|Polygalacturonase-inhibiting proteins (PGIPs) have been demonstrated to play a role in host defense in several plants. The PGIP now cloned from strawberry (Fragaria × ananassa) showed a high degree of homology to other fruit PGIPs. Seven different variants of the gene were identified in the five cultivars studied. The gene expression of strawberry PGIP was monitored in healthy leaves, flowers and fruit at different maturity stages. PGIP transcript levels were also analysed following fruit inoculation with the fungal pathogen Botrytis cinerea in strawberry cultivars displaying variation in susceptibility. Healthy mature berries showed the highest constitutive PGIP gene expression levels compared with leaves, flowers and immature fruit, indicating that the gene is developmentally regulated. One FaPGIP allele was preferentially expressed in leaf tissue, while two other alleles showed a fruit-specific expression pattern. Of the five cultivars (Elsanta, Korona, Polka, Senga sengana, Tenira) studied, Polka had the highest constitutive expression, whereas the PGIP genes were induced at a high level following B. cinerea infection in all cultivars. This high induction of the PGIP gene after inoculation with B. cinerea indicates that PGIP has a role in defense of strawberry. The upregulation after B. cinerea infection was accompanied by a significant change in FaPGIP allele frequencies as compared to non-treated fruits, and the similarities between the allele frequencies in genomic DNA and in cDNA following B. cinerea infection suggest uniform upregulation of all FaPGIP alleles present as a result of pathogenesis-related stress.
The real-time PCR proved to be the most sensitive assay for quantifying fungal colonisation of B. cinerea in strawberry and it detected the pathogen throughout the whole incubation period. In addition, the expression of six fungal polygalacturonases, Bcpg1-6, and the expression of three host defense genes, PGIP and two class II chitinases, were monitored throughout the incubation period with real-time RT-PCR. None of the fungal polygalacturonases were expressed in the ungerminated conidia, whereas Bcpg1 and 2 were already expressed at 8 hours post inoculation (hpi), at a time when most of the conidia had already germinated. The three host defense genes were all transiently induced by infection; PGIP and one of the chitinases were already induced at a high level at 8 hpi, while all the three genes showed maximum induction at 16 hpi. The possible roles of fungal polygalacturonases in the infection process and as elicitorsof host defense responses are discussed.