Abstract
M.Sc.
Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins
that inhibit endopolygalacturonases from phytopathogenic fungi. It has been
proposed that pgip encoding genes could be utilised for engineering increased
resistance in transgenic crops against important fungal pathogens such as Botrytis
cinerea.
During this study a pgip gene from Malus domestica cv Granny Smith apple fruit
was cloned by the degenerate and inverse polymerase chain reaction (PCR)
techniques. An alignment of the pear and bean PGIP sequences was used to design
degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the
amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA
sequence of ipgip was used to design inverse PCR primers. A Southern blot of apple
genomic DNA probed with the ipgip fragment was used to identify restriction
enzyme sites for inverse PCR. Inverse PCR enabled cloning of the remainder of the
gene, from which a composite pgip gene sequence was constructed.
The composite apple pgip gene comprised an open reading frame of 990bp that is
predicted to encode a 330 amino acid polypeptide. The polypeptide contains a
putative 24 amino acid N-terminal leader sequence that may function as a signal
peptide for secretion. The deduced apple PGIP contains nine cysteine residues and
seven potential N-linked glycosylation sites. Ten loosely conserved leucine-rich
repeat motifs characteristic of PG1Ps were identified in the apple PGIP sequence.
The apple PGIP showed 97% and 55% amino acid identity to the pear and bean
PGIPs, respectively.
The full-length apple pgip gene was re-isolated from genomic DNA by PCR using
primers designed to the 5' and 3' ends of the composite pgip gene. The apple pgip
gene was cloned into a plant transformation vector and transformed into tobacco by
Agrobacterium-mediated transformation. Phenotypically normal transgenic tobacco
plants were produced. Stable transgene insertion into the transgenic tobacco
genomes was verified by PCR and Southern blot analyses. Sequence analysis of the
pgip construct used for transformation revealed two potential mutations in the
deduced amino acid sequence. The substitutions of Asp residues with Asn and Tyr at
positions 43 and 196, respectively, could interfere with the secondary structure of the
expressed transgene protein.
To test whether the apple PGIP was effective against Botrytis cinerea, protein
extracts were prepared from apple fruit and transgenic tobacco and tested for
inhibitory activity against B. cinerea polygalacturonases. Biochemical assays showed
that a heat-denaturable PGIP extract prepared from apple fruit inhibited the
polygalacturonases produced by a virulent isolate of Botrytis cinerea grown on
pectin and apple cell walls. Protein extracts prepared from transgenic tobacco did
not show any inhibitory activity towards Botrytis polygalacturonases. This suggests
the absence of active PGIP in the extracts possibly due to inefficient transcription of
the transgene or due to the introduced mutations.