Ab-GALFA, A bioassay for insect gall formation using the model plant Arabidopsis thaliana

Insect galls are abnormal plant organs formed by gall-inducing insects to provide shelter and nutrients for themselves. Although insect galls are spatialized complex structures with unique shapes and functions, the molecular mechanism of the gall formation and the screening system for the gall inducing effectors remains unknown. Here, we demonstrate that an extract of a gall-inducing aphid, Schlechtendalia chinensis, induces an abnormal structure in the root-tip region of Arabidopsis seedlings. The abnormal structure is composed of stem-like cells, vascular, and protective tissues, as observed in typical insect galls. Furthermore, we confirm similarities in the gene expression profiles between the aphid-treated seedlings and the early developmental stages of Rhus javanica galls formed by S. chinensis. Based on the results, we propose a model system for analyzing the molecular mechanisms of gall formation: the Arabidopsis-based Gall-Forming Assay (Ab-GALFA). Ab-GALFA could be used not only as a model to elucidate the mechanisms underlying gall formation, but also as a bioassay system to isolate insect effector molecules of gall-induction.

Thus, gall-inducing insects secrete effector molecules into plant tissues using their mouthparts or ovipositors to manipulate plant development, thereby generating complex gall structures in host plants 1,2,13 . However, owing to the lack of model symbiotic systems for dissecting the molecular mechanisms of insect gall formation, the effector molecules and mechanisms underlying the gall formation process in plants remain largely unknown.
We hypothesize that the effectors control the developmental pathways in the model plant A. thaliana, as well as in the host plants, since the molecular machinery for development and the developmental pathways are highly conserved among higher plants.
Here, we develop the Arabidopsis-based Gall Formation Assay (Ab-GALFA), an effective method of analyzing the molecular mechanisms underlying gall formation using a model plant, Arabidopsis thaliana. This model system could be used to elucidate the molecular basis for gall formation in plants, and can be utilized as a bioassay for isolating the unknown insect effectors of gall formation in various host plants.

