Fungal toxin fusicoccin enhances plant growth by upregulating 14-3-3 interaction with plasma membrane H+-ATPase

doi:10.21203/rs.3.rs-3159720/v1

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Fungal toxin fusicoccin enhances plant growth by upregulating 14-3-3 interaction with plasma membrane H+-ATPase

https://doi.org/10.21203/rs.3.rs-3159720/v1

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Fusicoccin-A (FC-A) is a diterpene glucoside produced by a pathogenic fungus. FC-A is phytotoxic and induces stomatal opening and leaf wilting, eventually leading to plant death. Here, we report that FC-A enhances plant growth by stabilizing the protein-protein interaction between plasma membrane (PM) H⁺-ATPase and 14-3-3 in guard cells, promoting stomatal opening and thus photosynthesis. Long-term treatment of Arabidopsis plants with FC-A resulted in ~ 30% growth enhancement. Structurally similar fusicoccin-J (FC-J) showed a similar degree of growth-promotion activity as FC-A, whereas the more hydrophilic fusicoccin-H (FC-H) exhibited no effect on plant growth, indicating that the enhancement of plant growth observed with FC-A and FC-J involves upregulation of the protein-protein interaction between PM H⁺-ATPase and 14-3-3 in guard cells, which promotes stomatal opening and photosynthesis.

Biological sciences/Chemical biology/Natural products

Biological sciences/Plant sciences

Methods to increase the efficiency of food crop production are urgently needed in order to address the unprecedented demand placed on global food supplies brought about by continued population growth¹. In plant leaves, the stoma is a pore in the epidermis surrounded by a pair of guard cells that alternately swell and contract to regulate stomatal opening and closure. Stomatal opening facilitates the diffusion of CO₂ and O₂ gas as well as water vapor, which is essential for photosynthesis and transpiration². Chemical approaches for manipulation of stomatal opening may therefore hold tremendous promise for enhancing photosynthesis and plant growth, thereby increasing biomass and reducing CO₂production. Intensive research has focused on identifying compounds that induce phenotypic changes in stomata³, stomatal closure⁴ and opening⁵,alteration of stomatal density^6,7, and perturbation of asymmetric division⁸. However, the whole-plant effects of such compounds on growth remain to be evaluated.

Fusicoccin-A (FC-A, Fig. 1A) is a 5-8-5 tricyclic diterpene glucoside produced by the plant pathogenic fungus Phomopsis amygdali (syn. Fusicoccum amygdali)⁹. This fungal metabolite is known for its phytotoxicity, which promotes continuous stomatal opening resulting in over-transpiration, wilting of leaves, and eventual tree death^10–12. In the proposed molecular mechanism of action, FC-A stabilizes the protein-protein interaction (PPI) between plasma membrane (PM) H⁺-ATPase and 14-3-3 by forming a stable ternary complex in which FC-A binds to a hydrophobic cavity of the binary complex formed by the binding of 14-3-3 to the phosphorylated C-terminus of PM H⁺-ATPase¹³ (Fig. S1A). Appropriate cycling between association and dissociation of PM H⁺-ATPase and 14-3-3 in guard cells is critical for the regulation of stomatal movement in response to various signals, such as light^14,15. PM H⁺-ATPase consists of 10 transmembrane helices, with the N- and C-termini located on the cytoplasmic side of the plasma membrane¹⁶. The C-terminus of PM H⁺-ATPase possesses a 14-3-3–binding motif and acts as an autoinhibitory domain¹⁷. Photoexcitation of blue-light receptor phototropins triggers phosphorylation of the penultimate threonine (Thr) residue, and subsequent binding of 14-3-3 to the phosphorylated motif dissociates the inhibitory domain, thereby activating the enzyme^17–19. Activated PM H⁺-ATPase then releases protons and generates an inside-negative electrical potential across the plasma membrane, which in turn drives the accumulation of K⁺ through PM inward-rectifying K⁺ (K⁺_in)-channels²⁰, resulting in swelling of guard cells and subsequent stomatal opening. As a consequence of FC-A–mediated stabilization of the PPI, PM H⁺-ATPase becomes overactivated, thus promoting abnormal stomatal opening^21–25.

