Diversity of cutinases from plant pathogenic fungi: cloning and characterization of a cutinase gene from Alternaria brassicicola

Physiologicaland MolecularPlant Pathology (1994) 44, 81-92

81

Diversity of cutinases from plant pathogenic fungi" cloning and characterization of a cutinase gene from Alternaria brassicicola C. YAo a n d W. KOLLER*

Department of Plant PathologT,ComeU University, New York State Agriadtural ExperimentStation, Geneva, New York 14456, U.S.A. (Acceptedfor publication December1993)

Alternaria brassicicolaproduced two cutinase isozymes in the presence ofcutin monomers as specific inducers. A cDNA library was constructed from poly(A)+RNA isolated from mycelium incubated with cutin monomers. Specific cDNA clones were selected from the library according to their abilities to hybridize with first strand cDNA prepared from induced cultures, but not from glucose-grown cultures. Cutinase-specific cDNA clones were identified by Southern analysis of plasmid DNA, employing a mixture of two heterologous cutinase cDNAs and one cutinase gene as probes. The largest 984 bp insert found among positive clones contained the entire cutinase coding region composed of 209 amino acids. The amino acid sequence predicted from the cDNA nucleotide sequence contained amino acids and sequences highly conserved among fungal cutinases. The sequences of four additional positive cDNAs were identical and thus gave no indication for the presence of a second gene of origin. Southern analysis of genomic DNA of A. brassMcola yielded a similar result. The structural gene of cutinase (CUTAB1) was contained within a 1545 bp genomic DNA fragment. Nucleotide sequences of the cDNA and the gene were identical, with the exception of one intron of 56 bp. The location of the intron was identical with introns identified in other fungal cutinase genes. The potential role of CUTAB1 in pathogenicity will be determined by gene disruption.

INTRODUCTION T h e p l a n t cuticle, a h y d r o x y f a t t y acid polyester i m p r e g n a t e d with waxes, is the first defensive b a r r i e r e n c o u n t e r e d by m a n y p l a n t p a t h o g e n s [10, 12]. Several lines of evidence suggest that cutinase, a serine esterase specifically i n d u c e d by c u t i n monorders, is crucially involved in b r e a c h i n g the h y d r o p h o b i c cuticle layer d u r i n g infection [6, 10, 12, 15, 27]. M o r e recently, the roles o f c u t i n a s e released by g e r m i n a t i n g spores o f f u n g a l pathogens have been b r o a d e n e d beyond a f u n c t i o n in cuticle p e n e t r a t i o n . Release of cutinase from c o n i d i a ofE~ysiphe graminis f.sp. hordei a n d uredospores of Uromyces viciaefabae prior to appressorium f o r m a t i o n was i n t e r p r e t e d as a n i n d i c a t i o n for a role in surface adhesion [4, 19]." A n essential p a r t i c i p a t i o n of cutinase in s a p r o p h y t i c g r o w t h The sequence data reported in this paper has been submitted to the GenBank with accession number U03393. *To whom correspondence should be addressed. 0885-5765]94/020081 + 12 $08.00/0 6

© 1994 Academic Press Limited MPP 44

82

C. Yao and W. KOIler

rather than in cuticle penetration was concluded from the disruption of the cutinase gene in Fusarium solani f.sp. pisi (aVectria haematoccocca) [22]. However, there is growing evidence for considerable cutinase gene diversity. For example, the disruption of one cutinase gene from Magnaporthe grisea had no effect on the pathogenicity, but the cutinase activity released by respective transformants was only reduced and not eliminated, indicating the presence of a second but different cutinase isozyme potentially involved in penetration and thus infection [24]. Although two pathotypes of Colletotrichum gloeosporioides isolated from citrus produced cutinases, the cutinase gene cloned from the same fungus isolated from papaya hybridized only with genomic DNA isolated from one but not the other pathotype [16]. Alternaria brassicicola, a directly penetrating pathogen of Brassica spp., has been identified as a pathogen with different cutinase isozymes [25, 26]. Two cutinases differing in size and optimal pH of cutin hydrolysis were recently purified and characterized [26]. Both isozymes were induced in the presence ofcutin monomers, but were repressed by glucose [26]. This specific induction of cutinase gene transcription was utilized for the screening of a eDNA library prepared from mRNA isolated from induced cultures. The present report describes the cloning and characterization of one cutinase gene from the pathogen as a crucial step toward the elucidation of cutinase isozyme functions.

