Overexpression of a redox-regulated cutinase gene, MfCUTI, increases virulence of the brown rot pathogen monilinia Fructicola on prunus spp

Abstract

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUTl, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUTl carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUTl expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUTl gene expression in cutin-induced cultures, and in the present study, where H2O 2 enhanced MfCUTl gene expression. A β-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUTl under the control of the MfCUTl promoter, and this construct was then used to generate an MfCUTl-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUTl-GUS fusion protein was expressed correctly under the control of the MfCUTl promoter. MfCUTl-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUTl expression within 5 h of inoculation in both almond and peach petals. MfCUTlGUS transformants expressed MfCUTl transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUTl contributing to the virulence of M. fructicola. © 2010 The American Phytopathological Society.