Facultative symbiont virulence determines horizontal transmission rate without host specificity in Dictyostelium discoideum social amoebas.

In facultative symbioses, only a fraction of hosts are associated with symbionts. Specific host and symbiont pairings may be the result of host-symbiont coevolution driven by reciprocal selection or priority effects pertaining to which potential symbiont is associated with a host first. Distinguishing between these possibilities is important for understanding the evolutionary forces that affect facultative symbioses. We used the social amoeba, Dictyostelium discoideum, and its symbiont, Paraburkholderia bonniea, to determine whether ongoing coevolution affects which host-symbiont strain pairs naturally cooccur within a facultative symbiosis. Relative to other Paraburkholderia, including another symbiont of D. discoideum, P. bonniea features a reduced genome size that indicates a significant history of coevolution with its host. We hypothesized that ongoing host-symbiont coevolution would lead to higher fitness for naturally cooccurring (native) host and symbiont pairings compared to novel pairings. We show for the first time that P. bonniea symbionts can horizontally transmit to new amoeba hosts when hosts aggregate together during the social stage of their life cycle. Here we find evidence for a virulence-transmission trade-off without host specificity. Although symbiont strains were significantly variable in virulence and horizontal transmission rate, hosts and symbionts responded similarly to associations in native and novel pairings. We go on to identify candidate virulence factors in the genomes of P. bonniea strains that may contribute to variation in virulence. We conclude that ongoing coevolution is unlikely for D. discoideum and P. bonniea. The system instead appears to represent a stable facultative symbiosis in which naturally cooccurring P. bonniea host and symbiont pairings are the result of priority effects.

Diversity of retromer-mediated vesicular trafficking pathways in plants.

The plant endomembrane system is organized and regulated by large gene families that encode proteins responsible for the spatiotemporal delivery and retrieval of cargo throughout the cell and to and from the plasma membrane. Many of these regulatory molecules form functional complexes like the SNAREs, exocyst, and retromer, which are required for the delivery, recycling, and degradation pathways of cellular components. The functions of these complexes are well conserved in eukaryotes, but the extreme expansion of the protein subunit families in plants suggests that plant cells require more regulatory specialization when compared with other eukaryotes. The retromer is associated with retrograde sorting and trafficking of protein cargo back towards the TGN and vacuole in plants, while in animals, there is new evidence that the VPS26C ortholog is associated with recycling or 'retrieving' proteins back to the PM from the endosomes. The human VPS26C was shown to rescue vps26c mutant phenotypes in Arabidopsis thaliana, suggesting that the retriever function could be conserved in plants. This switch from retromer to retriever function may be associated with core complexes that include the VPS26C subunit in plants, similar to what has been suggested in other eukaryotic systems. We review what is known about retromer function in light of recent findings on functional diversity and specialization of the retromer complex in plants.

Facultative symbiont virulence determines horizontal transmission rate without host strain specificity.

In facultative symbioses, only a fraction of hosts are associated with a symbiont. Understanding why specific host and symbiont strains are associated can inform us of the evolutionary forces affecting facultative symbioses. Possibilities include ongoing host-symbiont coevolution driven by reciprocal selection, or priority effects that are neutral in respect to the host-symbiont interaction. We hypothesized that ongoing host-symbiont coevolution would lead to higher fitness estimates for naturally co-occurring (native) host and symbiont combinations compared to nonnative combinations. We used the Dictyostelium discoideum - Paraburkholderia bonniea system to test this hypothesis. P. bonniea features a reduced genome size relative to another Paraburkholderia symbiont of D. discoideum, indicating a significant history of coevolution with its host. Facultative symbionts may experience continued genome reduction if coevolution is ongoing, or their genome size may have reached a stable state if the symbiosis has also stabilized. Our work demonstrates that ongoing coevolution is unlikely for D. discoideum and P. bonniea. The system instead represents a stable facultative symbiosis. Specifically associated host and symbiont strains in this system are the result of priority effects, and presently unassociated hosts are simply uncolonized. We find evidence for a virulence-transmission trade-off without host strain specificity, and identify candidate virulence factors in the genomes of P. bonniea strains that may contribute to variation in benevolence.

A Centrifuge-Based Method for Identifying Novel Genetic Traits That Affect Root-Substrate Adhesion in Arabidopsis thaliana.

The physical presence of roots and the compounds they release affect the cohesion between roots and their environment. However, the plant traits that are important for these interactions are unknown and most methods that quantify the contributions of these traits are time-intensive and require specialist equipment and complex substrates. Our lab developed an inexpensive, high-throughput phenotyping assay that quantifies root-substrate adhesion in Arabidopsis thaliana. We now report that this method has high sensitivity and versatility for identifying different types of traits affecting root-substrate adhesion including root hair morphology, vesicle trafficking pathways, and root exudate composition. We describe a practical protocol for conducting this assay and introduce its use in a forward genetic screen to identify novel genes affecting root-substrate interactions. This assay is a powerful tool for identifying and quantifying genetic contributions to cohesion between roots and their environment.

