Gene. 120 (1992) 119-124 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

119

0378-l 119/92/$05.00

0669 1

Cloning and regulatory analysis of starvation-stress gene, ssgA, encoding a hydrophobin-like protein from the entomopathogenic fungus, Met arhizium

anisopliae (Insect

pathology;

Raymond

appressorium

formation;

glycosylation

site; nutrient

deprivation;

gene expression)

J. St. Leger, Richard C. Staples and Donald W. Roberts

Boyce Thompson Institute for Plant Research. Inc., Ithaca, NY 14853-1801,

USA

Received

May 1992; Received

by J. Kinghorn:

20 March

1992; Revised/Accepted:

20 May/24

at publishers:

22 June 1992

SUMMARY

The nucleotide (nt) sequence of a starvation-stress gene (ssgA) of the entomopathogenic fungus, Metarhizium anisopliae, and its deduced amino acid (aa) sequence were determined. The primary structure of the SSGA (96 aa; deduced M, = 9925; p1 = 4.1) protein shares extensive similarities with fungal wall proteins of the ‘hydrophobin’ class, and the eight Cys residues and putative signal sequences are conserved. Secondary structure predictions suggest an additional resemblance to low-M, toxins and agglutinins. Northern (RNA) blot analysis and nuclear run-on assays demonstrated transcriptional control of expression of ssgA during nutrient deprivation and during formation of infection structures. Hybridizations of M. anisopliae genomic DNA indicate that there is only one form of ssgA in the genome.

INTRODUCTION

A number of low-molecular-weight, hydrophobic, extracellular polypeptides called hydrophobins have been identified recently in the basidiomycete Schizophyllum commune and the ascomycete Aspergillus nidulans. Each have eight cysteines conserved at identical positions (Schuren and Wessels, 1990; Stringer et al., 1991). Schuren and Wessels (1990) described one S. commune hydrophobin-encoding gene, designated Sc3, that is expressed abundantly during

Correspondence to: Dr. R.J. St. Leger, Boyce Thompson

Institute for Plant

Research,

USA.

Inc., Tower Road,

Tel. (607) 254-1252; Abbreviations:

Ithaca,

NY 14853-1801,

Fax (607) 254-1242.

aa, amino acid(s); bp, base pair(s); kb, kilobase

bp; M., Metarhizium; MM, minimal media (0.1 ?0 KH,PO,/O.OS

or 1000 “i, MgSO,

pH 6); nt, nucleotide(s); Prl, M. anisopliae protease; prl, gene (DNA, mRNA) encoding Prl; SDB, Sabouraud dextrose broth; SDS, sodium dodecyl

sulfate;

SSC, 0.15 M NaCl/0.015

starvation-stress gene; SSGA, NaCI/O.Ol M NaH2P0,/0.00125

M Na,.citrate

pH 7.6; ssgA,

protein product of ssgA; SSPE, M EDTA pH 7.4.

0.15 M

the formation of aerial hyphae, whereas two related genes (Scl and Sc4) are regulated by mating-type genes and are expressed during the formation of fruit bodies. Apparently, the gene-encoded polypeptides are secreted into the growth medium by submerged hyphae and accumulate in the cell walls of emerged hyphae (Wessels et al., 1991). Stringer et al. (1991) described a hydrophobin gene in A. nidulans which is expressed in conidiophore cells, the protein product of which accumulates in the rodlet layer of the conidium wall. It has been proposed that by forming a hydrophobic coating on their surfaces, hydrophobins are important in the formation and function of aerial structures such as conidiophores and mushrooms. Here we describe a new member of this hydrophobin gene family produced by the commercially important entomopathogenic deuteromycete, Metarhizium anisopliae. The gene, designated ssgA, was obtained from a cDNA library representing a nutrient-deprived culture of M. anisopliae. Nutrient deprivation is one of the interactive phenomena which correlate formation of infection structures (appressoria) and production of insect-cuticle-

-- 324

817

ME1

2951

23

>HH>WH,HH>#H>WH wIulwxwwIwwIwwIw

Fig. 2.