Results
Extract of a gall-inducing insect can alter the morphology of the model plant, A. thaliana. We determined whether treating the seedlings of the model plant, A. thaliana, with an extract of a gall-inducing insect would induce morphological changes in the Arabidopsis tissues. As a first attempt, we tested S. chinensis as a model gall-inducing insect for the following reasons: (i) S. chinensis induces large gall over a short period, (ii) a large volume of clone insect bodies could be available in one gall tissue, (iii) we have previously investigated the expression profile of the early stage of gall (Fig. 1A,B) 6 .
Firstly, morphological changes in 4-day-old Arabidopsis seedlings soaked in various concentrations of the extract of gall-inducing aphid, S. chinensis, (hereafter referred to as Sc extract) were observed via light microscopy after 1 day of treatment (Fig. 1C). We found that several morphologies of the seedlings were significantly changed. For example, the root and elongation zones abnormally thickened, with several thick root hair-like structures that emerged from the root elongation zone ( Fig. 2A and B, Supplemental Movie 1). These morphological changes were not observed following treatment with extracts of the non-galling aphid species Acyrthosiphon pisum (A. p) and Megoura crassicauda (M. c) ( Fig. S1-S5), suggesting that the phenomenon was caused specifically by the extract of the gall-inducing insect.
We then examined the morphological changes that occur at the cellular level. We stained Sc extract-treated Arabidopsis seedlings with propidium iodide (Fig. 2C and D) and measured the vertical and horizontal lengths of the epidermal cells of the root-tip region. The width of the root epidermal cells from the division zone to the transition zone was increased, whereas the length of the root epidermal cells was only increased in the transition zone, when the seedlings were incubated with Sc extract. (Fig. 2E and F). From these results, we concluded that the morphological changes in the root-tip region induced by the the Sc extract were attributed to changes in the wider shape of the root epidermal cells from the division to the transition zones in the root.
The morphological changes in the root epidermal cells following treatment with the Sc extract might have been caused by changes in the cytoskeleton and/or organelle structures. A cytoskeletal structure, cortical microtubules, and the most prominent plant organelle, the central vacuole, are important for the construction of cell shape in plants 19,20 . Therefore, we investigated the mechanism by which the morphology of the cortical microtubules and central vacuoles were altered by the Sc extract treatment. The parallel alignment of the cortical microtubule array visualized using a fluorescent microtubule marker, GFP-MBD 21 . Measurement of the microtubule density of root epidermal cells of root elongation zone indicated that the cortical microtubule array was severely disordered by the Sc extract treatment ( Fig. 3A-C).
Plant vacuoles have a dynamic membrane system that undergoes complex architectural remodeling during developmental process 22,23 . In particular, dynamic morphological remodeling, in which fragmented small vacuoles are fused and form large central vacuolar structures, occurs during the development of mature root epidermal cells from root meristematic cells 24,25 . After treatment with the Sc extract, the bursiform and complex structures of the vacuoles in the root meristematic and elongation zones changed to more fragmented, smaller vacuolar structures (Fig. 3D-G) that resembled the vacuolar structures in root meristematic cells 24 . S. chinensis extract contains significant amounts of phytohormones. Gall-inducing insects are known to produce phytohormones in their bodies and inject them into host plants to induce gall-structure development 13,14,17,[26][27][28] . The application of appropriate combinations and concentrations of exogenous phytohormones can induce gall-like structures in plants 28 . These observations imply that the gall-like structure that emerged in the root region following treatment with the the Sc extract might be caused by exogenous phytohormones from the aphid bodies. Therefore, we measured the concentrations of typical phytohormones in S. chinensis and considerable quantities of abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), jasmonoyl-L-isoleucine (JA-Ile) were detected in addition to the presence of indole acetic acid (IAA), isopentyladenine (iP), and trans-zeatin (tZ) ( Table 1), suggesting that various types of phytohormones can function as insect effector molecules for gall-induction.
Typical characteristics of insect gall-like structures that emerged in the root-tip region after the aphid extract and phytohormone mixture treatments. Typical insect galls consist of three representative tissues: stem cells, vascular, and protective tissues 1,2 . We tested whether these three representative gall tissues (stem cells vascular, and protective tissues) could be observed in the gall-like structures in the root region of the Arabidopsis seedlings after the treatment with the Sc extract or an artificial phytohormone mixture (AHM), which mimicked the content and concentration of the phytohormones in the Sc extract.
As previously reported, several key regulatory molecules in stem cell generation are expressed during the initial stages of gall formation [5][6][7] . Therefore, we used a stem cell niche marker, PLETHORA1 (PLT1) 29 30 to determine whether the structure of the stem cell niche was altered following treatment with the Sc extract or AHM. PLT1-GFP fluorescence was also observed not only in the epidermal cells of the division zone but also of the root transition, elongation and division zones in the Sc extract treatment (Fig. 4). The ectopic expression of PLT1 in the cells of the root-transition and elongation zone was not observed following treatment with the extracts of A. pisum and M. crassicauda, AHM, and a control treatment (water) ( Fig. 4 and Fig. S1). A QC marker, WOX5p::WOX5-GUS-mNeonGreen exhibited similar expression pattern of the PLT1p::PLT1-GFP in root-tip region (Fig. S2). These results indicated that the stem cell niche spread from the root-meristematic region to the root-elongation zones only after the Sc extract treatment.
