Actin depolymerization is able to increase plant resistance against pathogens via activation of salicylic acid signalling pathway

The integrity of the actin cytoskeleton is essential for plant immune signalling. Consequently, it is generally assumed that actin disruption reduces plant resistance to pathogen attack. Here, we demonstrate that actin depolymerization induced a dramatic increase in salicylic acid (SA) levels in Arabidopsis thaliana. Transcriptomic analysis showed that the SA pathway was activated due to the action of isochorismate synthase (ICS). The effect was also confirmed in Brassica napus. This raises the question of whether actin depolymerization could, under particular conditions, lead to increased resistance to pathogens. Thus, we explored the effect of pretreatment with actin-depolymerizing drugs on the resistance of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae, and on the resistance of an important crop Brassica napus to its natural fungal pathogen Leptosphaeria maculans. In both pathosystems, actin depolymerization activated the SA pathway, leading to increased plant resistance. To our best knowledge, we herein provide the first direct evidence that disruption of the actin cytoskeleton can actually lead to increased plant resistance to pathogens, and that SA is crucial to this process.

establish that SA levels can increase upon actin depolymerization, we measured phytohormone content in A. thaliana seedlings treated with just 200 nM latrunculin B. Such a low concentration of latrunculin B proved sufficient to depolymerize actin filaments in the seedlings within 24 h (Fig. S1). Additionally, we showed that 24 h treatment with latrunculin B does not induce plant cell death (Fig. S2). Significantly, by that time there was a sevenfold increase in the free SA level of the treated seedlings compared with the control ones. The only other phytohormone to display an increase (twofold) was jasmonic acid (JA). Apart from Indole-3-acetamide (IAM), which showed a threefold decrease, the other tested phytohormones remained largely unaltered ( Fig. 1a; Table S1).
Having shown this dramatic rise in SA level in A. thaliana, we wondered which of its two SA biosynthetic pathways was responsible for this increase or whether they both contributed to it. One pathway involves phenylalanine ammonia-lyase (PAL, EC 4.3.1.24), which exists in four isoforms, while the other involves isochorismate synthase (ICS; EC 5.4.4.2), which occurs in two isoforms 18 . Analysis of the transcription of all AtPAL and AtICS genes in the seedlings revealed that only the AtICS genes were induced by latruculin B (Fig. 1b). This shows that drug-induced actin depolymerization activates the ICS-dependent pathway and that this pathway alone is responsible for SA biosynthesis under these conditions. Actin depolymerization leads to induced resistance of A. thaliana against Pst DC3000. Given that increased resistance to pathogens in A. thaliana is associated with SA biosynthesis through the ICS pathway 19,20 , is it possible that activation of the same pathway invoked by drug-induced actin depolymerization also results in increased resistance? To investigate this, we used Ishiga et al. 21 protocol as a basis for performing two in vitro A. thaliana-Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) flood-inoculation assays in liquid and solid media 21 . We treated the seedlings with latrunculin B 24 h before inoculation with Pst DC3000. Remarkably, under both conditions, the latrunculin B-pretreated seedlings were more resistant than the control ones ( Fig. 1c,d,e).
To ensure that this phenomenon is not just associated with in vitro conditions, we also performed experiments using four-week-old A. thaliana plants cultivated in soil, such plants typically being used for studies of A. thaliana resistance to Pst DC3000 22 . Unlike in the seedlings, 24 h treatment with 200 nM latrunculin B did not activate the SA pathway in the adult plants and, thus, no increased resistance was observed ( Fig. 2a,b). However, the transcription of SA marker genes (AtPR-1, AtICS1) was induced after 24 h treatment with 1 µM latrunculin B (Fig. 2a), leading to increased resistance to Pst DC3000 (Fig. 2b). This suggests that plant resistance is strongly dependent on latrunculin B concentration, probably due to differences between the efficiency of latrunculin B-induced actin depolymerization in seedlings and in adult plants (Figs S1 and S3). Similar to latrunculin B, pretreatment with cytochalasin E led to both SA-induced gene transcription (Fig. S4a) and increased plant resistance to Pst DC3000 (Fig. S4b), thereby strengthening the notion that such resistance is due to the depolymerizing activity of cytoskeletal drugs. It should be noted that we exclude the antibacterial effect of latrunculin B because Pst DC3000 grew in vitro in the presence of latrunculin B at a similar rate as in the control medium (Fig. S5).