The 14-3-3 proteins are a family of dimeric phospho-binding proteins that play central roles in the regulation of a diverse array of physiological processes mediated by serine/threonine kinases and phosphatases^26,27. Each 14-3-3 monomer possesses an amphiphilic binding groove ~25 Å in length that recognizes a phospho-consensus motif containing either phosphoserine or phosphothreonine²⁸. The 14-3-3 proteins are highly conserved and expressed in all eukaryotic cells, and hundreds of phosphorylated binding partners have been identified in plants²⁹ and humans²⁷. A study examining the crystal structure of the ternary complex (Fig. S1A) of tobacco 14-3-3c bound to FC-A and the PM H⁺-ATPase C-terminal phosphopeptide Gln-Ser-Tyr-pThr-Val (QSYpTV) demonstrated that FC-A binds in the hydrophobic cavity adjacent to the peptide, resulting in an increase in the affinity of both ligands for 14-3-3 of almost 2 orders of magnitude¹³. This cooperative binding to 14-3-3 is driven by hydrophobic interactions between the 5-8-5 fusicoccin skeleton and the isopropyl side chain of the C-terminal Val residue of the phospholigand, engaging significant van der Waals contacts. Analyses of the crystal structure revealed that FC-A forms a number of intermolecular interactions with the phospholigand and 14-3-3 (Fig. S1B): 1) the 12-hydroxy group forms a hydrogen bond with the C-terminal carboxylate of QSYpTV via a water molecule; 2) the 16-methoxy group varies in a narrow hydrophobic pocket composed of Phe126 and Met130; and 3) the 6’-pentenyl group binds to a hydrophobic surface region consisting of Leu50, Glu19, and Val45. This unique mode of PPI stabilization has prompted many researchers, including our group, to utilize FC-A not only for plant physiological studies but also for the development of new pharmaceuticals to treat various human diseases^30,31.

In 2014, Kinoshita and colleagues reported that overexpression of PM H⁺-ATPase specifically in guard cells of Arabidopsis thaliana promotes light-induced stomatal opening and photosynthesis³², resulting in significant enhancement of growth of the transgenic plants³³. In contrast, overexpression of phototropin or K⁺_in-channels in guard cells had no effect on growth, indicating that the number of PM H⁺-ATPase molecules (i.e., the enzyme activity in guard cells) determines the efficiency of photosynthesis. Importantly, the stomata of the transgenic plants were shown to close normally in response to darkness and abscisic acid, which is essential for normal plant growth. These results prompted us to address the following question: If plants are supplied with sufficient light and water to trigger photosynthesis and avoid drought, would treatment of plants with phytotoxic FC-A, which stabilizes the PPI between PM H⁺-ATPase and 14-3-3 and thus induces stomatal opening, in turn enhance plant growth? We tested this paradoxical hypothesis by leveraging structure-activity-relationship studies using FC-A and its biosynthetic intermediates fusicoccin-J (FC-J)^34,35 and fusicoccin-H (FC-H)³⁶ (Fig. 1A). Similar to FC-A, FC-J retains critical functionalities at the 12, 16, and 6’ positions that are required for stable complex formation, whereas FC-H lacks all three functional groups. We therefore anticipated that FC-J would stabilize the PM H⁺-ATPase–14-3-3 interaction, thereby resulting in a biological effect similar to that of FC-A, whereas FC-H would be less effective because it is potentially a weaker stabilizer. Here, we report that the phytotoxin FC-A and its biosynthetic intermediate FC-J (but not FC-H) enhance plant growth by upregulating the interaction between 14-3-3 and PM H⁺-ATPase to induce stomatal opening and promote photosynthesis.