Fig. 1. Sequence

of ssgA cDNA

and the deduced

[35S]dATP

and a double-stranded

constructed

in the pBluescript

glycosylation

site (Se?‘-Th?“)

template

aa sequence.

procedure

vector (Stratagene), are underlined.

The nt sequencing

provided

in the Sequenasc

was performed

and the entire inserts were sequenced

The stop codon

is marked

by the dideoxy

kit (U.S. Biochemicals, on both strands.

by three dots. GenBank

chain-termination

Cleveland,

The eight Cys residues

accession

number

procedure

OH). The ssgA subclones

using wcrc

and a possible O-linked

is M8528 1.

Fig. 2. Southern blot analysis of restricted chromosomal DNA from five isolates of M. anisopliae. Genomic DNAs (5 pg) prepared from m)cclia of five isolates (MEl, 23, 2951, 817, and 324) (St. Leger et al., 1992) were digested to completion with EcoRI (EI), EcoRV (EV), or Hind111 (HIII) and scparated by electrophoresis random

in 0.1 x SSC/O.l% plasmid

on a 1.Oob agarose

primed ssgA cDNA

fragment

gel. The fragments

under high stringency

SDS at 68”C, and subjected

using [3ZP]CTP

as described

were transferred conditions

to autoradiography

by Feinberg

and Vogelstein

to Gene Screen PlusTM membranes

(507; formamidc/h

x SSPE/ 1 x Denhardt’s

(4 h). The probe was prepared

(NEN) and probed with a “P-labeled solution/ 1“,, SDS at 42” C), washed

by oligo labeling of an EcoRI fragment

from the ssg,4

(1984).

degrading enzymes (St. Leger et al., 1989a). Nuclear run-on experiments confirmed that the gene is expressed synchronously with the gent encoding the cuticle-degrading protease. Pr 1. the only pathogenicity determinant so far described from an entomopathogenic fungus (St. Leger et al.,

1989b; 1991; 1992). One aim of our research is the molecular analysis of infection processes in entomopathogenic fungi. In this connection, a gene with a simple genomic organization that responds rapidly and strongly during the formation of infection structures is of great interest.

Number of Amino Acids 23 26 z: 46

ZB 80 50 96

1:: 117 89 146 100 111 125 159:

Fig. 3. Alignment

of the deduced

and Sc4 (S. commune) (Schuren (Lipman and Pearson, 1985).

SSGA aa sequence and Wessels,

(see Fig. 1) with other hydrophobin

aa sequences,

shown in a single letter code. Proteins:

1990) and RodA (A. nidulans) (Stringer et al.. 1991). The aa sequences

were aligned using the FASTP

SC 1, Sc3, program

121 EXPERIMENTAL

p1 = 4.1). The protein contains several hydrophobic quences including a highly hydrophobic N-terminal quence. The protein contains eight Cys residues and a tential O-glycosylation site ( Sers3-Thrs4) but lacks sites N-glycosylation.

AND DISCUSSION

(a) Sequence of ssgA and its putative protein Clones were isolated from the cDNA library (cloned into the EcoRI site of the vector igtl0) representing poly(A)‘RNA from mycelia of M. anisopliae deprived of nutrients for 3 h (St. Leger et al., 1992). We screened a total of 15000 plaques at a high stringency (50% formamide/ 6 x SSPE/l x Denhardt’s solution/0.3% SDS; washes ranged from 2 x SSC to 0.1 x SSC) and identified 45 clones differentially expressed during nutrient deprivation (St. Leger et al., 1992). The clones were rescued as Bluescript plasmids and segregated into four nonoverlapping clone classes by cross-hybridization. The 26 clones in class 1 were identified as prl (St. Leger et al., 1992). The 11 cDNA clones in class 2 ranged from 580 to 630 nt in length. Sequence analysis (Fig. 1) on three of the longest clones revealed identical putative coding regions. The ORF contained between the first ATG of the longest cDNA (ssgA, Fig. 1) and the first termination codon (TAA) is 288 bp long and encodes a 96-aa protein (deduced M,. = 9925,

sesepofor

(b) Genomic organization of the ssgA gene To determine whether ssgA is a member of a gene family, we performed DNA blot hybridization with genomic DNA prepared from five strains of M. anisopliae isolated from the USA (MEl, 23, 2951), Europe (817) and Australia (324) (Fig. 2). After digestion of the genomic DNA with appropriate restriction enzymes, only a single fragment hybridized to the ssgA cDNA, indicating that there is only one form of the gene in the genome. The same size fragments were present when DNA from the three American isolates was digested with EcoRI, EcoRV, and HindII1. Different fragments were obtained from the European and Australian isolates. The Australian isolate was distinctive in that two DNA fragments hybridized to the ssgA