MATERIALS AND METHODS

Fungal culture and materials Alternaria brassicicola (Schw.) Wilts. was provided by Dr P. Williams, University of Wisconsin, Madison, Wisconsin. Apple cutin was prepared as described elsewhere [14]. The plasmids of cutinase eDNA from Colletotrichum capsici and F. solani f.sp. pisi were obtained from Dr P. E. Kolattukudy, Ohio State University, Ohio. The clone of a cutinase gene from C. gloeosporioides was obtained from Dr M. A. Dickman, University of Nebraska, Lincoln, Nebraska. Restriction enzymes were purchased from Promega, Madison, Wisconsin. [~t-3~P]-dCTP, [~t-35S]-dATP and plaque hybridization membranes were obtained from Du Pont, Boston, Massachusetts. Hybond-N membranes for Southern and northern analyses were purchased from Amersham, Arlington Height, Illinois. All other chemicals were from Sigma Chemical Company, St Louis, Missouri, unless otherwise specified.

Construction and screening of a eDNA library Conidia obtained from 5-day-old cultures grown on potato dextrose agar were suspended in sterile water (1"4 x 10~conidia ml-1). Conidial suspensions were incubated in the presence ofcutin hydrolysate (250 gg m1-1) or glucose [0-1% (w/v)] for 84 h at 22 °C as described elsewhere [26]. Esterase activity in culture fluids was assayed with p-nitrophenyl butyrate as the substrate [26]. Total RNA was extracted from mycelium with guanidinium thiocyanate followed by centrifugation in caesium chloride solutions as described by Sambrook et al. [20]. Poly(A)+RNA was isolated with Promega's polyATract mRNA isolation system

Cloning and characterization of a cutinase gene

83

(Promega, Madison, Wisconsin). Poly(A)+RNA (5 ~tg) isolated from fungal mycelium incubated in the presence ofcutin monomers as cutinase inducer was used to synthesize eDNA. The ZAP-cDNA synthesis kit (Stratagene, La Jolla, California) was used for the construction of a eDNA library, cDNA was ligated into the vector Uni-ZAP XR, packaged with the Gigapack II Gold packaging extract, and transfected into Escherichia coli strain Sure for library amplification. The eDNA library was plated on E. coli strain XL1-Blue for screening. First strand eDNA used as probes for differential library screening was prepared from 1 gg poly(A)+RNA isolated from cultures induced with cutin hydrolysate or from glucose-grown cultures. Labeling reactions were carried out in 50 lal of 50 mM Tris HC1, pH 8"3, 140 mM KC1, 100 laM dATP, dGTP, dTTP, 4 mM DTT, 10 gg m1-1 oligo-dTls (Promega), 0"8 unit [/1-1 RNasin (Promega), 5 ~tCi ~tl-x [0c-32P]-dCTP, and 0"3 unit la1-1 AMV reverse transcriptase (Promega) at 42 °C for 2 h [20]. Seven plates containing approximately 1000 plaques each were used to prepare duplicate filter lifts. One set of filter lifts was hybridized with labeled first strand cDNA synthesized from poly(A)+RNA isolated from cutin hydrolysate induced culture, another set was hybridized with the respective probe prepared from glucose-grown culture (first screening). Plaques that specifically hybridized with the cDNA probe prepared from induced cultures were selected and converted to pBluescript S K ( - ) clones by using the Fl-mediated excision procedure. Two colonies from each converted phagemid clone were used for DNA isolation [28] and Southern hybridization [20] using the first strand cDNAs described above as probes (second screen). Both clones selected from each positive phage clone had the same molecular weight, and only one clone from each group was used for further analysis. Plasmid DNA was doubly digested with XhoI and EcoRI and subjected to Southern analysis, using the inserts of cutinase cDNA of F. solani f.sp. pisi and C. capsici and the insert of the cutinase gene from C. gloeosporioides as a mixture of heterologous cutinase probes.

Construction of a partial genomic library Genomic DNA of A. brassicicola was isolated according to the method described by Garber and Yoder [8]. Genomic DNA (10 gg) was digested with BamHI. The first two nucleotides of the BamHI site was filled in by Klenow fragments. One microgram of genomic DNA was ligated into 1 gg oflambda Fix II vector prepared by digestion with XhoI followed by fill in of the first two nucleotides of the XhoI site (Stratagene). The ligation product was packaged by using Gigapack II Gold packaging extract. The packaging mixture was used to transfect E. coli strain P2392. Hybridization conditions All prehybridizations and hybridizations in the study were performed in a solution of 5 x SSC, 5 x Denhardt's [20], 0"1% (w/v) SDS and 100 gg m1-1 denatured salmon sperm DNA. Hybridization temperatures for Southern analysis and differential hybridization were 65 °C for homologous and 60 °C for heterologous probes; the temperature for northern hybridization was 68 °C. Prehybridization periods ranged from 1-24 h; hybridizations were for 20-24 h. In the case of homologous probes, membranes were washed at hybridization temperature for 30 min in 1 x SSC and 6-2

