Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog

Gene, 171 (1996) 123-127 © 1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00

123

GENE 09629

Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog (Nucleotide sequence; 14-3-3 proteins; protein kinase regulators)

Sonja S. Klemsdal, Christopher K. Hayes*, Linda Hjeljord, Matteo Lorito*, Gary E. Harman* and Arne Tronsmo* Norwegian Crop Research Institute, Plant Protection, Department of Plant Pathology, Fellesbygget, N-1432,4s, Norway Received by J. Marmur: 17 April t995; Revised/Accepted: 23 October 1995; Received at publishers: 12 January 1996

SUMMARY

A full-length cDNA clone, Th1433, (GenBank accession No. U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum. The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue. The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the e isoform from sheep brain. Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family. At least two genes encoding 14-3-3-1ike proteins exist in T. harzianum. Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.

1. INTRODUCTION

The 14-3-3 family of proteins, originally identified by Moore and Perez (1967), have so far been studied in a wide range of eukaryotic organisms and tissues, including plants, insects, amphibians, yeast, and sheep, bovine and human brain tissue. An extraordinarily high sequence conservation is observed (Martens et al., 1992). A wide array of biological functions of the 14-3-3 protein family have been described (reviewed in Aitken et al., 1992; Morrison, 1994; Aitken, 1995). They inhibit phospholipid/Ca2÷-dependent protein kinase C (Toker Correspondence to: Dr. S.S. Klemsdal, Norwegian Crop Research Institute, Plant Protection, Department of Plant Pathology, Fellesbygget, N-1432~,s, Norway. Tel. (+47) 64949400; Fax (+47) 64949226; e-mail: [email protected] * Present addresses: (C.K.H.; G.E.H.) Department of Horticultural Sciences, Cornell University, Geneva, NY 14456, USA. Tel. (1-315) 787-2246; (M.L.) Istituto di Patologia Vegetale, Universit/l degli Studi di Napoli, Via Universitgt 100, 80055 Portici (NA), Italy. Tel. (39-81) 274752; (A.T.) Department of Biotechnological Sciences, Agricultural University of Norway, P.O. Box 5040, N-1432.~s, Norway. Tel. (+47) 64947738. PII S0378-1119(96)00031-5

et al., 1990), and activate tyrosine and tryptophan hydroxylases, which are enzymes involved in neurotransmitter synthesis (Ichimura et al., 1988). They associate with the products of proto-oncogenes and oncogenes and by an unknown mechanism stabilize or stimulate their kinase activity (Fantl et al., 1994). The yeast homolog seems to function in growth regulation (Van Heusden et al., 1992) and observations on fission yeast indicate the involvement of 14-3-3 proteins in cell cycle control (Ford et al., 1994). Here we describe the isolation and sequence analyses of a cDNA from Trichoderma harzianum (Th) encoding Abbreviations: aa, amino acid(s); Ab, antibody(ies); BMH1, 14-3-3 protein from Saccharomyces cerevisiae; BMHI, gene (DNA, RNA) encoding BMH1; bp, base pair(s), cDNA, DNA complementary to RNA; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; pAb, polyclonal Ab; SDS, sodium dodecyl sulfate; SSPE, 0.15 M NaC1/0.015 M Na.citrate/0.01 M NaH2PO4/1 mM EDTA pH 7.4; T., Trichoderma; Th, T. harzianum; Th1433, 14-3-3 protein from Th; Th1433, gene (DNA, RNA) encoding Th1433; UTR, untranslated region.

124 a protein h o m o l o g o u s to This is the first time this in a filamentous fungus. sponding gene following also described.

other k n o w n 14-3-3 proteins. c D N A has been characterized The expression of the correconidial spore germination is

