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Molecular genetics of
P. infestans: Transformation
Our laboratory has pioneered the development of transformation procedures for Phytophthora, starting in the early 1990s. By "transformation" we mean the introduction of DNA into an organism, for the purpose of expressing a novel gene, expressing higher levels of a native gene, or silencing the expression of a native gene.
One application of transformation in P. infestans.
Compared to some "model" organisms like S. cerevisiae and N. crassa, transformation technologies for P. infestans are still at an early stage. However, we have been making rapid progress over the last few years and transformation has become a routine tool for studying the biology of P. infestans and relatives. Transformants can be obtained using several approaches, such as treating protoplasts with DNA or by microprojectile bombardment.
Protocol for transforming P. infestans by microprojectile bombardment.
Three drug-resistance selectable markers exist, and visible markers such as -glucuronidase (GUS) and green fluorescent protein (GFP) are available. Expression vectors are developed, cotransformation occurs at high efficiency (>90%), and transgene stability is documented. We have assisted many laboratories in establishing P. infestans transformation in their own facilities, and helped others to successfully transform other oomycetes including P. palmivora, P. parasitica, P. porri, P. sojae, and Saprolegnia.
One current application of transformation in our laboratory is to fuse the promoter of a gene of interest to a reporter gene such as GUS; by staining for GUS activity, we can learn where the promoter is being expressed.
Transformant expressing a gene formed by fusing a promoter from a sporulation-induced gene with the GUS marker gene (blue color).
Gene silencing is another tool that we employ. By introducing sense or antisense copies of a P. infestans gene into a transformant, frequently the expression of the native gene can be silenced (i.e. homology-dependent transcriptional silencing). This can be used to test the function of a gene. Recently, we systematically tested several variables associated with silencing in order to optimize the method.
The Inf1 gene was silenced using either sense, antisense, or hairpin constructs, which were introduced into transformants using protoplast, electroporation, or bombardment methods.
Analysis of transformants silenced for bZIP. A, Use of RT-PCR to identify silenced strains in transformants containing sense or antisense copies of the bZIP transcription factor. RT-PCR with actin primers was used as a control (not shown). B, Traces from movies of zoospores from representative silenced and non-silenced controls, representing zoospore movement over 4 seconds. C, interpretation of normal and wild type swimming behaviors.
Another use of transformation is the expression of proteins fused to fluorescent protein tags, such as green fluorescent protein.
Cysts of strains expressing the green, cyan, and yellow fluorescent proteins fused to a protein phosphatase.
Another example of a technology that we are developing for oomycetes are inducible promoters, which can allow us to express genes in transformants at will.
Example of vector used for doxycycline-inducible expression.
Induction of reporter gene using the tet-on system.
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