84

C. Yao and W. KOller

0"1% SDS twice, then in 0"l x SSC once. For heterologous probes, the washing steps were carried out at a lower stringency. Membranes were washed at 60 °C for 15 min in 2 x SSC and 0"1% SDS three times. All membranes were exposed to Kodak X-OMAT AR film at - 7 0 °C (one intensifying screen) for differeht time periods according to the strength of hybridization signals.

DaVA sequencing The dideoxynucleotide chain termination method [21] was employed, using the Sequenase version 2 sequencing kit (USB, Cleveland, Ohio) according to the procedures recommended by the manufacturer. Nucleotide sequences were determined with double stranded plasmid DNA using T3 and T7 primers. For sequencing entire DNA inserts, the Erase-a-Base system (Promega) was employed to generate overlapping deleted subclones. Closed circular plasmid DNA (5 gg) purified by caesium chloride centrifugation was doubly digested with BstXI and XbaI. ExoIII digestion from 5' protruding ends generated by XbaI was carried out at 37 °C, the desired time points being separated by 30 s and set according to the size of target DNA. After single stranded DNA was deleted by S 1 nuclease, Klenow fragment ofDNA polymerase I was added to flush the ends. The ligation products were used to transform the competent cells of E. coli strain XL1-Blue. The preparation of bacterial competent cells followed the procedure described by Chung et al. [3]. Sequencing analysis was performed using programs from the University of Wisconsin Genetics Computer Group [5]. RESULTS

Cloning and analysis of cutinase eDNA A eDNA library was prepared from cultures induced with cutin monomers, and a total of 7000 plaques was used to prepare duplicate filter lifts. One of the filters was hybridized with first strand eDNA synthesized from mRNA derived from induced cultures, and the other filters were hybridized with eDNA derived from mRNA o f cultures grown in the presence of glucose. Fourteen plaques, which hybridized only with eDNA prepared from induced cultures, were converted to pBluescript S K ( - ) clones. The identity ofcDNAs reflecting cutin monomer-induced mRNA was confirmed for nine of these clones by Southern analysis of plasmid DNA with the discriminating cDNAs used for library screening. In a second step, plasmids isolated from the nine positive phagemid clones were doubly digested with XhoI and EcoRI and subjected to Southern analysis. A mixture of two cutinase cDNAs (F. solani f.sp. pisi and C. capsici) and a cutinase gene (C. gloeosporioides) served as heterologous probes for fungal cutinase cDNAs. Plasmid DNA from five clones showed hybridization, with the largest eDNA insert found for pCAB23. Plasmid pCAB23 and four overlapping subclones were used for sequence analysis. The total nucleotide sequence was 984 bp long with an open reading frame encoding a protein of 209 amino acids (Fig. 1). The peptide sequence of the respective protein showed the following degree of amino acid similarities and identities, respectively, when compared with the four other cutinases characterized thus far [7, 23]: C. gloeosporioides (74%, 64%), C. capsici (73 %, 61%), F. solani f.sp. pisi (69 %, 56 %) and M. grisea (71%, 57%). Sequence comparison revealed several high!y conserved

85

Cloning and characterization of a cutinase gene -549

TC•TC•••GTACCA•A•G•••TGT•CAGCC•ACCATCAGCGCTT•AT••ATGT•TGCTTAATATGCATGCCTAGACACAGATCCCAGGCA

-459

•AGAGTA•TGGA•CGAGGAAGCTCAGATG•AATCAAGATG••TACTGGT•AACT•GTCGACGGTCAA•AGCAAACA••AGATCT•GATCT

-36%

TCGAAAGATCGTATGAGAACGG•CGCAGTACTCTTTGGGGG•AGCATGTC•GCTCAc•AATATCAAGATGCATTC•AAGATCC•AGGCAA

-279

T~GGATACCGAGGTAGTTG~TGAAAATATGAGTGTGAAGACAG~ATCTTCTAcATGTAAAGAAGAG~ATAAAAG~AAGGGGATGcGTcA

-189

CGATTAGAGA•ACAGCACAGTCAAG•AG••GACTTC•AACCTTTTCAGA••A•TCTAC•CTCCAGAGCTATCAGGCA•AGT•TATCGTT•

-99

A••CAATCGC•AA•ATGAAGTCCATTG•AATCCTTTCTCTTTTCG•TGCTCT•G•TCTTACTTCG•CCAT•GC•GTCGCGGTA•CCGTCA

-9

CTGCGCCCAATGATGAACC TC AACTT~TTACTCTCGAAGC CC TGTCAGGCTA~TAC TACCAG AAACGAGCTTGAAA eGG GAAGCAG CGAT M M N L N L L L S K P C Q A S T T R N E L E T ~ S S D

