Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 257

If yeast mutants defective in the process of interest, and prefer-
entially exhibiting a conditional—usually temperature-sensitive,
but sometimes also pharmacologically enhanced—growth defect,
are available, such as the secretory pathway sec mutants [ 2 ], expres-
sion of a functionally equivalent plant gene may result in comple-
mentation of the mutant defect [ 3 ]. While even close sequence
similarity between genes from different organisms does not guar-
antee successful complementation, failure to complement may be
used as a starting point for identifi cation of functionally important
parts of the protein of interest by mutagenesis and/or construc-
tion of chimeric proteins and their subsequent testing for comple-
mentation in yeast [ 4 ].
While dead yeast is, on the fi rst glance, not an encouraging
starting point, even lethal loss-of-function mutations can be com-
plemented, since a lethal allele can be maintained in heterozygous
diploids. Introduction of a plasmid carrying a wild-type allele of
the gene in question into such a heterozygote will allow subse-
quent isolation of haploids carrying the loss-of-function mutation
and kept alive by the plasmid, which then cannot be lost.
Complementation of the mutation by a second plasmid carrying
the heterologous gene would enable plasmid loss, which can be
easily followed if the fi rst plasmid carries a marker affecting colony
color [ 5 ]. Since use of this technique includes isolation of haploids
by tetrad analysis, it goes beyond the scope of methods usually
available in a plant laboratory, and we recommend collaboration
with a specialized yeast laboratory.
In the absence of mutants suitable for complementation, novel
phenotypes may be elicited by heterologous expression of proteins
interfering with the intrinsic cellular machinery. If such phenotypes
result in a growth defect, they can be rescued by co-expression of
a second heterologous protein interacting with the fi rst one [ 6 ].
Complementation, phenotype induction, and rescue of induced
phenotypes can be used either for testing already available candi-
date plant clones preselected, e.g., on the basis of homology with
their yeast counterparts (i.e., in test mode), or for screening cDNA
libraries to isolate novel cDNAs (screen mode).
Especially in screening experiments, additional verifi cation of
the identifi ed clones by plasmid rescue from yeast and repeated
transformation of the initial yeast strain is crucial. An outline of a
general experimental strategy is shown in Fig. 1 .

2 Materials and Equipment

Besides standard equipment for molecular biology (pipettes, incuba-
tors and shakers for microbial cultures, an autoclave, a laminar fl ow
cabinet, a microcentrifuge, a vortex, a low-velocity centrifuge for
larger volumes, a spectrophotometer, a refrigerator, freezers,