title: A transposable element insertion in the susceptibility gene CsaMLO8 results in hypocotyl resistance to powdery mildew in cucumber.
abstract: BACKGROUND
  Powdery mildew (PM) is an important disease of cucumber (Cucumis sativus L.). CsaMLO8 was previously identified as a candidate susceptibility gene for PM in cucumber, for two reasons: 1) This gene clusters phylogenetically in clade V, which has previously been shown to harbour all known MLO-like susceptibility genes for PM identified in dicot species; 2) This gene co-localizes with a QTL on chromosome 5 for hypocotyl-specific resistance to PM.
  
  
  METHODS
  CsaMLO8 alleles from susceptible and resistant cucumber were cloned and transformed to mlo-mutant tomato. Cucumber seedlings were inoculated with Podosphaera xanthii, tissues were studied for CsaMLO8 expression at several timepoints post inoculation using qRT-PCR. The occurrence of the observed loss-of-function allele of CsaMLO8 in resequenced cucumber accessions was studied in silico.
  
  
  RESULTS
  We cloned CsaMLO8 alleles from susceptible and resistant cucumber genotypes, the latter carrying the QTL for hypocotyl resistance. We found that insertion of a non-autonomous Class LTR retrotransposable element in the resistant genotype leads to aberrant splicing of CsaMLO8 mRNA. Heterologous expression of the wild-type allele of CsaMLO8 in a tomato mlo-mutant restored PM susceptibility. However, heterologous expression of the CsaMLO8 allele cloned from the resistant cucumber genotype failed to restore PM susceptibility. Furthermore we showed that inoculation of susceptible cucumber with the PM pathogen Podosphaera xanthii induced transcriptional upregulation of CsaMLO8 in hypocotyl tissue, but not in cotyledon or leaf tissue. This coincides with the observation that the QTL at the CsaMLO8-locus causes full resistance in hypocotyl tissue, but only partial resistance in cotyledons and true leafs. We studied the occurrence of the loss-of-function allele of CsaMLO8 in cucumber germplasm by an in silico approach using resequencing data of a collection of 115 cucumber accessions, and found that this allele was present in 31 out of 115 accessions.
  
  
  CONCLUSIONS
  CsaMLO8 was characterised as a functional susceptibility gene to PM, particularly in the hypocotyl where it was transcriptionally upregulated upon inoculation with the PM pathogen P. xanthii. A loss-of-function mutation in CsaMLO8 due to the insertion of a transposable element was found to be the cause of hypocotyl resistance to PM. This particular allele of CsaMLO8 was found to occur in 27 % of the resequenced cucumber accessions.
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University & Research centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. jeroen.berg@wur.nl.
authors.forename: Jeroen A
authors.initials: JA
authors.lastname: Berg
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University & Research centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. michela.appiano@wur.nl.
authors.forename: Michela
authors.initials: M
authors.lastname: Appiano
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University & Research centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. miguel.santillanm@gmail.com.
authors.forename: Miguel
authors.initials: M
authors.lastname: Santillán Martínez
authors.affiliation: Bayer Crop Science Vegetable Seeds, P.O. Box 4005, 6080 AA, Haelen, The Netherlands. freddy.hermans@bayer.com.
authors.forename: Freddy W K
authors.initials: FW
authors.lastname: Hermans
authors.affiliation: Bayer Crop Science Vegetable Seeds, P.O. Box 4005, 6080 AA, Haelen, The Netherlands. wim.vriezen@bayer.com.
authors.forename: Wim H
authors.initials: WH
authors.lastname: Vriezen
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University & Research centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. richard.visser@wur.nl.
authors.forename: Richard G F
authors.initials: RG
authors.lastname: Visser
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University & Research centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. yuling.bai@wur.nl.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Wageningen UR Plant Breeding, Wageningen University & Research centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. henk.schouten@wur.nl.
authors.forename: Henk J
authors.initials: HJ
authors.lastname: Schouten
mesh_terms: D000483:Alleles; D001203:Ascomycota / Q000502:physiology; D001483:Base Sequence; D003001:Cloning, Molecular; D018553:Cucumis sativus / Q000235:genetics* / Q000382:microbiology*; D004251:DNA Transposable Elements / Q000235:genetics*; D060467:Disease Resistance / Q000235:genetics*; D005816:Genetic Complementation Test; D018745:Genome, Plant; D005838:Genotype; D018546:Hypocotyl / Q000235:genetics / Q000382:microbiology*; D018551:Solanum lycopersicum / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology*; D018515:Plant Leaves / Q000382:microbiology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D004251:DNA Transposable Elements; D010940:Plant Proteins