SEARCHING A FUNCTION FOR PPI1, AN INTERACTOR OF THE PLASMA
MEMBRANE H+-ATPase, USING INSERTIONAL MUTANTS
Piero Morandini1, Matteo Beretta Piccoli1, Giada Marino1, Chiara Consonni2, Ralph
Panstruga2 and Carlo Soave1
1
Dept. of Biology, University of Milan.
2
Max Planck Institut fuer Zuechtungsforschung, Dept. of Plant Pathogen Interactions, Cologne,
Germany
PPI1 (PROTON PUMP INTERACTOR 1) is a protein identified in a two hybrid screen
whose N-terminus binds to the Arabisopsis thaliana plasma membrane proton pump (PM
H+-ATPase). The entire protein, or fragments thereof, purified as fusion protein from E. coli
are able to stimulate the activity of the proton pump in purified membranes.
To study the function of the PPI1 protein we are using a reverse genetic approach, i.e.
identifying and characterizing Arabidopsis lines bearing a T-DNA inserted in the gene.
Three T-DNA insertions were identified (and confirmed by sequencing of the amplified
genomic DNA) in the available mutagenized collections (http://www.tmri.org or
http://signal.salk.edu) for Ppi1: an insertion in intron I, one in exon VI at aa 525 and one in
the promoter region (-235 from the transcription start site). For Ppi2, a gene with 52%
similarity to Ppi1, we identified insertions at aa34 and 550 (also confirmed by sequencing).
Homozygous plants for the various insertions were identified and back-crossed with the
wt Col-0. A double mutant ppi1,ppi2 was also generated by crossing individual knockouts. RT-PCR analysis on single insertion lines confirmed the transcript is either absent or
aberrant for insertions within both genes, while the insertion in the promoter of Ppi1 did
not affect the mRNA structure, as seen by RT-PCR.
Western blotting on homozygous knock-out plants are being performed to confirm that
also the protein is aberrant or absent for the insertions within the gene. Both the single and
the double mutant did not show evident phenotype when grown in pots. The single
mutants have been exposed to several pathogens (powdery mildew from barley, wheat
and Arabidopsis as well as Pseudomonas syringae), to wounding without showing
differences in respect to wt plants, both on the macroscopic or microscopic level (staning
with DAB or Trypan blue). Different growth conditions are being tested for identifying
phenotypic differences to wt.