in Ustilago maydis

Control of mating and development

Regine Kahmann, Tina Romeis, Michael Biilker and J&g KZmper

Ludwig-Maximilians-UniversiW

In the fungus

Mtinchen,

maydis, the ability to distinguish

L&i/ago

are of the same or of different

system. After cell fusion,
determines

homeodomain
which

allelic

recognition.

to form
not to

interact,

it is a specific

interact.

This

suggests

Opinion

Ustilqo ttiaydis, the causal agent of corn smut disease, is a

member of the diverse group of the basidiomycetes.
This
fungus is of particular
interest because its ability to mate
and to cause disease are interconnected.
U. maydis exists
in two morphologically
and developmentally
distinct
forms. The haploid form grows in a yeast-like
manner
by budding
and is unable to initiate disease. The other
stage is the dikaryon,
which results from fusion of two
compatible
haploid cells. This form shows filamentous
growth and is pathogenic
for corn, in which it induces
the typical
disease
symptoms
of neoplastic
growth
and tumors.
Mating
and pathogenic
development
are
genetically
controlled
by two unlinked
loci, termed
a
and b. To form an infectious
dikaryon,
two haploid
mating
partners
have to be different
in both
a and
in b [l-5].
The a locus controls
the cell fusion event
through
a pheromone-based
recognition
system [6,7].
The decision
to go on with the pathogenic
program
is made by the multiallelic
b locus on recognizing
that
the two nuclei carry different
b alleles. With at least
33 different
b alleles existing in nature, this implies that
hundreds
of b allele combinations
trigger pathogenic
and
sexual development,
and only 33 b allele combinations
are inactive ([1,3,8,9]; JE DeVay, unpublished
data cited
in [lo]).
Molecular
analysis of the b locus has provided
clues
as to how
this multiallelic
recognition
might
be
achieved.
It has been shown
that the locus encodes
a pair of homeodomain
proteins
designated
bE and
bW
[l l-131.
To trigger
development,
bE and bW
proteins
that originate
from different
alleles have to
be combined
[13]. Interestingly,
such
processes
of
intracellular
recognition
are not unique
to U. rrraydir,
but appear to control developmental
switches in several
0 Current

recognition

the proteins
feature
the

in

mechanism

in all different

of proteins

existence

of

from

the

a code for

recognition.

in Genetics & Development

Introduction

combinations

alleles of the locus. Recent

molecular

Whereas

protein-protein

Current

in pairwise

from different

heterodimers.

combinations
allele

a pheromone-based

Each allele encodes a pair of

are active only

suggest that the underlying

is the ability
same

that

the two partners originate

discoveries

by two mating-type

through

the other mating-type locus, which exists in multiple

intracellular

proteins

recognition

Germany

between partners that

mating type is controlled

loci. One locus allows extracellular

alleles,

Miinchen,

1995,

5:559-564

other
basidiomycetes
[14-161.
During
the past year,
it has become
clear that the molecular
mechanism
of self/non-self
discrimination
operates
on the level
of protein
dimerization.
Here,
we will review
these
studies and discuss their implications.
For more detailed
background
information
on mating-type
loci, the reader
is referred to several other recent reviews [17-lo].

Organization

of bE and bW proteins

The two genes of the b locus, bE and bl4( are divergently

transcribed
and encode proteins
of 473 and 644 amino
acids, respectively.
When allelic variants of bE and bW
are compared,
the proteins
can be seen to have three
distinct domains:
a highly polymorphic
amino-terminal
region (- 100 amino acids in bE and - 150 amino acids
in bW), a domain displaying characteristic
features of the
homeodomain
motif found in eukaryotic
transcription
factors, and a highly conserved
carboxy-terminal
domain
[l l-131 (Fig. la). Except for the homeodomain
motifs,
bE and bW are dissimilar.
bE will tolerate
carboxyterminal truncations
up to amino acid position
283; bW
can be shortened
to at least 493 amino acids without
apparent
loss or change in function
(Fig. la; M Urban,
J Kamper,
M Reichmann,
R Kahmann,
unpublished
data).