EXPERIMENTAL AND DISCUSSION

(a) Cloning and sequence analysis While looking for chitinases from Th strain P1 (ATCC 74058), we screened a c D N A library (Hayes et al., 1994) using polyclonal antibodies (pAb) prepared against a purified chitobiosidase ( T r o n s m o and H a r m a n , 1992; H a r m a n et al., 1993). Seven putative positive clones were isolated, all of them giving weak signals in the reaction with the pAb. The longest clone (B9) was sequenced and the E M B L nucleic acid database was searched for homology. N o h o m o l o g y was found to any D N A sequence corresponding to chitinolytic enzymes. However, the c D N A was found to be similar to those encoding the 14-3-3 family of regulatory proteins. Using the A L I G N p r o g r a m from the P C / G E N E software package (Intelligenetics) 69.8% identity was found between this nt sequence and the sequence of BMH1, the nt sequence encoding the 143-3 protein from Saccharomyces cerevisiae (Van Heusden et al., 1992). The c D N A clone was therefore given the name Th1433. Fig. 1 shows the entire nt sequence of Th1433 and the deduced aa sequence. T h e complete sequence consisted of 1101 bp, including a 34-bp leader UTR, an O R F encoding a putative protein of 262 aa, and a 278-bp Y UTR, followed by a poly(A) tail. The Swissprot database was searched for h o m o l o g y to the deduced aa sequence. The greatest h o m o l o g y was found to B M H 1 , which is the 14-3-3 protein from yeast, and the e isoform of the m a m m a l i a n 14-3-3 protein ( R o s e b o o m et al., 1992) isolated from sheep brain. 71% of the aa of these two proteins were identical to those of the Th protein, and 6.1% and 7.5% respectively were similar. W h e n the aa sequence of Th1433 and five 14-33 proteins from various species were aligned (Fig. 1 ), four highly conserved regions were found, interspersed with more variable regions. The predicted isoelectric point of the 30-kDa protein encoded by Th1433 was 5.5 ( P C / G E N E from Intelligenetics). This is in agreement with other 14-3-3 proteins, which were originally identified as a group of 30-kDa acidic proteins with an isoelectric point of a r o u n d 5 ( M o o r e and Perez, 1967). As in other 14-3-3 proteins (Aitken et al., 1992) a similarity to the 'pseudosubstrate' domain of protein kinase C (aa 53-56) (Fig. 1) was seen. Based on these observations we

CAC C A / ~ G T

T C ~ T

C TAC CG TC~AATGGG T CAC GAAGATGC T GT T TAC C T M G H E D A[V Y L

60 9

G G C CAAG C T C GC C GAG CAG GC T GAGC GATAT GAGGAGATGG T T GAGAACAT GAAGAT C GT A K L A E Q A E R Y E E M V E N M T K I V 2 9 * * * - * * * - * * _ * * * _ .

120

CGCCTCCGAGGACCGCGACCTGACCGTCGAGGAGCGCAACCTTC A S E D R DIL T V E E R N * _ * * * * *

TCTCCGTCGCCTACAA L S V A Y K * * * * * *

180 49

GAACGTCATTGGTGCTCGCCGCGCCTCT TGGAGAATAGTCAC T TCTATCGAGCAGAAGGA N V I G A R R A S W R I V T S I E Q K E • , * , _ _ * * _ * * * . * , * * ,

240 69

GGAGTCTAAGGGCAACTC T TCCCAGGTCACCCTTATC~AGGAGTACCGCCAGAAGATCGA E S]K G N S S Q V T L I K E Y R Q K I

300 89

GAACGAGCTTGCC~GATCTGCGACGATATCCTTGAGGT N E L A K I C D D I L E C TC T G C C A A G T C T G G C G A G T C C A A G G T C T S A K S G JE S K V _ * * * *

V

L *

TCTCGACCAGCACC L D Q H • _

E

TGATCCC L I P * _ .

TT T A C C A C A A G A T A A A G G G T G A C T A C C A C C G F Y H K I K G D Y H * * * * _ * * *

TTACCTCGCTGAGTTCGCTATTGGCGACCGCCGCAAGGACTCTGCCGACAAGTCCCTCGA Y L A E F IA I G D R R K D S A D K S

R *

L

E

GGCT TACAAGGCTGCCACCGAGGT TGCCCAGACTGAGCTGCCTCCCACCCACCCCATCCG A Y K A A T E V A Q T E [L P P T H P I _ _ * * * *

R *

360 109 420 129 480 149 540 169

CCT TGGAC T TGCCCTCAAC T TCTCCGTCTTCTAC TACGAGATCCTCAACGCTCCCGACCA L G L A L N F S V F Y Y E I L N A P D Q I 8 9

600

GGC TTGCCACCTCGCCAAGCAGGCCTTTGACGATGCTATTGC A C H L A K Q A F D D A I A E

660

*

CGAGGAGAGCTAC~GGAC E E S Y K D S •

.

•

.

•

,

_

*

TGAGCTCGACACCC TCAG D T L S 2 0 9

L *

*

*

*

*

_

*

TCCAC TCTCATCATGCAGCTGC TCCGTGACAACCTC~ACCCT T L I M Q L L R D N L T L 2 2 9 *

.

