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Five new genes belonging to the pathogenesis-related (PR) 4 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 very high sequence identity with  wPR4f-b  and constituting a new sub-family. Transcription start analysis was performed by RLM-RACE, leading to the isolation of a fifth gene, named  wPR4g , that is highly homologous to  wPR4e ; both encode putative vacuolar PR4 proteins (Wheatwin7 and Wheatwin5, respectively).  wPR4e  and  wPR4f  sub-family genes are induced by  F. culmorum  infection, by chemicals that lead to systemic acquired resistance and by wounding, showing different spatial and temporal induction pathways.  In silico  analysis of the 5′ untranslated regions of  wPR4e  and  wPR4f-b  revealed the presence of several abiotic and biotic stress-responsive elements.  wPR4e  and  wPR4f-b  putative promoters were fused to the β-glucuronidase ( GUS ) reporter gene, and transient and stable expression assays demonstrated that both are able to drive expression of  GUS . Characterisation of these new  PR4  genes and particularly of their 5′ untranslated regions, as well as the determination of their expression patterns, will contribute to our understanding of the responsiveness of this gene family to various stress conditions and of its role in plant defence.

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