PR-5 proteins investigated and other known deduced amino acid sequences of PR-5 proteins based on maximum parsimony (MP) and neighbor joining (NJ) analyses. Bootstrap values are placed above the branches (NJ) and below the branches (MP). Barperm1 (AF016327); BasPR-5, basic pathogenesis-related PR-5 protein (AJ001268); Thaumatin, preprothaumatin (J01209).

acidic PR-Hv1 proteins a, b and c [4]. All nodes in the topologies are well-supported by high bootstrap values. The dendrogram supports a close relationship among the proteins TLP6 and the basic pathogenesis-related HvPR5 protein (AJ001268), but also among TLP7 and barperm1 (AF016327). The proteins TLP4 and PRHv1a are only distantly related to the other proteins investigated. Both proteins have in common that they are considerably smaller compared to the other PR-5 proteins because of internal deletions which include seven of 16 conserved invariant cystein residues. These seven cystein residues are probably important for the secondary structure of the TL proteins [12] by the formation of eight disul®de bridges in the molecule to stabilize the external loops of this type of protein [11]. The N-termini of the deduced amino acid sequences are preceded by hydrophobic amino acid sequences of 22±25 residues, which are suggested to be signal peptides targeting the nascent polypeptides to the secretory system. The proteins lack speci®c information for retention in the endoplasmatic reticulum or for organellar targeting. For the deduced mature proteins several physicochemical characteristics were derived (Table 2). The

187

calculated molecular masses for TLP6, 7 and 8 seem to be a little too low. If PR-Hv1a, b and c [4] represent the three acidic proteins TLP1, 2 and 3, similar deviations exist for two of them. In all of these cases this may be due to the fact, that secreted proteins are often involved in various post-translational processes, which may result in changed molecular mass and changed electrophoretic mobility. Indications for this are the putative Nglycosylation sites found in TLP6 and 7. TLP8 lacks these sites, but the presence of O-glycosylation sites (data not shown) or other modi®cations like the glycosylation of hydroxylated prolins [10, 16] may contribute to the apparent molecular mass found by SDS±PAGE. A possible glycosylation was con®rmed by carbohydrate analyses of the barley seed proteins R and S [2]. These proteins correspond to TLP6/7 and to TLP8. The estimated carbohydrate contents were described as 2.8 and 5 %, respectively. In addition, on the 2D gel (Fig. 1) the spots for some of the TLPs are mirrored in spots representing proteins with the same pI but di€erent molecular mass. This phenomenon is known as typical for secreted proteins and may be another indication for posttranslational processes. Similar faster and slower forms were shown to exist for the two tobacco TL proteins PR5a ( protein R) [12, 17] and PR-5b ( protein S) [17]. The amino acid sequences estimated by N-terminal sequencing (Table 1) do not di€erentiate between TLP6 and TLP7. Nevertheless, using a single PCR fragment as a probe it was possible to isolate two cDNA clones that di€er in their sequence but represent the same N-terminal sequence for TLP6 and TLP7. The di€erent pI values calculated from the deduced amino acid sequences (Table 2) prompted the assignment to TLP6 and 7. This was based on the assumption that post-translational modi®cations do not change the pI ranking of these proteins. The TL proteins of barley (HvPR-5) are encoded by a small gene family. Future studies of the temporal and spatial expressions of the single members of the family by means of Northern analyses or RT-PCR using speci®c probes respectively primers from dissimilar regions of the cDNAs will contribute to a better understanding of the role of the PR-5 protein isoforms in defense reactions.

T A B L E 2. Data calculated from the sequences of the cloned novel cDNAs of barley PR-5 proteins using for MW, pI and Nglycosylation sites the algorithm of Expert Protein Analysis System (ExPASy) of the Swiss Institute of Bioinformatic [22]

TLP4 TLP6 TLP7 TLP8

Deduced peptide aa

Signal peptide aa

Deduced mature peptide aa

Cysteine residues

Molecular mass

pI

N-glycosylation

171 226 227 233

22 24 24 25

149 202 203 208

9 16 16 16

15 889 21 352 21 379 21 855

5.69 7.53 7.91 8.15

± 1 (N-164) 1 (N-165) ±

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E. Reiss and C. Horstmann

Overexpression of the PR-5 protein osmotin in potato resulted in increased disease resistance of the plants [7]. Likewise, the genetically engineered overexpression of di€erent HvPR-5 genes in barley or other plants may yield important information about the functions of these isoforms.

11.

12.

We wish to thank Dirk Mattern for producing the dendrogram. The skilled technical assistance of Uschi Apel is gratefully acknowledged.

13.

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14.

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