Promotion of stomatal opening by fusicoccin

First, chemical induction of stomatal opening was evaluated by directly spraying the compound on the surface of A. thaliana leaves in light prior to harvesting cotyledons at various time intervals for measurement (Fig. 1B). As previously reported^25,37, FC-A significantly promoted stomatal opening, with openings ~ 3 times larger than those of control plants at 6 h (Fig. 1B). Similarly, FC-J was also effective, but FC-H was apparently less potent than FC-A, indicating that the stomatal opening effect varies depending on the FC chemical structure. Infrared thermal images of Arabidopsis indicated a decrease in the temperature of the leaves after a droplet of FC-A was placed on the surface (Fig. S2), further supporting the apparent promotion of stomatal opening by FC-A in whole plants.

Stabilizing effect of fusicoccins on interaction between 14-3-3 and PM H⁺-ATPase

Next, we examined whether FCs stabilize the interaction between PM H⁺-ATPase and 14-3-3 in plants. Arabidopsis leaves were treated with each compound, and protein samples were then extracted and subjected to co-immunoprecipitation using an anti–PM H⁺-ATPase antibody; the relative amount of bound 14-3-3 was analyzed by Western blotting (Fig. 1C). The intensity of bands corresponding to 14-3-3 increased significantly in the presence of FC-A, FC-J, and FC-H, respectively, compared with the control, indicating that each FC stabilizes the complex formed by the binding of PM H⁺-ATPase to 14-3-3 (Fig. 1C). Notably, FC-H was less effective than FC-A, which was consistent with their observed effect in promoting stomatal opening. In addition, the level of PM H⁺-ATPase phosphorylation increased markedly in the presence of FC-A and FC-J, but not in the presence of FC-H (Fig. 1D), indicating that PM H⁺-ATPase activity is upregulated by stabilization of the interaction with 14-3-3, in which FC-H exhibited only a weak effect.

In order to quantitatively evaluate the effect of FCs in stabilizing the PM H⁺-ATPase–14-3-3 interaction, fluorescence polarization titration experiments in the presence/absence of FCs were performed using recombinant Arabidopsis 14-3-3φ and a fluorescein (FAM)-labeled phosphopeptide (FAM-TPSHYpTV) derived from the C-terminus of AHA2, a subtype of Arabidopsis PM H⁺-ATPase (Fig. 1E). Significant enhancement of the affinity of the peptide for 14-3-3 (K_d = 0.89 ± 0.19 µM) was observed in the presence of FC-A (K_d = 0.15 ± 0.03 µM) and FC-J (K_d = 0.16 ± 0.03 µM), whereas only weak affinity was observed in the presence of FC-H (K_d = 0.53 ± 0.89 µM). These results clearly show that the degree of upregulation of stomatal opening is correlated with the degree of stabilization of the PM H⁺-ATPase–14-3-3 PPI.

Transgenic Arabidopsis plants expressing superfolder GFP–AHA2 were prepared and subjected to fluorescent imaging analysis using BODIPY-TR reagents (Fig. S3). When epidermal tissues were treated with a simple fluorescent BODIPY-TR derivative (1, scheme S1), clear localization of AHA2 at the PM in guard cells was observed (Fig. S3, lane 2), whereas 1 was distributed in the cytosol and formed fluorescent puncta (Fig. S3, lane 3). This result was presumably due to the preferential accumulation of BODIPY dye in peroxisomes, as previously reported³⁸. On the other hand, the FC-attached BODIPY-TR derivative (2, scheme S1) showed a different cytosolic distribution than 1, forming much fewer puncta and localizing near the PM. These results support the PM localization of FC, which allows for interaction with AHA2 in guard cells.

Effect of fusicoccin-A on promotion of photosynthesis

We next examined whether FC-A improves the efficiency of photosynthesis. Intact leaves of four- to six-week-old Arabidopsis plants were sprayed twice with FC-A (30 µM) at 3 h and 2 h prior to irradiation with white light (600–700 µmol·m^− 2·s^− 1), and changes in the CO₂ assimilation rate and stomatal conductance were monitored over time using a gas-exchange system LI-6400XT (LI-COR). Both the CO₂ assimilation rate (Fig. 2A) and stomatal conductance (Fig. 2B) increased significantly in FC-A–treated plants compared with control plants, especially at the beginning of irradiation, indicating that FC-A enhances photosynthetic activity by promoting stomatal opening.

