Functional characterization of cucumber ( Cucumis sativus L.) Clade V MLO genes

Background Powdery mildew (PM) causing fungi are well-known pathogens, infecting over 10.000 plant species, including the economically important crop cucumber ( Cucumis sativus L.). Loss-of-function mutations in clade V MLO genes have previously been shown to lead to recessively inherited broad-spectrum resistance to PM in several species. In cucumber, one clade V MLO homolog ( CsaMLO8 ) was previously identified as being a susceptibility factor to PM. Two other closely related homologs ( CsaMLO1 and CsaMLO11 ) were found, but their function was not yet unravelled. Methods CsaMLO1 and CsaMLO11 were cloned from cucumber and overexpressed in a tomato mlo mutant. The transcript abundances of all three CsaMLO genes in different cucumber tissues were quantified using qRT-PCR and RNA-seq, with and without inoculation with the cucumber PM fungus Podosphaera xanthii . Allelic variation of CsaMLO1 and CsaMLO11 was screened in silico in sequenced cucumber germplasm. Results Heterologous overexpression of all three CsaMLO genes in the tomato mlo mutant restored susceptibility to PM caused by Oidium neolycopersici , albeit to a different extent: whereas overexpression of CsaMLO1 or CsaMLO8 completely restored susceptibility, overexpression of CsaMLO11 was only partially able to restore PM susceptibility. Furthermore, it was observed by qRT-PCR and RNA-seq that CsaMLO8 was significantly higher expressed in non-inoculated cucumber compared to the other two MLO genes. However, inoculation with P. xanthii led to upregulation of CsaMLO1 , but not to upregulation of CsaMLO8 or CsaMLO11 . Conclusions Both CsaMLO1 and CsaMLO11 are functional susceptibility genes, although we conclude that based on the transcript abundance CsaMLO8 is probably the major clade V MLO gene in cucumber regarding providing susceptibility to PM. Potential loss-of-function mutations in CsaMLO1 and CsaMLO11 have not been identified. The generation and analysis of such mutants are interesting subjects for further investigation.