91

G CTTGC CCACG CACCATCTTCATCTTC GCAAGGGGAAGCACTGAAG CTGGAAACATGGTGAG TACAAGC AAATTCTCTACAGACTCC TCC A C p R T ~ F ~ F al~ O ~ T E A G N M

171

ATAACAOCAATTAACCATC ACAGGGCGCACTCGTCGGTCCC TTCACAGCAAACGC CC TCGAGAGCG CGTATGGGGCATCCAATGTTTGG, G A L V G p F T A N A L E S A Y G A S N V ~ V

261

T••AGGG•GTAGGTGG•••CTATACCGC•GGTCT•GTAGAGAATG•••TT••AGCCGGTA•TT•T•AAG••GC•AT•CG•GAAGC••AG O G v G G P X T A G L v E H A L ~ A G T S 0 A A I R E A 0

351

G CCTCTTCAATC TCGC CGCAAG CAAATGC C CCAATACCCCCATCACTGCCGGTGGTTACTCTCAAGGTGCTGCAGTCATGTCCAACGCGA L ~ N L A A S K C D N T P I T A G G Y S O G A A V M S N A I

441

TC C CCGGTCTCAGCGCTGC CGTACAAG AC CAAATCAAGGGTG TCGTGTTGTTCGGCTACACTAAGAACTTGCAAAATGG AGG ~ G G A ~ P G L S A A V Q D Q 1 K C, V V L F G Y T R N L O N G G R

531

CAAACTTCCCTAC•AG•AAGACGACGATCTACTGTGAAA•CGGGGACTTGGTTTGCAA•GGTACA•TGATCATCACGCCTGCGCAT•TC• N

F

P

T

S

K

T

T

I

y

C

E

T

G

D

L

V

C

N

G

T

L

I

I

T

P

A

H

L

621

TG TACTCGGATGAAGCTGCTGTGCAGGCACCTACCTTCTTGAGGGC TCAGATTGACTCAGCATAGTCGGCTGTGAGAGATGTTTGGAAGG X S D E A A v Q ~ T r L R A Q I D S A •

711

GC A C T G T G C A A C A T G e A T G G A T A A A A C ~ ' G T A T G C

901

ACGCATTCTTTACAGTAGTCGCTAGTT~

891

ATAACTCAC~GA~AGTAGCTACTCGATAC~AAACTATTAGC~CATTcCT~TACGTCCTTGAACTGATCCTCAACATCATCAGGTATG

961

ccm'~'Gcc~'cccA

L

CACTATTTGGGTTGT TGG CAC GAG CAGATG CTTGACATTCTTGTTGGATGG AG~

~T* CATTGAGCCAATCGTAAACAGACTCAGTCCATCGTTCGCGCGACAATGTCCCAAACTC

Fxo. l. DNA sequence and predicted amino acid sequence of CUTABI. The amino acids conseryed among Alternaria brassicicola cutinase and the other four characterized fungal cutinases are underlined.

elements such as the sequence (-gly-gly-tyr-ser-gln-gly-) surrounding the active serine identified in cutinase from F. solani f.sp. pisi [7, 13], a conserved aspartic acid and histidine as additional part of the catalytic triad [7, 17], and four cysteine residues involved in disulphide bridges [7]. These similarities suggested that the cloned cDNA represented cutinase produced by A. brassicicola. Cutinase identity was further confirmed by northern analysis. The cloned cDNA hybridized with mRNA of 1.05 kb

86

C. Yao and W. KOller

extracted from cultures induced with cutin monomers, but no hybridization was observed for mRNA isolated from glucose-grown cultures, under conditions ofcutinase repression [26] (Fig. 2).

9.5 Z5

•

,'

4.4

2.4

1.4

0.,14

.

:~' : : .

FIo. 2. Northern blot of RNA from mycelium grown in glucose (lane 1) or induced with cutin monomers (lane 2). The probe is the insert of pCAB23. The numbers in the left lane represent RNA molecular weight standard in kb.

