Fig. 10

Studies of sporulation-induced protein kinase and and interactor Gβ-like protein. a Two-hybrid analysis showing tests of interaction between full-length kinase (PKS1), kinase catalytic domain (PKS1c; amino acids 135 to 466), Gβ-like protein (GβL), and canonical Gβ. An interaction between PKS1c and GβL is indicated by the strong growth in the center upper panel of the right culture plate. b Co-immunoprecipitation of GβL with PKS1c, showing input and co-precipitated amounts. c Expression patterns of PKS1 and GβL in mycelia grown on minimal media containing casamino acids (MY min) or rye media (MY rye), sporangia (SP), sporangia undergoing zoosporogenesis (CL), zoospores (ZO), germinated cysts (GC), and tomato leaves at 2 to 5 dpi (Tom). d Northern blot of selected transformants containing GβL silencing construct, using the gene for elongation factor 1-α (EF1) as a loading control. The ratio of GβL and EF1 relative to wild type is shown below the blots. Transformants T1, T2, and T6 show levels of GβL equivalent to wild-type, while T4 and T5 are knocked-down by four to five-fold. e RT-qPCR of wild-type strain 1306 with T4 and T5, using primers for GβL (PITG_09556) and flanking gene PITG_09555, which encodes a predicted ubiquitin-ribosomal protein fusion. This confirms the knockdown and indicates that flanking genes are not co-silenced. f Relative growth of 1306, control transformants, and T4 and T5 knockdown (KD) transformants on rye media (rich media), Plich media (semidefined), and in planta. The control transformants (CONT) are the average of non-silenced transformant T1 and two strains expressing the GUS transgene. Growth on media was determined on a dry weight basis, while growth in tomato was determined by qPCR using primers for a high-copy DNA locus [72]. Values are adjusted to 1306 equals 1.0