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  1. Author Response

    Reviewer #3 (Public Review):

    In this manuscript, authors are reporting identification of a compound, VAC1, which affects localization of vacuolar SNARE proteins in Arabidopsis root cells. Authors then examined the effect of VAC1 on other markers, and found that VAC1 affects localization/transport of plasma membrane proteins and FM4-64 but not early and late endosomal proteins, CLC, and actin microfilaments. Authors then examined the effects of VAC1 on expansion of the cell and vacuole and endocytic transport, based on which they concluded that endocytic transport from the PM to vacuole is enhanced during cell elongation, which could coordinate expansion of surface areas of the cell and vacuole.

    It is firmly demonstrated in this work that VAC1 treatment resulted in abnormal localization of vacuolar SNARE proteins. In pictures presented in Figure 2C, vacuolar SNAREs seem to be accumulating in aster-like structures. Given that the SNARE proteins are membrane-anchored proteins, the SNARE-positive aster-like structures should be membranous structures. However, the lipophilic dye FM4-64 does not seem to reach the SNARE-positive spikes, accumulating inside the structure. It would be helpful to understand which step is affected by VAC1 more precisely if the detailed structure of the VAC1 body is investigated, e.g., by TEM.

    We followed your suggestions and utilized electron microscopy to compare VAC1 and DMSO (solvent control) treated epidermal root cells. Thereby, we revealed that VAC1 induces accumulations of vesicles in the proximity of the vacuoles. Moreover, we observed morphological alterations of the vacuole, which appear to correlate to the mentioned “aster-like” structure. We added this set of data to the revised manuscript (see Figure 2).

    It would be also informative to test whether non-SNARE vacuolar proteins are also affected by VAC1 to see the effect of VAC1 is specific to SNARE proteins or not.

    We followed your suggestion and addressed the VAC1 effect on VHAa3-GFP, a commonly used tonoplast marker. VAC1 affected the distribution of VHAa3-GFP and the tonoplast marker was seemingly not excluded from the region of highest SNARE protein accumulation. We included this set of data into the revised version of this manuscript.

    Related to the comment above, in some figures (e.g., Figure 3E), VAC1 bodies seem to be located inside the vacuole. It would be good to explain whether this consistent with the proposed mode of action of VAC1.

    We also got the impression that VAC1 bodies are not only found near the vacuole, but possibly also inside the vacuole. We hypothesize that secondary effects, such as autophagic processes, could lead to “VAC1 body” clearance. Accordingly, we assume that VAC1 could possibly guide researchers to address SNARE-dependent and SNARE independent autophagy in plants.

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  2. Evaluation Summary:

    Plant cells can grow to extraordinarily large volumes; Arabidopsis root cells, for example, can expand beyond 50um long. Vacuole expansion is correlated with cell elongation, presumably to "fill up" the volume of the cell without requiring a tremendous volume of cytoplasm. Here, the authors carefully characterize a new small molecule inhibitor of endocytic trafficking to the vacuole. This new tool will be valuable to researchers studying endocytic trafficking and vacuole biogenesis in plants.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Plant vacuole expansion is correlated with cell elongation, presumable to fill the space within the cell and to limit the volume of cytoplasm required in mature, elongated plant cells, such as root cells. Previous work from this lab has shown that vacuole reshaping via actin and myosin interactions plays a role in defining vacuole size and that auxin can regulate cell size via posttranslational regulation of SNAREs.

    The authors conducted a screen of small bioactive molecules with known effects on plant cells (Drakakaki et al 2011) and isolated VAC1, a compound that causes aggregation of vacuolar SNARES (VAMP711, VTI11, SYP22) in an unknown compartment and changes to vacuolar morphology (Fig 2). The authors use HPLC-MS to analyze VAC1 metabolism in seedlings (Supplemental Figure 2) and test a few VAC1 analogues (Supplemental Figure 3).

    Using a variety of fluorescent subcellular markers, the authors find that endocytic trafficking to the vacuole is disrupted by VAC1 treatment, resulting intracellular in signal aggregation, rather than localization to the tonoplast (Fig 3). Localization of clathrin-mediated endocytosis machinery and TGN/early endosome markers are qualitatively unaffected by VAC1. However, treatment with BFA and WM decrease VAC1 aggregate signal, implying that the affected compartments are upstream of (early endosome) or upstream/parallel to (late endosome) the site of VAC1 action. Since different vacuolar SNAREs act in heterotypic (endosome-derived) and homotypic (vacuole-derived) fusion, the authors test whether affecting either of these pathways using inducible amiRNA lines that target these different complexes. The vps3 amiRNA lines were slightly resistant to VAC1, implying that VAC1 primarily targets endosome-derived vesicle fusion with the vacuole. These results imply that endocytosis is unaffected by VAC1, but that a later step in endocytic trafficking is affected, before cargo reaches the vacuole.