The predicted 209 amino acids translate into a protein with a molecular weight of 22-4 kDa. Molecular weights determined for cutinases Ao and Ba purified from A. brassicicola were 23 and 21 kDa, respectively [26]. Therefore, the size of the protein resembles cutinase Ba more closely than cutinase Ae, in particular because native cutinases lack a signal peptide [10]. According to the cloning strategy, which was based on a eDNA library prepared from induced cultures and a mixture of cutinase-specific heterologous probes, it could be expected that one of the additional positive phagemid clones represented the second cutinase isozyme produced under inductive conditions. However, sequence analysis of the inserts isolated from the four additional clones, which ranged from 433 to 951 bp in length, gave no indication of any departure from the nucleotide sequence determined for the cDNA from pCAB23 (data not shown).

Cloning of a cutinase gene Genomic DNA isolated from A. brassicicola was subjected to Southern analysis, using the cutinase eDNA (pCAB23) as probe. Homology was found for one BamHI, EcoRI or HindIII fragment; two fragments were identified after digestion with KpnI or XhoI (Fig. 3). A partial genomic library was constructed from genomic DNA digested with

Cloning and characterization of a cutinase gene

87

BamH]. Hybridization filters containing a total of approximately 100000 plaques were screened with the cutinase cDNA, and 250 positive plaques were identified during the first round. One phage was purified through an additiona| round of hybridization. Homology to the cDNA was found for a 3"7 kb EcoRI fragment of phage DNA. This fragment was subcloned into pBluescript S K ( - ) and designated pCUTAB]. The 5'and 3'-ends of the pCUTAB1 insert had no sequence homologies with the cutinase cDNA, indicating that the coding region was located in the middle of the insert. i

2

3

4

5

6

7

21

5.1 4.2

g

m

3.5

2.0

~:

I

F[o. 3. Southern blot ofgenomic DNA digested with BamHI (lane 2), EcoRI (lane 3), HindIII (lane 4), KpnI (lane 5), PstI (lane 6) and XhoI (lane 7). Lane 1 is the lambda DNA digested with HindIII and EcoRI. The probes are the insert ofpCAB23 and lambda DNA. The numbers in the left hand lane represent DNA molecular weight standard in kb. Subclones generated from ExolII- and S 1-nuclease deletions were used to determine the location of the cutinase gene, overlapping subclones for the region were isolated, and the nucleotide sequence of a 1"5 kb fragment was determined (Fig. 1). In.addition to the coding region, the segment included 549 and 310 bp of the 5' and 3' non-coding region, respectively. Comparison of the gene and cDNA sequences revealed a perfect match, except for the polyA tract and a 56 bp intron. The location of the intron (nucleotide positions 139-194; Fig. 1) was identical with the intron site found for all other cutinases [7, 23]. The cutinase gene was designated C U T A B 1 . In addition to the intron location, the nucleotide sequence of C U T A B 1 had significant similarities with other cutinase genes [7, 23]. The degree of base matches was 66 % for M. grisea, 64 % for C. gloeosporioides, 6 1 % for C. capsici and 60 % for F. solani f.sp. pisi. However, the 5' and 3' flanking regions of C U T A B 1 showed little homology with other cutinase genes. Within the 5' non-coding region, no homology

88

C. Yao and W. K~ller

was found to the - - 2 2 5 to - - 3 6 0 f r a g m e n t of the cutinase gene from F. solani f.sp. pisi. This segment was shown to act as p r o m o t e r i n v o l v e d in the i n d u c t i o n o f transcription by cutin m o n o m e r s [1, 11]. Similar to ocher cutinases [7, 23], the 5' n o n - c o d i n g region did not contain typical T A T A A and C A A T consensus regions. C U T A B 1 is the first gene sequenced from an Alterna~ia spp., and the c o d o n usage was therefore of interest ( T a b l e 1). O f the 61 n o n - t e r m i n a t i n g codons, eight codons are not used. O f the 209 a m i n o acids codons, 87 end in C, and 47, 41 and 34 end in T, G an d A, respectively. This preference for codons e n d i n g ha pyrimidines is similar to o t h e r filamentous fungi [2]. TABLE 1

Codon usagein CUTAB1 Amino acid

Codon

Gly Gly Gly Gly Glu Glu Asp Asp Val Val Val Val Ala Ala Ala Ala Arg Arg Ser Ser Lys Lys Asn Asn Met IIe IIe IIe Thr Thr Thr Thr