Effect of fusicoccins on plant growth

Based on results showing that treating plants with FC-A promotes photosynthesis, we examined the crucial question of whether a long-term FC-A treatment enhances plant growth. Arabidopsis plants were sprayed with various concentrations of FC-A (0.3–30 µM) once daily for 15 days and cultivated under conditions providing a sufficient supply of water and white light (80–100 µmol·m^− 2·s^− 1). The plants were then harvested and analyzed. Strikingly, FC-A treatment resulted in an apparent enhancement of plant growth by 15 days compared with control plants (Fig. 3A). Plants treated with FC-A at 3 to 30 µM exhibited a significant enhancement in the size of rosette leaves, and the fresh and dry weight of plants treated with 30 µM FC-A was ~ 30% greater than that of control plants (Fig. 3B, p < 0.05). The observed growth enhancement was dose dependent (Figs. 3B and S4). Importantly, FC-J also promoted plant growth, but FC-H did not (Figs. 3C and S5). These results were consistent with the ability of the compounds to activate PM H⁺-ATPase and induce stomatal opening.

To determine whether the increase in biomass was truly associated with the promotion of stomatal opening and upregulation of photosynthesis triggered by FC-A, the δ¹³C value of Arabidopsis plants was determined after FC-A treatment and cultivation. Higher CO₂ assimilation efficiency is known to result in a decline in the ¹³C/¹²C (δ¹³C) accumulation ratio because plants discriminate against ¹³C in favor of ¹²C during photosynthesis³⁹. Indeed, the chemically treated plants showed significantly lower δ¹³C values than nontreated controls (Fig. 3D), further confirming that FC-A–induced chemical activation of PM H⁺-ATPase in guard cells enhances plant growth, leading to increased biomass.

Changes in the effect of fusicoccin-A over time

A general concern with treatments to chemically control stomatal movement is whether the agents are degraded or inactivated in plants over time, such that the plants recover normal stomatal movement after a certain period of time post-treatment. A previous study showed that constitutive activation of PM H⁺-ATPase in guard cells of Arabidopsis results in constant opening of stomata, which does not enhance growth³². This suggests that stomatal closing at night is crucial when attempting to enhance plant growth by inducing stomatal opening. In order to determine whether the promotion of stomatal opening by FC-A is transient, leaves of Arabidopsis plants were treated with various amounts of FC-A, grown under normal conditions, and then detached from the plants. The change in weight of each detached leaf due to the upregulated transpiration caused by FC-A was then monitored and compared with the rate of weight loss of leaves from nontreated control plants. We anticipated that the rate of weight loss would decline if FC-A was degraded or inactivated in the leaf. When plants were incubated with FC-A for 12 h prior to detachment of leaves, the weight of chemically treated leaves declined significantly faster than that of control leaves in a dose-dependent manner (Figs. 4A and S6). However, the rate of weight loss of leaves from plants treated with FC-A for a longer period (24 h) declined, with no apparent difference compared with the control detectable by 48 h. These results indicate that FC-A remains effective and promotes transpiration on leaves for 12 to 24 h but becomes ineffective by 48 h under the conditions examined in this study. Consistent with these results, time-course analyses indicated an apparent increase in the adaxial stomatal aperture (not in the abaxial stomatal aperture) of Arabidopsis rosette leaves by 12 h after FC-A treatment, and the significant difference disappeared by 48 h (Figs. 4B and S7). These results indicate that the stomatal opening induced by FC-A is transient, presumably due to the inactivation of FC-A in the plant leaves.

