Growth Assay for the Stem Parasitic Plants of the Genus Cuscuta.

Cuscuta spp. are widespread obligate holoparasitic plants with a broad host spectrum. Rootless Cuscuta penetrates host stems with so called haustoria to form a direct connection to the host vascular tissue ( Dawson et al., 1994 ; Lanini and Kogan, 2005; Kaiser et al., 2015 ). This connection allows a steady uptake of water, assimilates and essential nutrients from the host plant and therefore enables Cuscuta growth and proliferation. To quantify the parasites' ability to grow on potential host plants one can use the quantitative growth assay ( Hegenauer et al., 2016 ) described herein, which exclusively utilizes fresh weight measurement as readout.

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease.

Subtilisin-like serine proteases (SBTs) are extracellular proteases that depend on their propeptides for zymogen maturation and activation. The function of propeptides in plant SBTs is poorly understood and was analyzed here for the propeptide of tomato subtilase 3 (SBT3PP). SBT3PP was found to be required as an intramolecular chaperone for zymogen maturation and secretion of SBT3 in vivo Secretion was impaired in a propeptide-deletion mutant but could be restored by co-expression of the propeptide in trans SBT3 was inhibited by SBT3PP with a Kd of 74 nm for the enzyme-inhibitor complex. With a melting point of 87 °C, thermal stability of the complex was substantially increased as compared with the free protease suggesting that propeptide binding stabilizes the structure of SBT3. Even closely related propeptides from other plant SBTs could not substitute for SBT3PP as a folding assistant or autoinhibitor, revealing high specificity for the SBT3-SBT3PP interaction. Separation of the chaperone and inhibitor functions of SBT3PP in a domain-swap experiment indicated that they are mediated by different regions of the propeptide and, hence, different modes of interaction with SBT3. Release of active SBT3 from the autoinhibited complex relied on a pH-dependent cleavage of the propeptide at Asn-38 and Asp-54. The remarkable stability of the autoinhibited complex and pH dependence of the secondary cleavage provide means for stringent control of SBT3 activity, to ensure that the active enzyme is not released before it reaches the acidic environment of the trans-Golgi network or its final destination in the cell wall.

Parasitic Cuscuta factor(s) and the detection by tomato initiates plant defense.

Dodders (Cuscuta spp.) are holoparasitic plants that enwind stems of host plants and penetrate those by haustoria to connect to the vascular bundles. Having a broad host plant spectrum, Cuscuta spp infect nearly all dicot plants - only cultivated tomato as one exception is mounting an active defense specifically against C. reflexa. In a recent work we identified a pattern recognition receptor of tomato, "Cuscuta Receptor 1" (CuRe1), which is critical to detect a "Cuscuta factor" (CuF) and initiate defense responses such as the production of ethylene or the generation of reactive oxygen species. CuRe1 also contributes to the tomato resistance against C. reflexa. Here we point to the fact that CuRe1 is not the only relevant component for full tomato resistance but it requires additional defense mechanisms, or receptors, respectively, to totally fend off the parasite.