GGG GGA GGT GGC GAG GAA GAT GAC GTG GTA GTT GTC GCG GCA GCT GCC AGG AGA AGT AGC AAG AAA AAT AAC ATG ATA ATT ATC ACG ACA ACT ACC

Number 3 4 10 4 3 5 2 3 2 3 2 4 3 8 8 10 3 I l 7 4 1 5 9 4 0 2 9 4 2 5 7

Amino acid

Codon

Trp End Cys Cys End End Tyr Tyr Leu Leu Phe Phe Ser Ser Ser Ser Arg Arg Arg Arg Gin Gin His His Leu Leu Leu Leu Pro Pro Pro Pro

TGG TGA TGT TGC TAG TAA TAT TAC TTG TTA TTT TTC TCG TCA TCT TCC CGG CGA CGT CGC CAG CAA CAT CAC CTG CTA CTT CTC CCG CCA CCT CCC

Number 1 0 2 3 1 0 2 4 5 1 0 7 2 I• 2 2 0 0 0 3 5 5 1 0 2 0 2 9 0 3 3 6

Cloning and characterization of a cutinase gene

89

DISCUSSION

The cutinase nature of the cDNA and gene described for A. brassicicola is based on several lines of evidence. Both the nucleotide and amino acid sequence have a relatively high degree of homology to the four other cutinase sequences described [7, 23], the gene is only transcribed under conditions ofcutinase induction by cutin monomers, and the position of the intron matches exactly the position found for all other cutinase genes. Additional proof is derived from a structural comparison of the predicted protein. The amino acids conserved in all cutinases comprise 32 % of the Alternaria sequence (Fig. 1). However, these highly conserved amino acids are not necessarily part of longer stretches. The lack of long conserved regions is reflected by nucleotide sequence comparisons. Although the degree of base matches of CUTAB1 with other cutinase genes ranges from 60 to 66%, the longest stretch of perfect matches is only 14 bp in length. The longest highly conserved amino acid sequences are found around the active site serine, and within two regions coinciding with the start of short loops in the threedimensional structure ofcutinase from F. solani f.sp. pisi [17]. These common elements, together with highly conserved locations of four cysteine residues, and a preserved histidine and aspartic acid involved in catalysis, appear to represent structural characteristics required for cutinase function. As indicated by the three-dimensional structure [17], and by the amino acid sequence surrounding the active serine [12], fungal cutinases comprise a unique class of enzymes. Based on these criteria, the enzyme encoded by CUTAB1 belongs to the class of fungal cutinases. M. Erisea C. capsici C. gloeosporioides F. solanf A. brassicicola

Met gln Met

leu val ser thr

ile set leu thr

ala leu ala ser ala leu val ala ala ala ala ile thr leu ala leu leu ala ala thr

pro val gly ala asn

ser ala val gln leu

glu asn ala glu leu

~ ~

ala ala thr ele~u ala l-eu ser lys

phe ile lys phe

set pro ala ala

~ ar~__~ arg gin ~ pro cys

thr leu Met

pro ile val glu ala ala set ala

leu set leu leu glu

val ser lys Met

ala val ile ile phe leu lys phe

ala thr val gly pro val leu pro

asp ... ...... asp ... gly arg ala ser

ser set ser thr thr

ala ser ser phe

leu leu val ala

thr ala leu leu

asn leu glu thr Met

val asp val ser met

gly thr gly ash asn

lys gly thr pro leu

val thr val thr thr

arg are ar~ ar~ ar 8

FIO. 4. The,Ar-terminalsequencesof~ngalcutinases~

The most pronounced difference between the Alternaria enzyme and all other cutinases is the sequence of the N-terminus (Fig. 4). Previously characterized sequences contain all or most of the typical elements of signal peptides typical for exported eukaryotic proteins [7, 23]: a basic amino acid close to the N-terminus; a central hydrophobic core; a more polar C-terminus; and amino acids with short neutral side chains in - 1 and - 3 position of the native N-terminus as recognition sites for signal