Next, to investigate the effect of the Sc extract on xylem development, we stained the xylem structure of the Sc extract-treated Arabidopsis seedlings with Auramine O, a fluorescent dye that mainly stains lignin and suberin 31 . Under normal growth conditions, the distance from the root tip to the protoxylem tracheary elements was approximately 1000 µm, whereas the distance after AHM or the Sc extract treatment was shortened to ~ 590 µm and ~ 300 µm, respectively ( Fig. 5A-G), indicating that both the AHM and the Sc extract have xylem development www.nature.com/scientificreports/ activity. Intriguingly, additional lignin deposition was observed in the endodermal cells in the root-transition zone after the Sc extract treatment (Fig. 5E,F).
To further confirm the effects of the Sc extract on xylem vessel formation, segments of the hypocotyl were cultured in xylem vessel-inducing medium 32 containing a 0-10% concentrations of the Sc extract stock solution (tenfold dilution of the stock solution). The frequency of xylem vessel formation was elevated at a 1% Sc extract concentration, and was relatively inhibited at concentrations above 2%. The application of up to 5% Sc extract inhibited tracheary element induction, suggesting that the Sc extract can promote or inhibit xylem vessel formation in a dose-dependent manner ( Fig. 5H and I).
We also confirmed that basic fuchsin-stained lignified tissues, a component of the secondary cell wall, increased more than fourfold after the Sc extract treatment compared with after the AHM treatment ( Fig. 6A and B). Notably, both protoxylem and metaxylem developed into the root elongation zone following the Sc extract treatment, whereas only protoxylem developed after AHM treatment, suggesting that the Sc extract has an ability to induce tracheary elements (Fig. 6B).
Finally, we determined whether the Sc extract could induce secondary cell wall in root-tip region. The secondary cell wall was stained with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA), which binds to  www.nature.com/scientificreports/ the 4-O-methylglucuronic acid of xylan and is an indicator of the secondary cell wall 32 . After the Sc extract treatment, secondary cell wall emerged in the epidermal cells of the root transition and elongation zone; they were not observed in the control, AHM, A. pisum or M. crassicauda extract treatments ( Fig. 6C and D, Fig. S3). Furthermore, we investigated whether the distribution of phytohormones, auxin, and cytokines is altered after the aphid extract treatments using fluorescent reporter lines of auxin (DR5rev::GFP) 33 and cytokines (TCS::GFP) 34 , respectively. Auxin, but not cytokines, was spread following the Sc extract treatment. No changes in the distribution of auxin and cytokines were observed after A. pisum or M. crassicauda extract treatments (Figs. S4 and S5). Collectively, only the Sc extract treatment induced gall-like structures in Arabidopsis seedlings, comprising secondary cell wall formation in the epidermal cells, the enhancement of xylem cell formation, and the dedifferentiation of epidermal and cortical cells in the root region. In contrast, AHM treatment only enhanced protoxylem elongation, suggesting that effectors other than plant hormones act to induce stem cell niche maintenance, secondary cell wall formation, and xylem cell formation.
RNA-seq analyses of aphid extract and hormone mixture treatments. We performed RNA-seq analysis of Arabidopsis seedlings treated with the Sc extract and AHM to determine whether the changes in the expression of Arabidopsis seedlings are induced by only phytohormones or phytohormones plus other effector molecules in the Sc extract. The Sc extract and AHM treatments upregulated 4129 and 3273, differentially expressed genes (DEGs), respectively. A Venn diagram analysis indicated that of these DEGs, 619, 1,024, and 469 were upregulated in the Sc extract treatment, AHM treatment, and commonly upregulated in these treatments, respectively. In contrast, 770, 1,175, and 356 DEGs were downregulated in the Sc extract treatment, AHM treatment, and commonly downregulated in both treatments, respectively. We performed a gene ontology (GO) enrichment analysis on the commonly upregulated DEG sets in Sc and AHM treatments and found that the upregulated DEGs were categorized into Hormone (GO-term categories: "response to abscisic acid" and "response to jasmonic acid"), Abiotic stress response (GO-term categories: "response to light stimulus", "response to alcohol", "response to lipid", "response to mannose", "response to water", "response to oxidative stress", "response to salt stress", "response to sucrose", "response to hypoxia" and "response to osmotic stress"), Development (GO-term categories: "leaf senescence", "aging"), Metabolite (GO-term categories: "suberin biosynthetic process", "terpenoid catabolic process" "isoprenoid catabolic process", "apocarotenoid metabolic process", "phenylpropanoid biosynthetic process" and "olefinic compound metabolic process") ( Fig. 7, Supplementary Tables S1-S3). Among the 469 commonly upregulated genes, 57 transcription factors were differentially expressed. These transcription factors were categorized as auxin signal transduction, ABA signal transduction, and circadian rhythm (Table S4). As the Sc extract contained IAA and ABA, the upregulation of these transcription factors could be accomplished through the effects of aphid-derived phytohormones. The Ab-GALFA results revealed that the Sc extract treatment induced the dedifferentiation of the epidermal and cortical cells in the transition zone, secondary cell wall formation in the epidermal cells in the differentiation zone, and the enhancement of xylem cell formation in the root. In accordance with these morphological changes, transcriptional regu-   (Table 2). Thus, although many genes were commonly upregulated in both the Sc extract and AHM treatments, the expression of significant number of genes were altered only in the Sc extract treatment (Fig. 7).
In the case of GO enrichment analysis of downregulated genes of Arabidopsis seedlings treated with Sc extract or AHM. The genes related to the photosynthetic process were downregulated both in the Sc extract and AHM treatments, indicating that the photosynthetic activity was changed by Sc and AHM treatments in chloroplasts of cotyledons, because both of positive and negative regulators are included in GO enrichment analysis. Intriguingly, two categories, "defense response" and "electron transport chain generation of precursor" were related to the genes increased and downregulated only in Sc treatment, as well as in the natural gall of R. javanica. These results suggest that not only phytohormones but also unknown effector molecules in the Sc extract regulated the gene expression of the Arabidopsis seedlings in this treatment.