III bE, a sequence

resembling
a nuclear
localization
sequence
has been identified
[12], but it is not located in
the essential protein domain.
The homeodomain
motifs
are related to the homeodomains
of the yeast al and a2
proteins, with the exception
that in bE, the spacer region
between
helix II and III contains
15 additional
amino
acids [l 1 ,131. The homeodomains
are crucial for function for both proteins,
and neither bE nor bW tolerate

Biology Ltd ISSN

0959-437X

559

560

Differentiation

and gene regulation

deletions or single-point
mutations at highly conserved
positions (R Schlesinger, J Kamper, unpublished
data).
With the help of chimeric alleles, the variable domains
have been shown to contain all elements determining
allele specificity [20,21]. Specificity regions were defined
as regions outside of which recombination
events create
alleles that retain the specificity of either parent but
within which recombination
events create alleles with
a specificity of neither parent [21].

convincingly
that bE and bW proteins act as pairs.
Thus, in a cross of two haploid strains, two of the
different active bE-bW
pairs generated are redundant
in their regulatory potential [13] (Fig. lb). This creates
the fascinating situation that .with 33 different b alleles
existing in nature, there are 33 inactive and 1056 active
bE-bW combinations.

Dimerization

What distinguishes
active and inactive bE-bW
pairs?
This question has recently been settled by using the yeast
two-hybrid
system in combination
with a biochemical
interaction
assay employing immobilized
proteins [22].
bE and bW are shown to form heterodimers
only
when they derive from different alleles. When both
proteins originate from the same allele, dimerization
cannot be detected [23**]. The interacting
domains
have been mapped to the variable amino termini
of bE and bW and shown to be distinct from the
adjacent homeodomains.
The smallest domains
still
allowing allele-specific
heterodimer
formation
are the

bE and bW proteins act as pairs

With each b locus encoding a pair of homeodomain
proteins, one could wonder why two different b loci
have to be combined
to trigger development.
This
issue has been resolved with the help of null mutants
with deletions for either bE, bW or both. Strains lacking
the complete b locus are unable to initiate development,
indicating an active role for the b proteins in this process
[13]. In strains in which bE or b W are deleted, b locus
function is seen only when bE and b W deriving from
different alleles are combined
(Fig. lb). This shows

(a)

of bE and bW

Homeodomain
644 aa

bW
446

:93

*,
Constant

Variable

473 aa

bE
25;

tL33

UP
Variable

Constant

Fig. 1. Organization
proteins
odomain,

Inactive
bE1

sequence

able

portions

and

Inactive

tion

given

allelic

and

E2) are shown

tive

with

lines

The

vari-

and W2,
the

same

El
tint-

as the genes. The six possible

(indicating

of protein

two being active

are observed)

nor sexual

(Wl

combinations

ing that dimerization

ment

(b)
and

vertical

differences.

domains

ucts are shown,

Inactive

dele-

acids.

for both bwkinted

bfi? with

protein

ing scheme

(+)

indicated.

(open bars) genes (bWI

and

able

pairwise

activity

are

in amino

are shown

bars) and bE
indicating

of carboxy-

retaining
(-)

and vari-

refer to the respective

endpoint,

bEl

localiza-

constant

activity

bW

interac-

of the home-

nuclear

and the extent

deletions

Two alleles

bE and

of their

helices

(NLS),

without

bW2,
a

of the
mode

putative

The numbers
E2

00

the

tion

terminal

-II

the

(a) The three

tion.

Active

and

that

and sexual
and four
neither

development

prod-

(indicatdevelop-

being

inac-

dimerization

can take place).

Control of mating and developmental in Ustilago may&

Kahmann et al.

amino-terminal
97 amino acids in bE2, <77 amino acids
in bE1 and <94 amino acids in bW2 [23**].

Self/non-self discrimination: a recognition code?

