Growth, Differentiation and Sexuality

314 R. Debuchy and B.G. Turgeon
B. Functional Analysis of MAT by Heterologous
Expression
1. Conversion of a Self-Incompatible to a Self-
Compatible Strain
To determine whether MAT genes alone can control
reproductive style, a sterile C. heterostrophus MATdeletion
strain was transformed with a construct
carrying the fused C. luttrellii MAT1-1-1; MAT1-2-1
gene (Yun et al. 1999). Transformants were selfed,
and crossed to albino C. heterostrophus MAT1-1
and MAT1-2 tester strains. Abundant pseudothecia
formed when the transformants were selfed or
crossed, most of which were fertile (1%–10% of
wild-type ascospore production). Pseudothecia
and progeny from selfs were pigmented, whereas
approximately half the pseudothecia and half the
progeny from crosses were albino, indicating that
self-incompatible C. heterostrophus expressing a
MAT from a self-compatible species can both self
and outcross. Thus, the C. luttrellii MAT1-1-1;
MAT1-2-1 gene alone conferred selfing ability
to normally self-incompatible C. heterostrophus,
without impairing its ability to cross. A similar
experiment was done with the C. homomorphus
MAT1-2-1; MAT1-1-1 gene. In this case, transformants
were able to make pseudothecia, but
these were barren. Since the only difference in the
C. luttrellii and C. homomorphus experiments is
the MAT gene itself (the genetic background of
C. heterostrophus is otherwise constant), we can
conclude that differences in these MAT sequences
determine fertility.
2. Conversion of a Self-Compatible to a Self-
Incompatible Species
a) Loculoascomycete Conversion
In a reverse of the above experiment, the C.
luttrellii MAT1-1-1; MAT1-2-1 gene was deleted
(Lu and Turgeon, unpublished data), rendering
the strain completely sterile. This strain was
then transformed, separately, with a MAT1-1-1
or MAT1-2-1 gene from self-incompatible C.
heterostrophus, yielding a MAT-deleted C. luttrellii
strain carrying C. heterostrophus MAT1-1-1, and
a MAT-deleted C. luttrellii strain carrying C.
heterostrophus MAT1-2-1. When these strains were
crossed, it was found that:
1. A C. luttrellii transgenic strain carrying
ChetMAT1-1-1 and a C. luttrellii transgenic
strain carrying ChetMAT1-2-1 can mate in
a self-incompatible manner, and the fertility
ofthecrossissimilartothatofawild-typeC.
luttrellii self.
2. A C. luttrellii transgenic strain carrying
ChetMAT1-1-1 can mate with the parental
wild-type C. luttrellii MAT1-1;MAT1-2 strain,
indicating that the latter is able to outcross,
a result that was expected but has not been
demonstrated previously.
3. Each transgenic C. luttrellii strain is also able
to self, although all pseudothecia produced are
smaller than those of wild type and fertility is
low (number of asci was about 5%–10% that
of the wild type, and full tetrads were found).
No recombinants were found in ascospores isolated
from these tiny pseudothecia, demonstrating
that all sexual structures originated
from a self.
ThesedatasupporttheargumentthatinCochliobolus
spp., and perhaps other Ascomycetes also, the
primary determinant of reproductive mode is MAT
itself, and that a self-incompatible strain can be
made self-compatible, or a self-compatible strain
canbemadeself-incompatiblebyexchangeofMAT
genes. This bolsters the argument that a change in
reproductive lifestyle is initiated by a recombination
event. The ability to self, observed in transgenic
C. luttrellii strains generated in this study,
also suggests that both MAT1-1-1 and MAT1-2-1
proteins of Cochliobolus spp. carry a set of equivalent
transcription regulatory activities capable of
promoting sexual development alone, in a suitable
genetic background.
b) Sordariomycete Conversion
In a parallel study, Lee et al. (2003) demonstrated
that G. zeae (MAT1-1;MAT1-2), a species that is naturally
able to self and also outcross, can be made
obligately self-incompatible by targeted deletion of
either the MAT1-1 or MAT1-2 sequence(s). Strains
(mat1-1;MAT1-2 and MAT1-1;mat1-2, respectively)
with these deletions are sterile – no perithecia are
formed when selfed. Subsequent crossing of MAT1-
1;mat1-2 by mat1-1;MAT1-2 strains results in fertile
perithecia. This experiment should be contrasted
with the Cochliobolus experiment, in which deletion
of the entire MAT locus from self-compatible
C. luttrellii results in sterility, but adding back a single
MAT gene from self-incompatible C. heterostrophus
can activate the full mating process. Reasons
forthis difference may reside inthe structures ofthe