+1.7 Janeson-Wolf (Antlgenlc Index) -1.7 GOR

GOR

/

/

turns

GIIR OL hekes

-

fl sheets

+1.7 Janeson-Wolf (Antlgenlc Index) ' -1.7 GLIR turns

KO

GOR

a hellces

GOR

B

n

e

w

sheets

h~l--_-

hydrophlllclty

rJ\,,

---

-5.0 +1.7 Janeson-Wolf (Antlgenlc 'nd~lx; ' GOR

Fig. 4. Computer-aided proteins. Hydrophilicity

o( hellces -

GOR

B sheets

I

\

n Al--I

secondary structure analysis of the deduced aa sequence of the SSGA (M. anisopliae), Scl (S. commune), and RodA (A. nidulans) was determined by the method of Kyte and Doolittle (1982). Hydrophobic regions are represented by negative numbers and

hydrophilic

regions

by positive

secondary

structure

predictions

were obtained

n

turns

GOR

from Schuren

ones, along the ordinate, according

and Wessels

to the algorithm

The antigenic of Garnier

(1990) and Stringer

index was calculated

according

et al. (1978) are also shown.

et al. (1991)

respectively.

to a formula

Primary

sequence

of Jameson

and Wolf (1988). The

data for Scl and RodA proteins

122 cDNA which may represent a different restriction pattern of a single gene or, less likely, multiple gene copies. (c) Sequence similarities and secondary structure of SSGA The predicted aa sequence of the SSGA shows 48”/,, 642, 67 %, and 48 y0 identity to the S. commune extracellular hydrophobins - Scl, Sc3, and Sc4 - and the rodlet protein from A. ~~~~Z~~~,respectively (Fig. 3). All five proteins contained a hydrophobic N-terminal sequence of about 20 aa resembling signal sequences for excreted proteins (see Fig. 4 for hydropathy plots). This suggests that the M. anisopliae SSGA is also secreted. The aa sequences of these leaders were quite homologous (8 out of 20 aa are shared between SSGA and Sc3 or Sc4). The eight Cys residues that were present in each of the five proteins are arranged in the same pattern, including the conserved tripkb

‘1

2

3

4

5

6

7

8

7.46 -

2.37-

eptide CCN. No other significant sequence similarities were identified in computer-assisted searches of the GenBank data bases. Computer-generated predictions of the secondary structure and hydrophilicity of S SGA were compared with data derived from the primary sequences of Scl (Schuren and Wessels, 1990) and RodA (Stringer et al., 1991) (Fig. 4). All three proteins contain regions of p-sheet with few, if any, regions of a-helix outside the N-terminal signal sequence. These features are shared with Sc3 and Sc4 (R.J.St.L., unpublished data). Apart from the signal sequences, hydrophobic regions in various domains of these molecules probably represent sequences passing through the interior of the protein (Kyte and Doolittle, 1982). The antigenic index [a surface probability plot (Jameson and Wolf, 1988)] also indicates that the O-glycosylation site in SSGA is located on a potentially exposed surface peak of the protein, making glycosylation possible. If the eight Cys residues formed disuhide bonds within the SSGA molecule, then its shape and contours would be altered in a manner dependent on bridging patterns. These cysteines may also be involved in cross-linking of the polypeptides to form polymeric structures (Shuren and Wessels, 1990; Stringer et al., 1991). However, as they were small, secreted hydrophobic proteins composed primarily of a p-sheet secondary structure and containing eight Cys residues, the hydrophobins closely resemble a wide range oftoxins and agglutinins (Drenth et al., 1980). The disullide bridging patterns which vary between toxin molecules result in four loops cont~ning a toxin-aggIutinin fold. Further biochemical and structural studies are necessary to identify the polypeptide structure of the ssgA product and relate it to the function.