Synergy among Exocyst and SNARE Interactions Identifies a Functional Hierarchy in Secretion during Vegetative Growth.

Vesicle exocytosis underpins signaling and development in plants and is vital for cell expansion. Vesicle tethering and fusion are thought to occur sequentially, with tethering mediated by the exocyst and fusion driven by assembly of soluble NSF attachment protein receptor (SNARE) proteins from the vesicle membrane (R-SNAREs or vesicle-associated membrane proteins [VAMPs]) and the target membrane (Q-SNAREs). Interactions between exocyst and SNARE protein complexes are known, but their functional consequences remain largely unexplored. We now identify a hierarchy of interactions leading to secretion in Arabidopsis (Arabidopsis thaliana). Mating-based split-ubiquitin screens and in vivo Förster resonance energy transfer analyses showed that exocyst EXO70 subunits bind preferentially to cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distribution and suppressed vesicle traffic similarly to the dominant-negative truncated protein SYP121ΔC, which blocks secretion at the plasma membrane. These phenotypes are consistent with the epistasis of exo70A1 in the exo70A1 syp121 double mutant, which shows decreased growth similar to exo70A1 single mutants. However, the exo70A1 vamp721 mutant showed a strong, synergy, suppressing growth and cell expansion beyond the phenotypic sum of the two single mutants. These data are best explained by a hierarchy of SNARE recruitment to the exocyst at the plasma membrane, dominated by the R-SNARE and plausibly with the VAMP721 longin domain as a nexus for binding.

Micro-scale interactions between Arabidopsis root hairs and soil particles influence soil erosion.

Soil is essential for sustaining life on land. Plant roots play a crucial role in stabilising soil and minimising erosion, although these mechanisms are still not completely understood. Consequently, identifying and breeding for plant traits to enhance erosion resistance is challenging. Root hair mutants in Arabidopsis thaliana were studied using three different quantitative methods to isolate their effect on root-soil cohesion. We present compelling evidence that micro-scale interactions of root hairs with surrounding soil increase soil cohesion and reduce erosion. Arabidopsis seedlings with root hairs were more difficult to detach from soil, compost and sterile gel media than those with hairless roots, and it was 10-times harder to erode soil from roots with than without hairs. We also developed a model that can consistently predict the impact root hairs make to soil erosion resistance. Our study thus provides new insight into the mechanisms by which roots maintain soil stability.

Vacuolar Protein Sorting 26C encodes an evolutionarily conserved large retromer subunit in eukaryotes that is important for root hair growth in Arabidopsis thaliana.

The large retromer complex participates in diverse endosomal trafficking pathways and is essential for plant developmental programs, including cell polarity, programmed cell death and shoot gravitropism in Arabidopsis. Here we demonstrate that an evolutionarily conserved VPS26 protein (VPS26C; At1G48550) functions in a complex with VPS35A and VPS29 necessary for root hair growth in Arabidopsis. Bimolecular fluorescence complementation showed that VPS26C forms a complex with VPS35A in the presence of VPS29, and this is supported by genetic studies showing that vps29 and vps35a mutants exhibit altered root hair growth. Genetic analysis also demonstrated an interaction between a VPS26C trafficking pathway and one involving the SNARE VTI13. Phylogenetic analysis indicates that VPS26C, with the notable exception of grasses, has been maintained in the genomes of most major plant clades since its evolution at the base of eukaryotes. To test the model that VPS26C orthologs in animal and plant species share a conserved function, we generated transgenic lines expressing GFP fused with the VPS26C human ortholog (HsDSCR3) in a vps26c background. These studies illustrate that GFP-HsDSCR3 is able to complement the vps26c root hair phenotype in Arabidopsis, indicating a deep conservation of cellular function for this large retromer subunit across plant and animal kingdoms.

Measuring Plant Protein Secretion.

Fluorescent protein fusions are widely used for visualizing the subcellular localization and mobility of intercellular proteins. There is now a variety of colors, expression vectors, and photoactivated molecules to choose from, each with their own strengths and limitations. In this chapter, the methodologies for expressing and quantifying protein secretion with fluorescent protein fusion constructs using two separate protocols-one in which the retention of a transiently expressed fluorescent marker is measured in seedling roots to quantify a block in secretion, and one in which the secretion of a fluorescent marker into the space of the apoplast is measured to quantify secretion in plant leaves-are described. In the first protocol, seedling roots are transiently transformed with multicistronic constructs; and in the second protocol, markers can be stably expressed and controlled under an inducible promoter in mature plants. Both methods provide tools for quantifying protein secretion and visualizing defects in secretion pathways in Arabidopsis.