The induced resistance caused by actin depolymerization is dependent on salicylic acid.
To further demonstrate the dependence of such resistance on the SA pathway, we performed assays using mutants known to have an impaired SA pathway and thus be more susceptible to Pst DC3000: nahG, which induces low endogenous SA levels through the expression of SA-hydroxylase 23 , and sid2, a knock-out mutant of the AtICS1 gene 24 . As expected, latrunculin B did not induce resistance in the nahG plants (Fig. 2d,e). Sid2 plants treated with latrunculin B were more resistant compared to sid2 controls. However, latrunculin B treated sid2 plants were still more susceptible than WT controls (Fig. 2e). The SA level is not induced in sid2 plants (Fig. 2f) which correlates with the fact that none of SA biosynthetic genes does have induced transcription (Fig. S6b). Contrarily in seedlings, ICS2 transcription is induced by latB (Fig. S6a). Altogether these results clearly confirm the crucial role of SA for actin depolymerization-induced resistance. However, increased resistance of latB treated sid2 mutants uncover a new possible unknown SA independent mechanism triggering immunity. www.nature.com/scientificreports www.nature.com/scientificreports/  and mutants with impaired SA pathways (nahG and sid2) were used (d, e, f). Tissue was harvested 3 days after inoculation with Pst DC3000. The values represent mean and error bars (SEM) from four (a,f) and six (b,c,e) independent samples. The asterisks represent statistically significant changes in latB-treated samples compared with controls (**P < 0.01; two tailed Student's t-test) and statistical differences between the samples (d,f) were assessed using a one-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison post hoc test. Different letters indicate a significant difference, Tukey HSD, P < 0.01, n = 6. (2019) 9:10397 | https://doi.org/10.1038/s41598-019-46465-5 www.nature.com/scientificreports www.nature.com/scientificreports/ as with adult A. thaliana, the effect of latrunculin B on B. napus was concentration dependent (Fig. 3a). The increased transcription of BnPR-1 also occurred 72 h after latrunculin B treatment. On the other hand, BnICS1 was not induced, indicating a transient effect of actin depolymerisation on BnICS1 transcription (Fig. S7). The treatment of B. napus with 10 µM latrunculin B 3 days before inoculation with a hemibiotrophic fungal pathogen, L. maculans, efficiently inhibited hyphal colonisation and necrosis formation in the infected cotyledons ( Fig. 3b,c,d). Treatment with 1 µM latrunculin B led to much weaker and variable resistance against L. maculans (Fig. 3b), corresponding to the weaker transcription of defence-related genes (Fig. 3a). These data are in accordance with our previous study characterizing the importance of SA in the defence of B. napus against L. maculans 25 . In addition, we observed significant cytochalasin E-induced resistance to L. maculans in B. napus (Fig. S8), which suggests that the effect is not compound-specific. Furthermore, neither latrunculin B nor cytochalasin E displayed antifungal activity on L. maculans growth in vitro (Fig. S9). Interestingly, the co-inoculation of B. napus cotyledons with a joint solution of 1 and 10 µM latrunculin B and L. maculans conidia also induced resistance (Fig. 3b).