In support of the claim that heterodimerization

is a
prerequisite
for activity of the bE-bW
complex are
the results obtained
in two different mutagenesis experiments. Mutants for bE2 selected for interaction with
bW2 in the two-hybrid
system were subsequently shown
to trigger pathogenic development
in combination
with
bW2 in U. muydir. The other set of mutants was first
screened for function
with bW2 in L? rnaydis and
subsequently
tested for interaction
with bW2 in the
two-hybrid
system. Nearly all of the mutant proteins
from this collection showed interaction with bW2. This
demonstrates convincingly
that measurable interaction of
bE and bW in the two-hybrid
system correlates with
function
in U. may& and suggests furthermore
that
even weak interactions
that cannot be detected in the
two-hybrid
system may be sufficiently strong to allow
function in U maydis [23].

To accommodate
the situation
that a given bE or
bW polypeptide
can form heterodimers
with at least
32 different partners but not with the partner that is
encoded by the same allele, one has to assume that
a cohesive surface for promiscuous
interactions
exists;
in addition, there must be specific determinants
that
prevent interaction between bE and bW from the same
allele. This is supported by the observation
that single
amino acid substitutions
are sufficient to allow dimerization. The nature of these amino acid substitutions
indicates that an increase in hydrophobicity
or a change
of charge is responsible for the observed heterodimer
formation
[23]. Thus, the ability of bE and bW
to interact must be governed mainly by hydrophobic
and/or polar interactions, a situation that is reminiscent
of proteins that engage in combinatorial
interactions
through coiled-coil motifs [24,25]. A schematic model

bE1

bE2

bE3

bE4

bW1

bw4

1995

(Current Opinion

in Cenet~cs & Development

Fig. 2. Model for allele-specific dimerization. Schematic drawings of the interaction domains are shown for the bW proteins on the left and for the bE proteins
on top, with each protein providing four
potential cohesive contact points. The 12
pairwise combinations of bE and bW in
open boxes can form heterodimers and
are active. The four combinations of proteins shown in tinted boxes derive from
a single allele and are inactive because
they cannot interact.

561

562

Differentiation and gene regulation

Unfortunately,
it is not yet known whether bE and
bW act as transcriptional
repressors or activators; their
primary
structure
does not provide definite
clues,
nor is a specific target gene known. As the bE-bW
heterodimer
is needed to initiate sexual development,
one can envisage three non-mutually
exclusive scenarios
for how the b polypeptides
regulate these steps. In
the first, the bE-bW
heterodimer
could function as a
transcriptional
activator for one or more pathogenicity
gene. In the second, the heterodimer
could be a
repressor of certain haploid-specific
genes analogous to
the al-a2
heterodimer
in yeast [26]. Some evidence
in support of this possibility comes from the finding
that cell fusion is affected in strains expressing an
active bE-bW
heterodimer
([27,28,29*]; M Urban,
R Kahmann,
M Biilker, unpublished
data). There is
no indication,
however, that this effect is the direct
consequence
of the bE-bW
heterodimer
in repressing
the respective promoters.
In the third scenario. the

proposes that bE and bW proteins

carry multiple
exposed hydrophobic
or polar residues that prevent
self-interaction
in haploid cells [23**] (Fig. 2). Assuming
that a single cohesive contact suffices for dimerization,
as suggested by bE2 point mutants
[23*-l, non-self
interaction
can be easily explained for many different
combinations
(Fig. 2). Interestingly, just four contact
points (as shown in Fig. 2) can create so much variability
that nearly all different b specificities that occur in nature
can be accommodated.

The regulatory

functions

of the bE and bW

polypeptides
The presence of homeodomain
motifs indicates that
the b polypeptides
act as DNA-binding
proteins.

bE3

bW3

X
b3

b2

1
First
recombination

a
bW2

bE2

event

bE3*

bW3

b3

bz
Second
recombination
event

a
bE3*

bW3*

Fig. 3. Model

of a b al-

for the generation

lele that displays

a new specificity.

