title: A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens.
abstract: OBJECTIVE
  Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1.
  
  
  RESULTS
  The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants.
  
  
  CONCLUSIONS
  The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species.
  
  
  CONCLUSIONS
  D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started.
authors.affiliation: Department of Genetics, Stellenbosch University, Matieland, South Africa.
authors.forename: M
authors.initials: M
authors.lastname: Visser
authors.forename: D
authors.initials: D
authors.lastname: Stephan
authors.forename: J M
authors.initials: JM
authors.lastname: Jaynes
authors.forename: J T
authors.initials: JT
authors.lastname: Burger
mesh_terms: D060054:Agrobacterium / Q000187:drug effects*; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D042521:Burkholderiaceae / Q000187:drug effects*; D054807:Cecropins; D060467:Disease Resistance; D018274:Electroporation; D005822:Genetic Vectors; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*; D010935:Plant Diseases / Q000382:microbiology / Q000517:prevention & control; D018515:Plant Leaves / Q000235:genetics / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction; D027843:Vitis / Q000235:genetics / Q000382:microbiology*
chemical_list: D000900:Anti-Bacterial Agents; D054807:Cecropins; C443753:D4E1 peptide; D010455:Peptides