The dimension of the plants largest organelle—the vacuole—plays a major role in defining cellular elongation rates. The morphology of the vacuole is controlled by the actin cytoskeleton, but molecular players remain largely unknown. Recently, the Networked (NET) family of membrane-associated, actin-binding proteins has been identified. Here, we show that NET4A localizes to highly constricted regions of the vacuolar membrane and contributes to vacuolar morphology. Using genetic interference, we found that deregulation of NET4 abundance increases vacuolar occupancy, and that overexpression of NET4 abundance decreases vacuolar occupancy. Our data reveal that NET4A induces more compact vacuoles, correlating with reduced cellular and organ growth in Arabidopsis thaliana.

Cell division and cell elongation are fundamental processes for growth. In contrast to animal cells, plant cells are surrounded by rigid walls and therefore loosening of the wall is required during elongation. On the other hand, vacuole size has been shown to correlate with cell size and inhibition of vacuolar expansion limits cell growth. However, the specific role of the vacuole during cell elongation is still not fully resolved. Especially the question whether the vacuole is the leading unit during cellular growth or just passively expands upon water uptake remains to be answered. Here, we review recent findings about the contribution of the vacuole to cell elongation. In addition, we also discuss the connection between cell wall status and vacuolar morphology. In particular, we focus on the question whether vacuolar size is dictated by cell size or vice versa and share our personnel view about the sequential steps during cell elongation.

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish on living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish on cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-ASSOCIATED KINASE 1 (BAK1) and SUPPRESSOR OF BIR1-1 (SOBIR1) Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a non-canonical defence response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of ciaR and variants, acetyl phosphate was identified in vivo as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of ciaH and the acetate kinase gene ackA led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of S. pneumoniae cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the Escherichia coli two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent in vitro CiaR phosphorylation experiments confirmed in vivo observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in S. pneumoniae or even in other organisms.

The dimension of the plants largest organelle -the vacuole, plays a major role in defining cellular elongation rates. The morphology of the vacuole is controlled by the actin cytoskeleton but the mechanistic connection between them remains largely elusive. Recently, the NETWORKED (NET) family of membrane-associated, actin-binding proteins has been identified and represent potential candidates to impact on vacuolar morphology. Here, we show that NET4A localizes to highly constricted regions in the vacuolar membrane and contributes to the compactness of the vacuole. Using genetic interference, we found that deregulation of NET4 abundance impacts on vacuole morphogenesis and overexpression leads to more compact vacuoles. We moreover show that the NET4A-induced changes in vacuolar shape correlates with reduced cellular and organ growth in Arabidopsis thaliana. Our results demonstrate that NET4 modulates the compactness of vacuoles and reveal higher complexity in the regulation of actin-reliant vacuolar morphology. Results and Discussion