90

C. Yao and W. KOller

.peptidases [18]. Furthermore, all four cutinases characterized previously contain a conserved region (-leu-glu-ala/thr-arg-gin-leu/ser-) in close vicinity to the putative signal cleavage site (Fig. 4). The jV-term~nal sequence ofcutinase from A. brassicicola is clearly different. Assuming that arginine (Fig. 4) as the first highly conserved amino acid is close to or at the signal cleavage site, the signal peptide is considerably shorter than in other cutinases, Furthermore, the conserved sequence (-leu-glu-ala/thr-arggln-leu/ser-) is lacking from the Alternaria signal peptide. Although different from other cutinases, the N-terminus contains a short hydrophobic core, followed by more polar residues and several short and neutral amino acids (-ala-ser-thr-thr-) as putative signal cleavage'sites and, thus, can be rated as a putative signal peptide. As indicated by the export of yeast invertase with randomly replaced signal sequences, the recognition of signals for export purposes is not highly specific [9]. However, other parameters such as recognition by signal peptidases and levels ofgene expression are influenced by the signal peptide structure [9]. Although speculative at the present stage, the profound difference in signal peptide sequence identified for the Alternaria cutinase might reflect a difference in regulatory aspects of gene expression of precursor processing. The lack of any sequence homologies in the non-coding 5' region of CUTAB1 with the inducible promoter identified for the cutinase gene of F. solani f.sp. pisi [1] might also indicate differences in gene regulation. Although the predicted size of the Alternaria cutinase (22"4 kDa of the full length protein and 20"7 kDa without the putative signal peptide) indicates a closer relationship to cutinase Ba (21 kDa) rather than to cutinase A~ (23 kDa) [26], direct evidence for a precise isozyme designation of CUTAB1 is lacking at the present stage. Computerassisted restriction map analysis of CUTAB1 revealed one restriction site for KpnI, PstI and XhoI, but no restriction site for BamHI, EcoRI and HindIII. Southern analysis of genomic DNA digested with the set of restriction enzymes yielded the expected fragment patterns (Fig. 3), with the exception of the one fragment obtained with PstI. The respective cleavage site, however, is located in the middle of the gene, and two fragments of similar size might have been generated. The Southern analysis gave n o indication for the presence of a second cutinase gene with sequence homologies to CUTAB1, regardless of the two isozymes purified from A. brassicicola [26]. The conclusion is supported by the identical sequences obtained from five different cDNAs, which were selected according to hybridization to a mixture of three heterologous cutinase probes. The mRNA used for preparation of the eDNA library was isolated from cultures excreting both isozymes, and it is likely that a second cutinase eDNA would have been found if sufficient sequence homologies existed. The situation described here resembles results reported for M. grisea [24]. The disruption of one cutinase gene with homologies to other cutinases did not abolish the secretion of cutinase activity by the respective transformant. Similar to results with A. brassicicola, Southern analysis gave no indication of the presence of a second gene with homologies to CUT1 in M. grisea. Two possibilities exist for the origin of the two isozymes in A. brassicicola. The second cutinase might be encoded by a different gene with a very low degree of homology to CUTAB1 and other cutinase genes, or both isozymes might originate from one gene, with isozyme differences due to posttranscriptional or post-translational modifications. The disruption of CUTAB1 and the expression of cutinase by respective transformants may differentiate between these

Cloning and characterization of a cutinase gene o p t i o n s . T h e role o f c u t i n a s e e n c o d e d b y C U T A B I the host, Brassica oleracea u s i n g C U T A B l - d i s r u p t e d

91 in p a t h o g e n i c i t y will be tested o n t r a n s f o r m a n t s as i n o c u l u m . T h i s

w o r k is in progress. T h e a u t h o r s wish to t h a n k D r P. E. K o l a t t u k u d y for p r o v i d i n g c u t i n a s e c D N A s a n d D r M . B. D i c k m a n for the g e n e o f C. gloeosporioides. T h i s w o r k was s u p p o r t e d b y the National Science Foundation (Grant No. DCB-9018076).