0.24Fig. 5. Detection Poly(A)

of the

.q@

mRNA

during

+RNA isolated from mycehum of M.

in SDB and transferred transferred

nutrient

deprivation.

nksopliaegrown for 32 h

to different culture media for 3 h. Cultures

to fresh

SDB

N-acetylglucosamine/0.5’!0 3); to MM plus 0.5’:; chitin (3 h) followed by a second transfer plus either lo0 glucose

(2 h) (lane 4) or

(lane 5). SDB cultures

transferred

lug ~~-acetyl~lucosamine

to unsupplement~d

MM supplemented

with starch (O.S’:,) (lane 7) or cellulose (l”,) were grown in SDB and transferred

previously,

and total RNA was extracted

to MM (2 h)

MM (lane 6). to

Methods: Cultures described

were

(lane I), to MM supplemented with I “,, chitin (lane 2); to MM plus chitin (0.5”“) (lane

(lane 8).

to fresh media as

from fungal samples

using guanidine HCI (St. Leger et al., 1991). The poly(A)‘RNA fraction was purified by oligo(dT)-cellulose chromatography (Sambrook et al., 19X9). RNAs (0.5 &lane) for Northern blots were denatured with giyoxal and dimethylsulfoxide (Sambrook et al., 1989). The I ‘.,, agarose gels were blotted to nylon and hybridized lowing autoradiography

as for Southern

(12 h), the membranes

blots (see Fig. 2). Folwere incubated

in 0.4 N

NaOH at 7O’C for 30 min and rehybridized with a radiolabeled r-tubulin gene fragment ofM. aniwpfiaeto confirm the even transfer of RNA during blotting.

The a-tub&in

gene was obtained

in the igtl I vector with (R.J.St.L., unpublished).

monoclonal

by screening anti-~-tubuiin

a cDNA (Sigma,

library T9026)

(d) Expression of ssgA The ssgA gene was selected in a screen for genes expressed during nutrient deprivation. The pattern of the ssgA transcript accumulation was examined by Northern hybridization analysis using ssg_4 cDNA as a probe (Fig. 5). The transcript was present at low or undetectable levels in mycelia growing rapidly in SDB medium (Difco) or when the mycelia were transferred to medium containing N-acetylglucosamine, a readily utilized carbon and nitrogen source. By contrast, a hybridizing band of approx. 600 nt accumulated when cells were transferred to MM. Production was enhanced when the MM was supplemented with polymers (cellulose, chitin) at levels too low to produce carbon catabolite repression. A similar phenomenon has been recorded for Pr 1 production (St. Leger et al., 1988; 1991). The sJ;rA mRNA ceased to accumulate when chitin medium was supplemented with glucose or IV-acetylglucosamine for 2 h, confimling that transcript synthesis was catabolite repressed. We performed nuclear run-on

123 experiments

to confirm that regulation

of synthesis

N

D

C

was at

the level of transcription, and we compared the transcription rates of ssgA with those for mRNA of prl which had

bp

been previously determined (St. Leger et al., 1992) (Fig. 6). Neither transcript (ssgA or prl) was produced by nuclei from nutrient-rich cultures, but they were produced rapidly (< 2 h) when the cells were deprived of nutrients, reaching a maximum concentration after 24 h. Although the pattern of production of prl and ssgA transcripts was coordinated, levels of the prl transcript always exceeded those of ssgA, consistent with Pr 1 being the major starvation-specific protein. Pr 1 is produced at high levels by M. anisopliae germlings induced to differentiate infection structures by nutrient deprivation (St. Leger et al., 1989b). Coordinated regulation of ssgA with prl implies that the .ssgA gene would be expressed during infection processes. Indeed, the transcript was present at low or undetectable levels in conidia or undifferentiated germlings but at high levels in germlings producing infection structures (appressoria) (Fig. 7).