Clathrin Heavy Chain Subunits Coordinate Endo- and Exocytic Traffic and Affect Stomatal Movement.

The current model for vesicular traffic to and from the plasma membrane is accepted, but the molecular requirements for this coordination are not well defined. We have identified the hot ABA-deficiency suppressor1 mutant, which has a stomatal function defect, as a clathrin heavy chain1 (CHC1) mutant allele and show that it has a decreased rate of endocytosis and growth defects that are shared with other chc1 mutant alleles. We used chc1 alleles and the related chc2 mutant as tools to investigate the effects that clathrin defects have on secretion pathways and plant growth. We show that secretion and endocytosis at the plasma membrane are sensitive to CHC1 and CHC2 function in seedling roots and that chc mutants have physiological defects in stomatal function and plant growth that have not been described previously. These findings suggest that clathrin supports specific functions in multiple cell types. Stomata movement and gas exchange are altered in chc mutants, indicating that clathrin is important for stomatal regulation. The aberrant function of chc mutant stomata is consistent with the growth phenotypes observed under different water and light conditions, which also are similar to those of the secretory SNARE mutant, syp121 The syp121 and chc mutants have impaired endocytosis and exocytosis compared with the wild type, indicating a link between SYP121-dependent secretion and clathrin-dependent endocytosis at the plasma membrane. Our findings provide evidence that clathrin and SYP121 functions are important for the coordination of endocytosis and exocytosis and have an impact on stomatal function, gas exchange, and vegetative growth in Arabidopsis (Arabidopsis thaliana).

Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in Arabidopsis.

BACKGROUND: The Arabidopsis root hair represents a valuable cell model for elucidating polar expansion mechanisms in plant cells and the overall biology of roots. The deposition and development of the cell wall is central to the root hair expansion apparatus. During this process, incorporation of specific wall polymers into the growing wall architecture constitutes a critical spatio-temporal event that controls hair size and growth rate and one that is closely coordinated with the cell's endomembrane, cytoskeletal and signal transduction apparatuses. RESULTS: In this study, the protocol for live cell labeling of roots with monoclonal antibodies that bind to specific wall polymers is presented. This method allows for rapid assessment of root hair cell wall composition during development and assists in describing changes to cell wall composition in transgenic mutant lines. Enzymatic "unmasking" of specific polymers prior to labeling allows for refined interpretation of cell wall chemistry. Live cell immunofluorescence data may also be correlated with transmission electron microscopy-based immunogold labeling. CONCLUSIONS: Live Arabidopsis root hairs may be labeled with cell wall polymer-specific antibodies. This methodology allows for direct visualization of cell wall dynamics throughout development in stable transgenic plant lines. It also provides an important new tool in the elucidation of the specific interactions occurring between membrane trafficking networks, cytoskeleton and the cell wall deposition/remodeling mechanism.

SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis.

BACKGROUND AND AIMS: Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described. METHODS: Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)-VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy. KEY RESULTS: VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP-VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP-VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells. CONCLUSIONS: These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.

Polysaccharide krestin is a novel TLR2 agonist that mediates inhibition of tumor growth via stimulation of CD8 T cells and NK cells.

PURPOSE: Polysaccharide krestin (PSK) is a mushroom extract that has been long used in Asia and recently in Western countries as a treatment for cancer due to its presumed immune potentiating effects. Although there have been reports of clinical responses after patients have ingested PSK, the mechanism of action of the agent remains undefined. The current study was undertaken to investigate the mechanism of the antitumor actions of PSK. EXPERIMENTAL DESIGN: The immunostimulatory effect of PSK was first evaluated in vitro using splenocytes from neu transgenic mice and Toll-like receptor (TLR) 2 knockout (TLR2(-/-)) mice. Then the immunostimualtory and antitumor effect of PSK was determined using tumor-bearing neu transgenic mice, TLR2(-/-), and wild-type C57BL/6 mice. RESULTS: We demonstrate that PSK is a selective TLR2 agonist, and the activation of dendritic cells (DC) and T cells by PSK is dependent on TLR2. Oral administration of PSK in neu transgenic mice significantly inhibits breast cancer growth. Selective depletion of specific cell populations suggests that the antitumor effect of PSK is dependent on both CD8(+) T cell and NK cells, but not CD4(+) T cells. PSK does not inhibit tumor growth in TLR2(-/-) mice suggesting that the antitumor effect is mediated by TLR2. CONCLUSION: These results demonstrate that PSK, a natural product commonly used for the treatment of cancer, is a specific TLR2 agonist and has potent antitumor effects via stimulation of both innate and adaptive immune pathways.