REFERENCES 1. Bajar A, Podila GK, Kolattukudy PE. 1991. Identification of a fungal cutinase promoter that is inducible by a plant signal via a phosphorylated trans-acting factor. Proceedingsof the National Academy of Sciences U.S.A. 88: 8208-8212. 2. Ballanee JD. 1991. Transformation systems for filamentous fungi and an overview of fungal gene structure. In: Leong SA, Berka RM, eds. Molecular Industrial Mycology: Systems and Applicationsfor Filamentous Fungi. New York: Marcel Dekker, 1-30. 3. Chung CT, Niemela SL, Miller RH. 1989. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedingsof the National Academy of Sciences U.S.A. 86: 2172-2175. 4. Deising H, Nicholson ILL, I--Iaug M, H o w a r d RJ, Mendgen K. 1992. Adhesion pad formation and the involvement of cutinase and esterases in the attachment of uredospores to the host cuticle. The Plant Cell 36: 495-508. 5. Devereux J, Haeberli P, Smithies O. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12: 387-395. 6. Dickman MB, Podila 6 K , Kolattukudy PE. 1989. Insertion ofcutinase gene into a wound pathogen enables it to infect intact host. Nature 342: 446-448. 7. Ettinger WF, Thukral SK, Kolattukudy PE. 1987. Structure of cutinase gene, eDNA, and the derived amino acid sequence from phytopathogenic fungi. Biochemistry 26: 7883-7892. 8. Garber RC, Yoder OC. 1983. Isolation of DNA from filamentous fungi and separation into nuclear, mitochondrial and plasmid components. Analytical Biochemistry 135, 416--422. 9. Kaiser CA, Preuss D, Grisafi P, Botstein D. 1987. Many random sequences functionally replace the secretion signal sequence of yeast invertase. Science 235 : 312-317. 10. Kolattukudy PE. 1985. Enzymatic penetration of the plant cuticle by fungal pathogens. Annual Review of Plant Pathology 23: 223-250. 11. Kolattukudy PE. 1991. Plant-fungal communications that trigger genes for breakdown and reinforcement of host defensive barriers. In: Verma DPS, ed. Molecular Signals in Plant-Microbe Communication. Boca Raton: CRC Press, 65-83. 12. K f l l e r W. 1991. Plant cuticles: a barrier to be overcome by plant pathogens. In: Cole GT, Hoch HC, eds. The Fungal Spore and Disease Initiation in Plants and Animals. New York: Plenum Press, 219-240. 13. K f l l e r W, Kolattukudy PE. 1982. Mechanism of action of cutinase: chemical modification of the catalytic triad characteristic for serine hydrolases. Biochemistry 21 : 3083-3090. 14. K f l l e r W, P a r k e r DM. 1989. Purification and characterization of cutinase from Venturia inaequalis. Phytopathology 79: 278-283. 15. K f l l e r W, P a r k e r DM, Beeker CM. 1991. Role of cutinase in the penetration of apple leaves by Venturia inaequalis. Phytopathology 81 : 1375-1379. 16. Liyanage HD, K f I l e r W, MeMillan RT, Kistler C. 1993. Variation in cutinase from two subpopulations of Golletotrichumgloeosporioides from citrus. Phytopathology 83 : I 13-116. 17. Martinez C, De Geus P, Lauwereys M, Matthyssens G, Carnbillatl C. 1992. Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent. Nature 356: 615-618. 18. Muller M. 1992. Proteolysis in protein import and export: signal peptide processing in eu- and prokaryotes. Experientia 48:118-129. 19. Paseholati SF, Yoshioka H, Kunoh H, Nicholson RL. 1992. Preparation of the infection court by Erysiphegraminis f.sp. hordei. Cu tinase is a component of the conidial exudate. Physiologicaland Molecular Plant Pathology 41 : 53-59. 20. S a m b r o o k J, Fritsch EF, Maniatis T. 1989. ~Iolecular Cloning, A Laboratory Manual, 2nd edn. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 21. Sanger F, Nieklen S, Coulson AR. 1977. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences U.S.A. 74 : 5463-5467.

92

C. Yao and W. KOIler

22. Stahl DJ, Sch/ifer W. 1992. Cutinase is not required for fungal pathogenicity on pea. The Plant Cell 4: 621-629. 23. Sweigard JA, C h u m l e y FG, V a l e n t B. 1992. Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisen. Molecular and General Genetics 232: 174-182. 24. Sweigard JA, C h u m l e y FG, V a l e n t B 1992. Disruption ofa Magnaporthegrisen cutinas~: gene. Molecular and General Genetics 232: 183-190. 25. T r a i l F, K611er W. 1990. Diversity ofcutinases from plant pathogenic fungi : evidence for a relationship between enzyme properties and tissue specificity. Pl~siological and Molecular Plant Pathology 36: 495-508. 26. T r a i l F, K611er W. 1993. Diversity of cutlnases from plapt pathogenic fungi: purification and characterization of two cutinases from Alternaria brasskkola. "Physiological and Molecular Plant Pathology 42 : 205-220. 27. Vaxley DA, Podila GK, H i r e m a t h ST. 1992. Cutinase in Cryphonectria rarasitica, the chestnut blight fungus. Suppression of cutinase gene expression in isogenic hypovirulent strains containing double-stranded RNAs. Molecular and Cellular Biology 12: 4539--4544. 28. Zhou C, Yang Y, Jong AY. 1990. Mini-prep in ten minutes. BioTechniques 8: 172-173.