The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response.

Highlights d SOSEKI proteins are deeply conserved polar proteins in land plants d A DIX domain mediates polymerization and polarization of SOSEKI proteins d SOSEKI polymerization allows polar recruitment of an effector protein d DIX-dependent polymerization is shared between animal and plant polarity proteins

The phragmoplast, a plant-specific apparatus that mediates cytokinesis, mainly consists of microtubules (MTs) arranged in a bipolar fashion, such that their plus ends interdigitate at the equator. Membrane vesicles are thought to move along the MTs toward the equator and fuse to form the cell plate. Although several genes required for phragmoplast MT organization have been identified, the mechanisms that maintain the bipolarity of phragmoplasts remain poorly understood. Here, we show that engaging phragmoplast MTs in a bipolar fashion in protonemal cells of the moss Physcomitrella patens requires the conserved MT cross-linking protein MICROTUBULE-ASSOCIATED PROTEIN65 (MAP65). Simultaneous knockdown of the three MAP65s expressed in those cells severely compromised MT interdigitation at the phragmoplast equator after anaphase onset, resulting in the collapse of the phragmoplast in telophase. Cytokinetic vesicles initially localized to the anaphase midzone as normal but failed to further accumulate in the next several minutes, although the bipolarity of the MT array was preserved. Our data indicate that the presence of bipolar MT arrays is insufficient for vesicle accumulation at the equator and further suggest that MAP65-mediated MT interdigitation is a prerequisite for maintenance of bipolarity of the phragmoplast and accumulation and/or fusion of cell plate-destined vesicles at the equatorial plane.

Live-cell imaging of plant-pathogen interactions is often hampered by the tissue complexity and multicell layered nature of the host. Here, we established a novel pathosystem with the moss Physcomitrella patens as host for Phytophthora. The tip-growing protonema cells of this moss are ideal for visualizing interactions with the pathogen over time using high-resolution microscopy. We tested four Phytophthora species for their ability to infect P. patens and showed that P. sojae and P. palmivora were only rarely capable to infect P. patens. In contrast, P. infestans and P. capsici frequently and successfully penetrated moss protonemal cells, showed intracellular hyphal growth and formed sporangia. Next to these successful invasions, many penetration attempts failed. Here the pathogen was blocked by a barrier of cell wall material deposited in papilla-like structures, a defence response that is common in higher plants. Another common response is the upregulation of defence-related genes upon infection and also in moss we observed this upregulation in tissues infected with Phytophthora. For more advanced analyses of the novel pathosystem we developed a special set-up that allowed live-cell imaging of subcellular defence processes by high-resolution microscopy. With this set-up, we revealed that Phytophthora infection of moss induces repositioning of the nucleus, accumulation of cytoplasm and rearrangement of the actin cytoskeleton, but not of microtubules.

SummaryDifferent from animal cells that divide by constriction of the cortex inwards, cells of land plants divide by initiating a new cell wall segment from their centre. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands towards the parental cell wall [1][2][3]. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network [4][5][6]. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly [7][8][9]. How vesicle immobilisation and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell plate formation is lacking. Here we show that membranous material for cell plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4 mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins [10,11], we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis. Results and DiscussionEarly ultrastructural work in the flowering plant Haemanthus katherinae has revealed that microtubules from the two halves of the phragmoplast interdigitate at the midplane where cell plate vesicles aggregate [12]. The same configuration exists in the phragmoplast of the moss Physcomitrella patens [13], indicating that it might be universally relevant for the spatial orchestration of cytokinesis in land plants. We therefore investigated whether regions of antiparallel microtubule overlap could have a direct role in determining the location of cell plate assembly in the genetically amenable moss P. patens ( Figure 1A). For this we imaged the conserved microtubule bundling protein MAP65c labelled with citrine (a GFP variant) as a marker for microtubule overlaps [14][15][16] in combination with the lipophilic dye FM4-64 that stained the membrane of the forming cell plate in life caulonema apical cells ( Figure 1A Whether kinesin-4 has a direct role in division plane reorientation is currently unclear because the underlying process is mechanistically not understood.The mechanism by which kinesin-4 induces overlap shortening was investigated using a purified recombinant N-terminal fragment of moss Kin4-Ic. We observed that it supressed microtubule growth velocities in a dose dependent manner with a minimal effect on catastrophe rate ( Figure S2E-H Having found a way to increase overlap length, we next examined the consequenc...

SummaryFor dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signalling networks including G-protein and phospholipid signalling. Oomycetes possess a family of novel proteins called GPCR-PIPKs (GKs) that are composed of a seven-transmembrane spanning (7-TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G-protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologues in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P. infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7-TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full-length and truncated constructs in P. infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.

The Mycobacterium tuberculosis Beijing genotype, consisting of the more ancient (atypical) and modern (typical) emerging sublineage, is one of the most prevalent and genetically conserved genotype families and has often been associated with multidrug resistance. In this study, we employed a 2D-LC-FTICR MS approach, combined with dimethylation of tryptic peptides, to systematically compare protein abundance levels of ancient and modern Beijing strains and identify differences that could be associated with successful spread of the modern sublineage. The data is available via ProteomeXchange using the identifier PXD000931. Despite the highly uniform protein abundance ratios in both sublineages, we identified four proteins as differentially regulated between both sublineages, which could explain the apparent increased adaptation of the modern Beijing strains. These proteins are; Rv0450c/ MmpL4, Rv1269c, Rv3137, and Rv3283/sseA. Transcriptional and functional analysis of these proteins in a large cohort of 29 Beijing strains showed that the mRNA levels of Rv0450c/MmpL4 are significantly higher in modern Beijing strains, whereas we also provide evidence that Rv3283/ sseA is less abundant in the modern Beijing sublineage. Our findings provide a possible explanation for the increased virulence and success of the modern Beijing sublineage. In addition, in the established dataset of 1817 proteins, we demonstrate the pre-existence of several, possibly unique, antibiotic efflux pumps in the proteome of the Beijing strains. This may reflect an increased ability of Beijing strains to escape exposure to antituberculosis drugs. Molecular & Cellular

The increasing occurrence of multidrug resistant tuberculosis exerts a major burden on treatment of this infectious disease. Thioridazine, previously used as a neuroleptic, is active against extensively drug resistant tuberculosis when added to other second- and third-line antibiotics. By quantitatively studying the proteome of thioridazine-treated Mycobacterium tuberculosis, we discovered the differential abundance of several proteins that are involved in the maintenance of the cell-envelope permeability barrier. By assessing the accumulation of fluorescent dyes in mycobacterial cells over time, we demonstrate that long-term drug exposure of M. tuberculosis indeed increased the cell-envelope permeability. The results of the current study demonstrate that thioridazine induced an increase in cell-envelope permeability and thereby the enhanced uptake of compounds. These results serve as a novel explanation to the previously reported synergistic effects between thioridazine and other antituberculosis drugs. This new insight in the working mechanism of this antituberculosis compound could open novel perspectives of future drug-administration regimens in combinational therapy.