Discussion
Our results indicate that depolymerized actin can trigger resistance to bacterial or fungal pathogens.
Thus, we have shown that plant immunity is strongly activated by depolymerised actin and that this phenomenon appears to be generally valid; namely, it seems not to be species specific, pathogen-type specific or drug-type specific. These findings do not negate those of previous studies that showed the susceptibility of plants treated  www.nature.com/scientificreports www.nature.com/scientificreports/ with cytoskeletal drugs to pathogens [5][6][7]12,13,15 . Rather, they reveal that the plant disease resistance is strongly dependent on whether the plant has sufficient time to activate SA-mediated immunity (Fig. 4). This was clearly shown by our experiments with Pst DC3000, in which pre-infection treatment with cytoskeletal drugs resulted in resistance whilst co-inoculation did not (Fig. 2b,c,e). The co-inoculation of cytochalasin D and Pst DC3000 also had no effect on resistance according to Shimono et al. 11 . Other previous studies using actin-depolymerizing drugs showed higher susceptibility to Pst DC3000 when co-inoculation was used [5][6][7] (Fig. 4). It is also important to mention that actin filaments response to plant immunity is strongly dependent on conditions used in the study. A good example are effects of different MAMPs (flg22 and elf26) on actin reorganization. Using 24 day-old plants infiltrated with MAMPs, Henty-Ridilla et al. 5 showed that treatment with flg22 induces actin reorganization, while elf26 does not 5 . Contradictorily to that, in epidermal cells of hypocotyl grown in the dark, Henty-Ridilla et al. 4 showed that elf26 induces reorganisation and flg22 does not 4 (an explanation could be that under these conditions, FLS2 receptor of flg22 is not expressed). In this study, we excluded the effect of different conditions on induced resistance of A. thaliana against Pst DC3000 by testing three different setups (Figs 1c,d and 2b,e). The result was in all cases similar, whereby pretreatment with latrunculin B induced resistance of A. thaliana against Pst DC3000.
Interestingly, treatment with latrunculin B resulted in increased resistance in both L. maculans setups: pretreatment (Fig. 3b) and co-inoculation (Fig. 3b). This suggests that the rapidity of pathogen growth is a crucial factor. In contrast to Pst DC3000, which strongly damaged the inoculated leaves within three days, almost no multiplication of L. maculans occurred during the same period 25 . Thus, it appears that the slow growth of L. maculans enabled B. napus to establish the SA pathway, which was induced within 24 hours of cytoskeletal drug treatment (Fig. 3a). Overall then, while it is true that plant resistance to pathogens is decreased by a disrupted actin cytoskeleton, our results show that, given sufficient time, plants are able to trigger SA-based defence mechanisms to overcome such threats. This could be due to SA antimicrobial activity, accompanied by the SA-induced production of antimicrobial compounds. These powerful SA properties have been nicely demonstrated in relationship to so-called age-related resistance [26][27][28] . Our study shows that SA pathway, specifically induced by actin depolymerization, is more powerful despite the missing actin dynamics. www.nature.com/scientificreports www.nature.com/scientificreports/ Up to date, some other works suggest the possible positive effect of depolymerization of actin cytoskeleton on plant immunity. Kobayashi and Kobayashi 16 showed that treatment with cytochalins induce NtPR1 transcription in tobacco. Additionally, cytochalasin E primed tobacco cells to induce HR-like cell death in presence of Erysiphe cichoracearum. We can speculate that it could lead to higher resistance against this biotrophic pathogen but it was not explicitly tested 16 . We confirmed the induction of AtPR-1 gene upon treatment with cytoskeletal depolymerizing drugs in A. thaliana 17,29 . Recently it was shown that overexpression of AtPRF3, which leads to depolymerization of actin filaments, increased ROS production upon flg22 treatment 30 . However to our best knowledge, we herein provide the first direct evidence that disruption of the actin cytoskeleton can actually lead to increased plant resistance to pathogens, and that SA is crucial to this process. We strongly believe that our work opens a new and important direction for further research. Is the influence of the actin cytoskeleton on vesicle trafficking involved in SA biosynthesis? For example, when PRRs (pattern recognising receptors) on the plasma membrane recognize MAMPs, it triggers PRRs endocytosis which, in turn, might activate the SA pathway 31,32 . A well characterised example is the internalization of FLS2, which is dependent on the actin-myosin complex 33 . Thus, could an imbalance in PRRs result in constitutively activated immunity and, thereby, induce SA biosynthesis? A hint in support of such a hypothesis is provided by a double mutant with impaired phosphatidylinositol-4-kinase β1 and β2 (pi4kβ1β2), which has been shown to alter vesicle trafficking and constitutively increase SA concentration 34,35 . It is also possible that plants have evolved a system for detecting actin cytoskeleton disruption and that the activation of such a system triggers SA-specific immune responses. However, as yet, we are not able to determine if chemically-depolymerised actin is really the triggering event for immune signalling or whether a pleiotrophic event, such as endoplasmic reticulum stress, results in SA induction. For this reason, further research should be focused on deciphering the specific mechanism by which actin depolymerization triggers SA biosynthesis and the ensuing increased plant resistance to pathogens.