Discussion
The wide variation in the unique shapes of insect galls has attracted attention. However, the molecular mechanisms underlying gall development remain poorly understood owing to the lack of a gall-inducing insect-host plant model. Gall-inducing insects produce effectors that can control the developmental programs of their host plants. In this study, we found that abnormal structures in the root tip region of A. thaliana induced by an extract of the gall-inducing aphid, S. chinensis have the typical characteristics of the insect gall structures [9][10][11] .

Typical characteristics of the insect galls is induced by the treatment of S. chinensis extract.
We found that the shape of the root region of Arabidopsis seedlings treated with the Sc extract significantly changed. The morphological changes in the root region of the seedlings can be characterized as follows: the essential elements of secondary cell wall (lignin, xylan, and suberin) were deposited in the epidermal cells of the root transition and elongation zone, indicating that secondary cell wall structures were constructed  www.nature.com/scientificreports/ in the secondary cell wall-free cells of the root-transition zone . This characteristic appears to reflect the typical structure of the outer layer of gall tissue. From the meristematic zone to the elongation zone, the width of the root epidermal cells thickened, while the parallel alignment of the cortical microtubule array was severely disrupted by Sc extract treatment. Furthermore, the bursiform and complex structures of vacuoles in the root division and transition zones changed to more fragmented, smaller vacuolar structures after Sc extract treatment. In addition, a stem cell niche marker (PLT1-GFP or PLT1-mNeonGreen and WOX5mNeonGreen fluorescence) was detected in the epidermal cells of the root-transition zone, in addition to the meristematic cells. From these results, we concluded that the cells of the root-transition zone became dedifferentiated and formed callus-like structures after Sc extract treatment. Furthermore, Sc extract treatment induced xylem vessel differentiation in the root-transition zone. The ability of TE differentiation was also confirmed by an in vitro xylem vessel formation induction system.
Collectively, we concluded that the Sc extract treatment could induce gall-like structures in the root tip region. The gall-like structures are composed of the secondary cell wall in the epidermal cells of the root elongation and differentiation zones, corresponding to the outer shells of galls; the enhancement of xylem vessel formation, corresponding to the newly formed vascular tissues inside galls; the dedifferentiation of epidermal and cortical cells in the root meristematic and elongation regions of the Arabidopsis seedlings, corresponding to the calluslike cells in galls (Fig. 8).
The gall-like structures induced only in the root-tip region are possibly because the cells in the root elongation zone still possess pluripotency. In fact, in root-knot nematode gall formation, the second-stage juvenile (J2) of the root-knot nematodes enters the inner root at the tip, and they migrates intercellularly to the vascular cylinder to reprogram root tissues of the root elongation zone into giant cells 35 . Moreover, the treatment with J2 crude extracts from outside of the seedlings induces abnormal structures in the root elongation zone 36 . Table 2. The genes associated with stem cell formation, xylem development, and the secondary cell wall were up-relulated in Arabidopsis seedlings treated with Sc extract or Hormone mixture, compared with in Rhus javanica. www.nature.com/scientificreports/