Fig. 7. Northern germlings

phal growth duced

analysis

producing

of the ssgA mRNA

appressoria

(N). Methods:

by germinating

Infection

conidia

glass petri dishes as described

in conidia

(D) or producing structures

(appressoria)

in yeast extract previously

(C) or from

nondifferentiated

media (0.0125%

w/v) in

(St. Leger et al., 1989a). Yeast

extract media (0.08% w/v) was used to obtain polar hyphal growth. RNA

(3 pgilane)

1991), denatured

was extracted with glyoxal

with a labeled random

primed f&RI

in Figs. 2 and 5. Following

branes were incubated

in 0.4 N NaOH

Stock Center) to confirm

Time

(h)

of prl and ssgA transcription

during nutrient

tion. Methods: Nuclei were isolated from nutrient-rich from cultures

transferred

to 72 h. Transcription isolated and assayed 1992). Assay MnCl,/200

depriva-

(SDB) cultures and

from SDB to MM (see legend of Fig. 5) for up

of prl and ssgA was measured by previously

mixes contained

described

in vitro. Nuclei were

procedures

(St. Leger et al.,

10 mM Tris pH S/l0 pM MgCl,/2

PM KC1/0.5 mM dithiothreitol/lO”;

glycerol/O.05

PM

mM each

ATP, CTP, and GTP/200 PCi of [3ZP]UTP (3000 Ci/mmol)/S x 10’ nuclei. Incubations lasted 30 min at 30°C. Incorporation of [32P]UMP into a trichloracetic-acid-precipitable et al., 1989). The synthesized

product was determined (Sambrook RNA was extracted and ethanol precipi-

tated, and 5 x IO’ cpm was used for hybridization natured

DNA (ssgA cDNA

ters as described

(Sambrook

plasmid

inserts)

to 10 pg of alkali de-

bound

to nitrocellulose

et al., 1989). Hybridizations

fil-

were carried out

in 50”,, formamide/ x SSCjl x Denhardts solution/O. 1 Of, SDS. Washes ranged from 2 x SSC to 0.1 x SSC. Counting error (two standard deviations) was 5% or less. cc-Amanitin added to the transcription that hybridized system.

(an inhibitor

of RNA polymerase

mix (0.5 mg/ml) blocked

to the ssgA gene, demonstrating

the synthesis the specificity

II)

of RNA of the

rDNA

Total

(St. Leger et al.,

and dimethylsulfoxide (Sambrook et al., w/v agarose gel), and blotted and probed

(1.25,

ized with a radiolabeled

Fig. 6. Regulation

using guanidine,HCl

1989), electrophoresed described

hy-

were in-

fragment

from the ssgA plasmid

autoradiography

as

(24 h), the mem-

at 70°C for 30 min and rehybrid-

fragment

the even transfer

(clone

1:7D, Fungal

Genetics

of RNA during blotting.

(e) Conclusions (1) Although the SSGA protein is not an enzyme and therefore presumably incapable of directly providing nutrients, the ssgA gene expression is coordinately regulated with prl, and it is transcribed when fungal growth is limited by nutrient deprivation, as during the formation of infection structures (appressoria). (2) As a hydrophobin, the SSGA peptide is presumably involved in building the walls of these infection structures and could assist with hydrophobic attachments to the cutitular surface and preventing desiccation. The resemblance of SSGA to toxins also merits further investigation to identify function. (3) The role of nutrient levels in regulating hydrophobin production by other fungi has not been determined. Nutrient deprivation plays a role in stimulating conidiation in many fungal species (Griffin, 1981) and aerial hyphae represent a potential nutrient sink. The effect of nutrient levels on the gene expression is therefore consistent with the location of other hydrophobins on aerial hyphae (Wessels et al., 1991) or conidial walls (Stringer et al., 1991).

124 Schuren,

ACKNOWLEDGEMENTS

F.H.J.

in fruiting

We thank David C. Frank for technical assistance. This work was supported in part by a grant (89-37263-4463) from the United States Department of Agriculture Competitive Research Grants Office.

and Wessels, dikaryons

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R.C.: Molec-

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