Materials and Methods
Plant material. For the A. thaliana experiments, the following genotypes were used: Columbia-0 (WT); sid2-3 (SALK_042603) 24 ; nahG 23 and pUBC::Lifeact-GFP 36  For the Pst DC3000 resistance assay, the seedlings grown in 24-well plates were treated by replacing the pure liquid MS medium in the plate wells with medium containing 200 nM latrunculin B or 0.01% DMSO control. The seedlings cultivated on the solid medium were treated 24 h by flooding with 10 mL of MS/2 medium containing 200 nM latrunculin B or 0.01% DMSO control. Fully-developed leaves from four-week-old A. thaliana grown in soil were infiltrated either with 200 nM or 1 µM latrunculin B (0.01% or 0.05% DMSO as respective controls) or with 1 µM or 10 µM cytochalasin E (0.025% or 0.25% DMSO as respective controls) 24 h before Pst DC3000 infection using a needleless syringe.
For the transcriptomic assay, the seedlings of A. thaliana grown in 24-well plates were treated 24 h with 200 nM latrunculin B (0.01% DMSO control) or 10 µM cytochalasin E (0.25% DMSO control). Four-week old A. thaliana were infiltrated with 200 nM (0.01% DMSO) or 1 µM latrunculin B (0.05% DMSO) for 24 h. The 10-day-old cotyledons of B. napus were infiltrated either with 1 µM or 10 µM latrunculin B or with 10 µM cytochalasin E (in all cases with corresponding DMSO controls) using a needleless syringe. For infection assay 3 days before infection with L. maculans, for transcriptomic assay 24 and 72 h before harvesting tissue.
Inoculation of A. thaliana seedlings with Pst DC3000. After the A. thaliana seedlings had been cultivated in 24-well plates in the liquid MS medium for 10 days, the cultivation medium was exchanged for one containing latrunculin B or cytochalasin E, and incubated for 24 h. On day 11, the medium was replaced with a bacterial suspension of Pst DC3000 in 10 mM MgCl 2 (OD 600 = 0.01). The seedlings were incubated in this bacterial suspension for 1 min. After incubation, the suspension was replaced with the liquid MS medium. On day 12, the seedlings were harvested, each sample taken containing all of the seedlings from three wells. The seedlings were then homogenized in tubes with 1 g of 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). The resulting homogenate was serially diluted and pipetted onto King B plates. The colonies were counted after 1-2 days of incubation at 28 °C.
www.nature.com/scientificreports www.nature.com/scientificreports/ The seedlings cultivated on solid medium were flooded with 200 nM latrunculin B solution in water on day 13. Control plants were treated with a corresponding solution of DMSO. On day 14, the solutions were replaced with a suspension of overnight culture of Pst DC3000 (OD 600 = 0.01) containing 0.025% Silwet. Samples were harvested at 0, 1 and 2 dpi, with each sample containing the plants from five plates. The seedlings were homogenized in tubes with 1 g of 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). The resulting homogenate was serially diluted and pipetted onto LB plates containing rifampicin. The colonies were counted after 1-2 days of incubation at 28 °C.

Inoculation of four-week-old A. thaliana with Pst DC3000. Pst DC3000 was grown overnight on
King B agar plates at 28 °C, resuspended in 10 mM MgCl 2 , and diluted to an OD 600 of 0.001. Using a needleless syringe, the bacterial suspension was infiltrated into three fully-developed leaves from one plant. After 3 days, the infected tissue was collected as cut leaf discs (one disc per leaf, 0.6-cm diameter); three leaf discs from one plant represent one sample. The discs were homogenized in tubes with 1 g of 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). The resulting homogenate was serially diluted and pipetted onto King B plates. The colonies were counted after 1-2 days of incubation at 28 °C.
After harvesting, conidia obtained according to Šašek et al. 25 were washed once with distilled water, diluted to 10 8 spores/ml, and stored at -20 °C for up to 6 months. The cotyledons of 14-day-old plants were infiltrated by conidial suspension (10 5 conidia/ml), with at least 12 plants being used for each inoculation. The leaves were assessed for lesions 10 days after inoculation. The leaf area and the lesion areas therein were measured by image analysis using APS Assess 2.0 software (American Phytopathological Society, USA). The relative lesion area was then calculated as the ratio of lesion area to whole leaf area. For the microscopy studies, the cotyledons infected with GFP-tagged v23.1.3 isolate 38 were observed at 10 dpi using a Leica DM5000 B microscope.
Gene expression analysis. The whole seedlings from three independent wells were immediately frozen in liquid nitrogen. The tissue was homogenized in tubes with 1 g of 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). Total RNA was isolated using a Spectrum Plant Total RNA kit (Sigma-Aldrich, USA) and treated with a DNA-free kit (Ambion, USA). Subsequently, 1 μg of RNA was converted into cDNA with M-MLV RNase H − Point Mutant reverse transcriptase (Promega Corp., USA) and an anchored oligo dT21 primer (Metabion, Germany). Gene expression was quantified by q-PCR using a LightCycler 480 SYBR Green I Master kit and LightCycler 480 (Roche, Switzerland). The PCR conditions were 95 °C for 10 min followed by 45 cycles of 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 20 s. Melting curve analysis was then conducted. Relative expression was normalized to the housekeeping genes AtSAND and BnTIP41. Primers were designed using PerlPrimer v1.1.21 39 . A list of the analysed genes and primers is available in Table S2.