•

•

.

.

.

,

.

.

.

.

CTGGACCTCCTCCGAGGCCGAGACTCCGGCCAGGCTGATGCCCCTGCTAAGGAGGAGACC W T S ] S E A E T P A R L M P L L R R

720

*

*

R

P

CCTGC TGAGGC TGCCGTCCCGCCGCCGAAGAGCCC~a~GGCTGAGTAAGTTCAGGGGAGCA L L R L P S R R R R A Q G # CGGTATAAATCTCTAGGCTTGGGTCGCTTTGTTATCGGATGGGCTGGTGTCTACGGTGTG G A T GG)~AGAC T T C G T T T T T GATCa~-~LGAGC T T C G G C A T C G T T T T A G G T ~ C _ ~ x C G C ~ C TAT T T TCG GG~GGAATCGAT T T GGTTCCCCC T T TAC G~CTGGCAGGGAGGATGAAGAA GAAGAGCATAT TGGAGG T TC T GC TTT TAGAC TC T TGAT GCG TAGCCG TC T GGAC~TAA ~u%AC T A C T ~ T T G C T T T T T

780 249 840 262 900 960 1020 1080 1101

Fig. 1. The nt sequence of the cDNA clone Th1433,encoding a protein homologous to the 14-3-3 family of regulatory proteins. In the deduced aa sequence, the stop codon is denoted by #. The region similar to the 'pseudosubstrate' domain of protein kinase C is in bold letters. The sequence is deposited in GenBank, Accession No. U24158. The Th1433 aa sequence was aligned with those deduced from cDNA clones encoding the yeast BMH1 (Van Heusden et al., 1992), sheep e isoform (Roseboom et al., 1992), OenotheraPHP-O protein (Hirsch et al., 1992), barley HV1433 protein (Brandt et al., 1992) and Arabidopsis RCI14B (Jarillo et al., 1994). Positions where all six aa are identical are marked by an asterisk and positions where five of six aa are identical are denoted by dashes (-). Four regions with more than 86% identity are boxed. The alignment was done with the ALIGN program of PC/GENE (Intelligenetics). The arrow indicates the Sz°9 position essential for Caz + binding properties.

conclude that the isolated c D N A represents a gene encoding a 14-3-3 like Th protein. Some plant 14-3-3 proteins have been shown to contain a region similar to an E F - h a n d Ca2+-binding motif (Lu et al., 1994). A Gly residue essential for this motif can be found in most plant 14-3-3 proteins (Fig. 1), but it is absent from yeast and m a m m a l i a n 14-3-3 proteins and from Th1433. A computer-generated d e n d r o g r a m comparing representatives of 14-3-3 proteins of fungal, plant and m a m m a lian origin is presented in Fig. 2. The ~ isoform sequence from sheep has been described to be evolutionarily closer to the plant and yeast proteins than to the other m a m m a lian isoforms (Aitken et al., 1992). This report identifies

125 Bo q Bo y

kb 9.4-

BEHP

Bo p Bo

'I

6.6-

Ara

L

l

Bar

4.4-

Den

Sh 8 Sac Tri

Fig. 2. Dendrogram for 14-3-3 proteins. The different aa sequences were aligned by using the simultaneous alignment and phylogeny CLUSTAL program of PC/GENE (Intelligenetics). The aa sequences are as follows: bovine (Bo rl) (Ichimura et al., 1988), Bo T and Bo I] (Isobe et al., 1991), Bo ~ (Isobe et al., 1992), Arabidopsis RCI14B (Ara) (Jarillo et al., 1994), barley HV1433 (Bar) (Brandt et al., 1992), Oenothera PHP-O (Oen) (Hirsch et al., 1992), sheep (Sh e) (Roseboom et al., 1992), yeast BMH1 (Sac) (Van Heusden et al., 1992) and Trichoderma Th1433 (Tri) (this work).

the 14-3-3 protein from the filamentous Th fungus as a member of the same homology group.

