Identification and characterization of the defensin-like gene family of grapevine

Defensins are a class of small and diverse cysteine-rich proteins (CRPs) found in plants, insects and vertebrates, which share a common tertiary structure and usually exert broad-spectrum antimicrobial activities. We used a bioinformatic approach to scan the Vitis vinifera genome and identified 79 defensin-like (DEFL) sequences corresponding to 46 genes and allelic variants, plus 33 pseudogenes and gene fragments. Expansion and diversification of grapevine DEFLs has occurred after the split from the last common ancestor with Medicago and Arabidopsis. Grapevine DEFLs localization on the Pinot Noir genome revealed the presence of several clusters likely evolved through local duplications. By sequencing reverse transcription PCR products we could demonstrate the expression of grapevine DEFLs with no previously reported record of expression. Many of these genes are predominantly or exclusively expressed in tissues linked to the plant reproduction, consistently with findings in other plant species, and that some of them accumulated along fruit ripening. The transcripts of five DEFLs were also significantly upregulated in tissues infected with Botrytis cinerea, a necrotrophic mold, suggesting a role of these genes in defense against this pathogen. Finally, three novel defensins were discovered among the identified DEFLs. They inhibit Botrytis cinerea conidia germination when expressed as recombinant proteins