Effect on growth of other dicots

Finally, the potential of FC-A to enhance the growth of other dicots was investigated. Japanese mustard spinach plants (Komatsuna; Brassica rapa var. perviridis, a member of the same family as Arabidopsis) were treated with FC-A once daily and cultivated in a hydroponic system in an outfield greenhouse for 28 days prior to harvesting and analysis. Treatment with FC-A resulted in a notable enhancement of plant growth, and the biomass of both fresh and dry plants increased significantly in a concentration-dependent manner up to 48 pmol·plant^− 1·day^− 1 (Figs. 5 and S8A), indicating that FC-A also promotes the growth of Komatsuna plants. It should be noted that the size and number of leaves decreased at a dose of 480 pmol·plant^− 1·day^− 1 (Fig. 5), and partially wilted leaves were observed (Fig. S8B). These results suggest that FC-A is toxic to Komatsuna plants at high doses.

In the 60 years since its discovery, fungal toxin FC-A has been widely studied as a potent phytotoxin that promotes continuous stomatal opening and over-transpiration, thereby eventually leading to plant death. In this work, we found that FC-A stabilizes the PPI between 14-3-3 and PM H⁺-ATPase in guard cells, promoting stomatal opening and photosynthesis, thus resulting in approximately 30% enhancement of growth in Arabidopsis plants. A decrease in the carbon isotope ratio (δ¹³C) in chemically treated plants further supports the observation that the increase in biomass is due to the chemical activation of PM H⁺-ATPase in guard cells.

A structure-activity relationship study using FC-A and its biosynthetic intermediates FC-J and FC-H showed that the structurally similar FC-A and FC-J stabilize the binding of the phosphorylated C-terminal peptide of PM H⁺-ATPase to 14-3-3 by approximately 6-fold in terms of K_d value as compared with controls (Fig. 1E), enhance the PPI in plants (Fig. 1C), and enhance stomatal opening by approximately 200% (Fig. 1B). In contrast, hydrophilic FC-H was apparently less effective in both enhancement of the PPI and stomatal opening compared with FC-A and FC-J, indicating that the chemical functionality at the 12, 16, and 6’ positions is important for the activity. These results are consistent with those of a previous study showing that FC-A stabilizes the binding of human 14-3-3σ to the TASK-3 C-terminal phosphopeptide 10 times more effectively than FC-H⁴⁰. The X-ray crystal structure of the ternary complex of 14-3-3σ bound to the TASK-3 phosphopeptide and FC-A showed that the 16-methoxy group of FC-A binds to a narrow hydrophobic pocket, anchoring the whole molecule to 14-3-3σ in a similar manner to that seen in the structure of tobacco 14-3-3 bound to FC-A (Fig. S1). In the case of the ternary complex of FC-H, which lacks the O-methyl group (3ux0), a highly defined water molecule was found to occupy this position⁴⁰, suggesting that binding of the O-methyl group to the pocket squeezes out the water molecule, thus achieving entropically favored binding to 14-3-3. Indeed, whole-plant evaluations demonstrated that FC-A and FC-J enhance the growth of Arabidopsis plants to a similar degree, whereas FC-H is less effective (Fig. 3C). These results confirmed that the enhancement of plant growth is caused by enhancement of the PPI between 14-3-3 and PM H⁺-ATPase by FCs in guard cells.

It remains unclear whether FC-A reversibly or irreversibly upregulates stomatal opening in whole plants. Earlier studies based on in vitro evaluations concluded that FC-A irreversibly activates PM H⁺-ATPase^19,41. In these studies, upregulation of the binding of 14-3-3 to PM H⁺-ATPase was evaluated by surface plasmon resonance spectroscopy¹⁹ or by submerging guard-cell protoplasts⁴¹ in a buffer solution containing a relatively high concentration of FC-A (10 µM). In the present study, a sub-nmol amount of FC-A was applied to plant leaves, and at least under these conditions, the effect of FC-A on stomatal opening was found to be transient, being active up to 24 h and becoming ineffective by 48 h (Fig. 4). These results strongly suggest that inactivation of FC-A occurs in the plant leaves. At least two α-glucosidase genes and more than 40 β-glucosidase genes have been identified in Arabidopsis^42,43. Thus, metabolic degradation and inactivation involving deglycosylation are possible, as removal of the glucoside moiety from FC-A reduces its apparent affinity for 14-3-3 by approximately one order of magnitude⁴⁰.