    The authors hypothesize that endocytic trafficking from the cell surface to the vacuole might be involved in vacuolar expansion during elongative growth. In agreement with this, they found a correlation between intensity of FM4-64 uptake (representing endocytosis) to VAC1 bodies, BFA bodies, or the tonoplast and cell elongation. This correlation could not be explained simply by the larger surface are of elongating cells (Figure 5). They also found that VAC1 temporarily disrupted this vacuole growth and elongative growth (although it recovers at later stages), further confirming that VAC1 targets endocytic trafficking to the vacuole (Fig 6). Although it is difficult to interpret FM4-64 tracking experiments quantitatively across different samples, these data imply that endocytic trafficking to the vacuole is correlated with cell expansion. Although the authors present these results as a paradox, it is unsurprising that increased endocytic trafficking is correlated with cell expansion from the perspective of cell wall secretion, since the volume of the cell wall must increase much more than the surface area of the plasma membrane during elongative growth.

    Overall, this work carefully characterizes a small molecule that disrupts endocytic trafficking to the vacuole and places it within the molecular context of vacuolar trafficking, which will make this a useful tool to many researchers studying endocytic trafficking and vacuole biogenesis in plants.

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  4. Reviewer #2 (Public Review):

    Dünser et al. investigate how membrane delivery to the vacuole affects cellular growth in the model plant Arabidopsis thaliana. The starting hypothesis of this paper is that interference with vacuolar N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein function would specifically allow addressing the importance of membrane delivery to the vacuole for overall growth. A chemical screen was deployed to look for small compounds affecting SNARE accumulation and vacuolar morphology. Several vacuolar affecting compounds (VACs) were identified and the authors focused on one of these, VAC1, which induced an ectopic accumulation of the vacuolar SNARE complex adjacent to the main vacuole. Structure activity relationship analysis revealed that the compound itself, rather than its degradation or conversion products, was responsible for the observed cellular effect and that several chemical groups of VAC1 were essential for its function. Using cell biological, genetic and pharmacological tools, the authors conclude that VAC1 interferes with SNARE-dependent vesicle fusion events at the tonoplast. Application of VAC1 allowed the authors to identify a differential rate of membrane internalization between elongating and meristematic root cells. Quantification of plasma membrane and tonoplast surface area revealed to be closely balanced during rapid cellular expansion and VAC1 quickly disturbed this balance, while at the same time, this correlated with diminished root growth.

    Strengths:
    -The authors report here on a very original and novel concept in plant endomembrane trafficking and the experiments performed support their hypotheses.
    -The authors performed a clever initial screen to identify vacuolar affecting compounds via accumulation of GFP-tagged SNARE proteins, followed by detailed confocal analysis of the effects of these compounds to in the end focus on a single compound for in depth analysis, which included a very detailed analysis of its degradation and conversion products as well as its derivatives to rule out that the observed effects were non-specific to the administered compound.
    -The subcellular imaging is of very high quality and overall the quantifications are rigorously performed and visualized with adequate statistical analysis.

    Weaknesses:
    -Although the authors conclude that VAC1 likely affects SNARE-dependent membrane fusion at the tonoplast, its molecular target(s) remain unknown. Mechanistic insight into the observed effects therefore remains to be determined.
    -Support for the absence of any adverse effects of VAC1 on endocytic trafficking and actin dynamics could be more optimally be achieved using dynamic imaging.
    -The correlation between enhanced endocytic trafficking and cellular expansion, which is a key finding of this manuscript, could be further supported by the differential internalization of integral membrane proteins instead of membrane dyes.

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  5. Reviewer #3 (Public Review):

    In this manuscript, authors are reporting identification of a compound, VAC1, which affects localization of vacuolar SNARE proteins in Arabidopsis root cells. Authors then examined the effect of VAC1 on other markers, and found that VAC1 affects localization/transport of plasma membrane proteins and FM4-64 but not early and late endosomal proteins, CLC, and actin microfilaments. Authors then examined the effects of VAC1 on expansion of the cell and vacuole and endocytic transport, based on which they concluded that endocytic transport from the PM to vacuole is enhanced during cell elongation, which could coordinate expansion of surface areas of the cell and vacuole.

    It is firmly demonstrated in this work that VAC1 treatment resulted in abnormal localization of vacuolar SNARE proteins. In pictures presented in Figure 2C, vacuolar SNAREs seem to be accumulating in aster-like structures. Given that the SNARE proteins are membrane-anchored proteins, the SNARE-positive aster-like structures should be membranous structures. However, the lipophilic dye FM4-64 does not seem to reach the SNARE-positive spikes, accumulating inside the structure. It would be helpful to understand which step is affected by VAC1 more precisely if the detailed structure of the VAC1 body is investigated, e.g., by TEM. It would be also informative to test whether non-SNARE vacuolar proteins are also affected by VAC1 to see the effect of VAC1 is specific to SNARE proteins or not.

    Related to the comment above, in some figures (e.g., Figure 3E), VAC1 bodies seem to be located inside the vacuole. It would be good to explain whether this consistent with the proposed mode of action of VAC1.

    Was this evaluation helpful?