(b) Southern analysis The Th1433 cDNA does not contain any sites for the restriction enzymes BamHI, EcoRI, HindIII or PstI. One band giving a strong hybridization signal can be seen in all lanes of the genomic Southern blot analysis (Fig. 3). In addition one or two faint bands (difficult to see in the PstI lane) can be seen. Introns have been found in more than half the filamentous fungal genes studied. These introns are predominantly short, most being around 50bp in length (Clutterbuck, 1995). It is therefore unlikely that restriction sites for all the tested restriction enzymes should exist within the introns of the corresponding gene. This suggests that the Th genome contains at least two genes encoding 14-3-3 like proteins, the weak signals representing the other gene(s). This is also the case in plants and mammalian tissues, where multiple isoforms have been found (Aitken et al., 1992), but is in contrast to the situation in yeast where only one gene was found (Van Heusden et al., 1992). (c) Northern analysis To study the level of expression, mRNA was isolated from Th grown on a basal medium using glucose as the carbon source. In Northern analysis (Fig. 4) it can be seen that in the time interval studied, 1-4 days after inoculation of the cultures with conidial spores, the Th1433 cDNA is constitutively expressed at a high level.

2.32.0-

0.6Fig. 3. Southern analysis of genomic DNA. Total DNA (5 ~tg) from Th strain P1 was digested with BamHI (B), EcoRI (E), HindlII (H), or PstI (P) and separated on 0.8% agarose gels. The DNA was vacuum blotted onto Hybond-N+ (Amersham, Arlington Heights, IL, USA) membranes according to the manufacturer's directions. The complete 32p-labelled Th1433 clone was used as a probe. The final washing was in 0.1 x SSPE/0.1%(w/v) SDS at 65°C (high stringency).

Furthermore, the highest level of Th1433 expression was found 24h after inoculation. The spores germinate 12-14 h after inoculation. The time with the highest level of expression therefore corresponds to the active growth period 10-12h after germination. The barley 14-3-3 protein is induced during infection by the powdery mildew fungus (Erysiphe graminis) (Brandt et al., 1992). Trichoderma spp. have been used in biological control of plant pathogenic fungi (Papavizas, 1985; Cook and Baker, 1983; Tronsmo, 1986; Jin et al., 1990). Proteins involved in the defense response generally are induced by pathogens, but have no or only a low constitutive expression. Our result may suggest that unlike the pathogenesisrelated barley 14-3-3 protein, the Th protein could be involved in a process similar to that found in fission yeast (Ford et al., 1994), where 14-3-3-1ike proteins participate in cell cycle control and are essential for cell proliferation. More investigations have to be done to determine the biological function of these Th genes.

(d) Conclusions (1) A full length cDNA, Th1433, was isolated from the filamentous Th fungus.

126

24 48 72 96 h

Fig. 4, Northern analysis. Th strain P1 was inoculated as a spore suspension (ca. 107 spores/ml final concentration) into liquid SM basal medium (Tronsmo and Harman, 1992) supplemented with 15%(v/v) vegetable juice (Granini@, Deutsche Granini, 4800 Bielefeld 1, Germany) and 0.4% glucose, and incubated on a reciprocating shaker (150 rpm) at 22°C. The mycelium was harvested by filtration through a Whatman GF/C filter after 24, 48, 72 and 96 h, frozen in liquid nitrogen, and stored at - 80°C. Total RNA was extracted by the guanidinium thiocyanate method for tissue and purified by CsCI ultracentrifugation (Kingston, 1991). 10 lag total RNA was separated on a 1.5% formaldehyde-1% agarose gel, vacuum blotted to a Hybond N + membrane, and hybridized ON at 65'~C to the same probe as used in the Southern analysis. The final wash was 0.1 x SSPE/0A% SDS at 65'C (high stringency). Autoradiographic film was exposed for 12 h.

(2) We s e q u e n c e d this c D N A

and found an ORF

e n c o d i n g a p u t a t i v e p r o t e i n of 262 aa. (3) S e a r c h t h r o u g h d a t a b a s e s r e v e a l e d h o m o l o g y to the 14-3-3 p r o t e i n s , a f a m i l y of p u t a t i v e k i n a s e r e g u l a t o r s showing an extraordinary high sequence conservation. Best h o m o l o g y was f o u n d to the c o r r e s p o n d i n g p r o t e i n in yeast a n d sheep. (4) S o u t h e r n analysis i n d i c a t e t h a t 14-3-3 like p r o t e i n s are e n c o d e d by at least t w o Th genes. (5) T h e h i g h e s t level of m R N A

e x p r e s s i o n was f o u n d

the first d a y after i n o c u l a t i o n o f the c u l t u r e w i t h c o n i d ial spores.

ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d by the N o r w e g i a n R e s e a r c h C o u n c i l , in p a r t by B A R D

g r a n t U S - 1 7 2 3 - 8 9 , a n d by

N A T O g r a n t C R G 931173.

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