Most importantly, the activity of FC-A in terms of plant growth enhancement found in this work suggests that the promotion of stomatal opening is not the sole cause of plant death and that there are other unelucidated mechanisms underlying the phytotoxicity of FC-A. Extensive studies have shown that FC-A affects a number of physiological processes in plants^12,30, including the induction of abscission^44,45, proton extrusion⁴⁶, cell enlargement and cotyledon growth⁴⁷, and seed germination⁴⁸. These activities are often explained by the “acid-growth theory”⁴⁹, in which overactivation of PM H⁺-ATPase by FC-A results in cell-wall acidification, followed by activation of expansin, which loosens the cellulose network for growth. The diverse activities of FC-A may also reflect the wide range of physiological roles of PM H⁺-ATPase⁵⁰ as well as 14-3-3 signaling networks in plants⁵¹. For instance, in Arabidopsis, PM H⁺-ATPase is encoded by 12 genes⁵², and 13 isoforms of 14-3-3 (10 characterized and 3 putative isoforms) have been identified that are expressed in many organelles in addition to the cytoplasm, such as chloroplasts⁵³ and the nucleus⁵⁴. Other studies have shown that 14-3-3 plays roles in a diverse array of physiological processes, including nitrogen metabolism⁵⁵, ATP synthesis⁵⁶, potassium ion diffusion⁵⁷, flowering⁵⁸, and hormone-driven transcriptional regulation⁵⁹. The protein sequences of the 14-3-3 isoforms are highly conserved, and the residues involved in binding to FC-A are identical across all of the isoforms (Fig. S9); thus, FC-A could potentially stabilize any 14-3-3 interaction if the binding partner includes an appropriate consensus motif capable of forming a ternary complex with FC-A. Our present results, therefore, do not exclude any additional mechanism(s) underlying the enhancement of growth triggered by FC-A. Further research will be necessary to elucidate more details regarding the mechanism leading to growth enhancement as well as the molecular basis of the phytotoxicity of FC-A.

In the present work, we found that the fungal toxin FC-A and its biosynthetic intermediate FC-J enhance plant growth. This effect can be attributed to stabilization of the PPI between 14-3-3 and PM H⁺-ATPase in guard cells, which promotes stomatal opening and photosynthesis. This unexpected property should stimulate further research examining potential agricultural applications of FC-A and FC-J.

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Yes there is potential Competing Interest. J. Ohkanda, H. Kiriyama, S. Kasuga, H. Irieda, and T. Kinoshita (JP patent 2022-132203).

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Fungal toxin fusicoccin enhances plant growth by upregulating 14-3-3 interaction with plasma membrane H+-ATPase

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Abstract

Figures

Main text

Results

Promotion of stomatal opening by fusicoccin

Stabilizing effect of fusicoccins on interaction between 14-3-3 and PM H⁺-ATPase

Effect of fusicoccin-A on promotion of photosynthesis

Effect of fusicoccins on plant growth

Changes in the effect of fusicoccin-A over time

Effect on growth of other dicots

Discussion

Declarations

Online content

References

Additional Declarations

Supplementary Files

Status:

Version 1

Fungal toxin fusicoccin enhances plant growth by upregulating 14-3-3 interaction with plasma membrane H+-ATPase

Status:

Version 1

Abstract

Figures

Main text

Results

Promotion of stomatal opening by fusicoccin

Stabilizing effect of fusicoccins on interaction between 14-3-3 and PM H+-ATPase

Effect of fusicoccin-A on promotion of photosynthesis

Effect of fusicoccins on plant growth

Changes in the effect of fusicoccin-A over time

Effect on growth of other dicots

Discussion

Declarations

Online content

References

Additional Declarations

Supplementary Files

Status:

Version 1

Stabilizing effect of fusicoccins on interaction between 14-3-3 and PM H⁺-ATPase