Sym-

bols used are as in Fig. 2, and the specificity

of the alleles

respective

is indicated

bE-bW

ing the two parental

crossed.

b new

nation

During
event

bW3.

(b)

with
bW3*

bE3*

bW3*

bE2

bw3*

bE3

When
the

nation

b2 parent,
can

bW3*-bE3*

new specificity,

a second
bW3*.

designated

and

crossed
recombi-

between

bW2

Note that the

pair

as b new.

is crossed with

b3 specihcity,

at least one active

formed.

bE3

is again

and has thus acquired

If the b new strain

of b2 and

carrying

strain carries an inactive

of b polypeptides

b new

a first recombi-

occur

and bW3 generating

the

carry-

bE3 and bE2 gen-

strain

this strain

event

below

Strains

b2 and b3 are

alleles

meiosis,

between

erates a haploid

(a)

pair.

bE-bW

in both

(b)

strains
cases

heterodimer

is

Control of mating and developmental in Ustilago maydis

Kahmann

et a/.

bE-bW
heterodimer
might act as a repressor of a
repressor of pathogenicity
genes. This latter scenario
has gained support from the isolation of a recessive
mutation
that bypasses the requirement
for an active
bE-bW complex [30], but to our knowledge, the nature
of this mutation has not been further investigated.

of these proteins may help us to decipher the general

code for protein-protein
recognition. Understanding
the
mechanism by which the key players work should also
allow the identification
of downstream
targets of the
b polypeptides
and the assessment of their functional
significance in the pathogenicity
process.

Generation

Note added in proof

of new alleles

The issue remains of how this intricate

system of
multiallelic
recognition
has evolved in nature. This
can be addressed by considering
how alleles with new
specificity can be generated. A new allele must behave
in accordance with the following rule: the bE and bW
proteins of the new allele must not dimerize with each
other, but in combination
with all other existing alleles,
at least one active bE-bW
pair should be produced.
To generate bE or bW genes with altered specificities,
few amino acid substitutions
or single recombination
events seem to be sufficient [21,22,23].
This creates
haploid strains expressing a self-interacting
protein pair
through which the pathogenicity
pathway is activated.
To advance from there to a new b allele, it is required that
compensatory
mutations occur in one or both partners
that lead to non-interaction.
At least in the laboratory,
haploid strains with an active bE-bW
complex are
viable and produce spores [13]. Such strains have not
yet been isolated from nature. As strains that carry
an active bE-bW
heterodimer
cannot undergo
cell
fusion [27,28;29*], however, they are excluded from
meiotic recombination
and may thus have a selective
disadvantage.
A second possibility for the generation
of new alleles
is suggested from sequence comparisons of the b genes.
The nucleotide sequences exhibit a patchwork structure
that probably
resulted from multiple
recombination
events [l l-131. If one assumes an initial recombination
event between different alleles, there is a good chance
of creating a chimeric
bE or bW gene that retains
its specificity despite substantial
sequence
exchanges
[20,21]. Such an allele could be maintained
in the
population
without obvious disadvantages.
If we now
assume a second recombination
event in the b gene that
was not engaged in the first recombination,
leading again
to a non-interacting
pair, there is a good chance that the
allele now created has acquired a new specificity (Fig. 3).

Conclusions

A paper published
after the writing
of this review
shows that the process of self/non-self
discrimination
of homeodomain
proteins in the mushroom
Coprinus
cinereus also involves heterodimerization
of compatible
partner proteins [3 l].

Acknowledgements
This
the

W;LF supported

Leibniz

by

through

grants

from

the

IIeutsche

SFU 190, SFU 369 and through

program.

References and recommended

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Kahmann,

Romeis,
M %lker
and J Kimper,
lnstitut
Mikrobiologie
der Universitat
Miinchen,
la, 80638 Miinchen,
Germany.

for correspondence:
R Kahmann.
uj4421O@sunmail.lrz-muenchen.dr