Phytohormonal analysis.
Hormone analysis was carried out on four samples, each of which contained all seedlings from six of the 24 wells or from the four-week-old A. thaliana three leaf discs from every single plant were sampled, three individual plants were sampled as one sample. Plant hormone levels were determined as described by 40 . Briefly, samples were homogenized in tubes with 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). The samples were then extracted with a methanol/H 2 O/formic acid (15:4:1, v:v:v) mixture, which was supplemented with stable isotope-labeled phytohormone internal standards (10 pmol per sample) in order to check recovery during purification and validate the quantification. The clarified supernatants were subjected to solid phase extraction using Oasis MCX cartridges (Waters Co., USA). The eluates were evaporated to dryness and the generated solids dissolved in 30 μl of 15% (v/v) acetonitrile in water. Quantification was performed on an Ultimate 3000 high-performance liquid chromatograph (Dionex, USA) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, USA) as described by 41 . Metabolite levels were expressed in pmol/g fresh weight (FW).

Confocal microscopy of actin filaments.
For in vivo microscopy, a Zeiss LSM 880 inverted confocal laser scanning microscope (Carl Zeiss AG, Germany) was used with either a 40× C-Apochromat objective (NA = 1.2 W) or a 20x Plan-Apochromat objective (NA = 0.8). GFP fluorescence (excitation 488 nm, emission 489-540 nm) was acquired in z-stacks (20-25 µm thickness). The maximum intensity projections obtained from the z-stacks were created using Zeiss ZEN Black software. Actin filaments density analysis was calculated by Fiji software (https://fiji.sc/) 42 as the percent occupancy of GFP signal in each Maximum intensity projection. Image threshold was set to include all actin filaments and area fraction was measured. We analysed 7-11 cutouts from 6-7 plants for each variant. Representative images were selected from photos from at least 6 independent plants.

Growth of Pst DC3000 and L. maculans in vitro in presence of latrunculin B or cytochalasin E.
Pst DC3000 grew overnight on solid LB medium containing rifampicin. From this, a fresh bacterial suspension was prepared (OD 600 = 0.01) in liquid LB or liquid MS medium. To this suspension, latrunculin B (200 nM or 1 µM) or DMSO (0.05% or 0.01%) was added. The OD 600 was measured 6 and 24 h after suspension preparation. Four independent samples were prepared for each type of treatment.
Conidia of the GFP-tagged v23.1.3 isolate of L. maculans 38 were grown in vitro in Gamborg B5 medium (Duchefa, Netherlands) supplemented with 0.3% (w/v) sucrose and 10 mM MES monohydrate, and adjusted to pH 6.8. This medium contained latrunculin B (1, 10 µM), cytochalasin E (1, 10 µM) or DMSO control (0.5%), and had a final concentration of 2500 conidia per well. The plates (black 96-well plate, Nunc R), covered with lids and sealed with Parafilm ® , were incubated in darkness at 26 °C. On day 4, fluorescence was measured using a Tecan F200 fluorescence reader (Tecan, Switzerland) equipped with a 485/20 nm excitation filter and 535/25 nm emission filter. Eight wells were measured for each treatment.