Phytohormones in the aphid extract could partially induce gall-like structures in Arabidopsis.
Phytohormones produced by gall-inducing insects play key roles in gall formation 27 . Active phytohormones such as IAA and CKs have been identified in several gall-inducing insects 17,27,28 . We previously identified amounts of IAA and CK in S. chinensis. In this study, in addition to IAA and CKs 6 , we observed abscisic acid, SA, and JAs inside the bodies of S. chinensis. Given that these phytohormones in insects are involved in the gall-like structure formation processes, including abnormal cell division, cellular enlargement, and dedifferentiation in Arabidopsis seedlings, the application of an AHM mimicking the phytohormone constituents of the Sc extract might contribute to the induction of the gall-like structures observed in the Arabidopsis seedlings. However, our results showed that the morphological changes (extra xylem vessel formation, the dedifferentiation of epidermal and cortical cells, and the formation of secondary cell wall in the epidermal cells), occurred only by the Scextract treatment in the root region of the Arabidopsis seedlings, suggesting the existence of effector molecules other than phytohormones in the Sc extract. www.nature.com/scientificreports/ Common characteristics of gene expression profiles of gall formation in S. chinensis and gall-like tissue formation in Arabidopsis. We found several commonly enriched the gene ontology (GO)-term categories of the genes expressed in the developmental gall of Rhus javanica and the gall-like tissue in Arabidopsis root-tip region. The corresponded GO-term categories are "response to hypoxia", "defense response", "phenylpropanoid metabolic process", "response to oxidative stress", response to osmotic stress", "electron transport chain", and "photosynthesis". These categories are closely related to the typical characteristics of gall tissue of R. javanica 6 , e.g., the genes categorized to "phenylpropanoid metabolic process" is related to the synthesis of components of secondary cell wall of outer shell of gall tissue, "response to jasmonic acid" and "defense response" are related to the defense reaction of gall-tissue to the biotic stresses, and "response to hypoxia", "response to oxidative stress", "response to osmotic stress" are related to the biotic stress reaction of gall-tissue. Our RNA-seq analysis of Arabidopsis seedlings after treating S. chinensis extract revealed that characteristic genes involved in the formation of the typical gall-structure are significantly increased. For instance, the stem cell maintenance genes, (CLAVATA1, RGFR2, WOX4, and WOX5), a suppressor of plant stem cell differentiation (CLE44), an essential factor for QC specification and stem cell activity (PLT2), root meristem growth factors required for root stem cell niche maintenance (RGF1-RGF4), the vascular development-related genes (ACL5 and VND7), the genes involved in the secondary cell wall biosynthesis (KNAT7, MYB105, MYB42, MYB63, SND2, and SND3) were upregulated by the treatment using of S. chinensis extract (Table 2).
Collectively, we concluded that the expression profile of the gall-like structure induced by the Sc-extract treatment is similar to that of the developmental gall of R. javanica produced by S. chinensis.

Conclusion
In this study, we demonstrated that gall-like structures can be induced in the root regions of Arabidopsis seedlings by the application of an extract of the gall-inducing aphid, S. chinensis. This phenomenon can be used as a model system to analyze the molecular mechanisms of gall formation processes. We referred to this model system as the Arabidopsis-based gall-forming assay (Ab-GALFA). The Ab-GALFA can be utilized as a bioassay for isolating the unknown insect effectors of gall formation in various host plants. Histological staining of Arabidopsis seedlings. The cell walls of the root cells were stained with propidium iodide, as previously described 35

Measurements of cell length, width, and microtubule density. After staining the root cells with
propidium iodide, the length and width of the cells was determined using ImageJ software. The microtubule density in the GFP-TUB6-expressing line with or without the Sc-extract treatment was measured, as previously described 41 .

Measurements of intensity and statistical analyses.
Subsets of data with defined x-, y-, and z-dimensions were acquired using LASX software (Leica), and all subsets were transformed into 2D images using the maximum intensity projection function of LASX software. In all images, uniform brightness and contrast correction was performed before being exported for image analysis. All quantitative data were produced using the publicly available software Image Studio (LI-COR). The values were normalized relative to the average fluorescence intensity measured in the control group. Final statistical data evaluation and plot preparation were performed using R software.
RNA extraction, library construction, and RNA sequencing. Total RNA was extracted from Arabidopsis seedlings using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). All samples were pretreated with DNase I, using the Qiagen RNase-Free DNase Set (Qiagen, Hilden, Germany), to eliminate DNA contamination. RNA quality was checked by determining the RNA integrity number using an RNA 6000 bioanalyzer and RNA Nano Chip (Agilent Technologies). RNA-seq libraries were prepared using the Illumina TruSeq® Stranded RNA LT kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Three independent RNA samples from each tissue were used for the analysis. Pooled libraries were sequenced using NextSeq 500 (Illumina), and single-end reads, 75 bp in length, were obtained. The obtained reads were mapped to the reference A. thaliana genome (TAIR10) using TopHat2 42 . The htseq-count script in the HTSeq library was used to count the reads 43 . Count data were subjected to trimmed mean of M-value normalization using EdgeR 44,45 . DEGs were defined using the EdgeR GLM approach 45 , and genes with FDRs < 0.05 were classified as DEGs. Scaled expression values were used for clustering, based on the SOM 46,47 .