Molecular and functional analysis of new members of the wheat PR4 gene family

Five new genes belonging to the PR4 family have been cloned and characterised in Triticum aestivum. Two full length genes, named wPR4e and wPR4f-b, were isolated by library screening demonstrating the presence of a small intron only in wPR4f-b. Two other PR4 genes (wPR4f-a and wPR4f-c) were isolated by PCR showing a very high sequence identity with wPR4f-b and constituting a new sub-family. Transcription start analysis was performed by RLM-RACE leading also to the isolation of a fifth gene, named wPR4g, highly homologous to wPR4e, both encoding putative vacuolar PR4 proteins (Wheatwin7 and Wheatwin5, respectively). wPR4e and wPR4f sub-family genes are induced upon F. culmorum infection, SAR chemical inducer treatments and wounding showing different spatial and temporal induction pathways. In silico analysis of 5' untranslated region of wPR4e and wPR4f-b allowed us to characterise both putative promoter regions in terms of regulative elements revealing the presence of several abiotic and biotic stress-responsive elements. wPR4e and wPR4f-b putative promoters were fused to the ?-glucuronidase (GUS) reporter gene and used in transient and stable expression assays demonstrating that both are able to drive expression of the GUS reporter gene. Characterisation of these new PR4 genes and particularly of their 5' untranslated region as well as the determination of their expression patterns would contribute to the understanding of the responsiveness of this gene family to various stress conditions and of its role in plant defence.