A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium

Abstract

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development.

Plant pathogens affect both the magnitude and quality of agricultural production; thus, one of the most important goals in agricultural science is to understand the induction mechanisms of plant disease. Plant pathogens can cause diverse disease symptoms in roots, leaves, flowers, fruits, stems, and tubers. In particular, symptoms of disease involving cell death, such as necrotic spots, vascular necrosis, blight, and cankers, have been studied in detail (1). Several virulence factors known to induce cell death in plants and the components of their related signaling cascades have been identified (1). However, there are many other types of disease symptoms in nature. For example, leaf malformation, leaf distortion, leaf rolling, small leaves, stunted growth, dwarfism, witches' broom (the development of numerous shoot branches), shortened internodes, bolting (the growth of elongated stalks), flower virescence (the greening of floral organs), or phyllody (leaf-like petals and sepals), all of which induce abnormal organ formation in plants, can cause devastating agricultural losses.

One group of plant pathogens causes the development of numerous small and short branches that look like the nest of Tengu, a mythical, long-nosed Japanese goblin who lives in the mountains and flies through the sky (see Fig. S1A). Therefore, the disease caused by these pathogens is referred to as Tengu-su (Tengu's nest) disease in Japan. The distinctive symptoms associated with this disease have fascinated and troubled the Japanese people for some time, especially those involved in agricultural production. For example, Paulownia witches' broom (“Paulownia Tengu-su” in Japanese) disease, which is caused by a plant-pathogenic bacterium (phytoplasma), was first described more than 140 years ago in Japan (Fig. S1 B–D). The symptoms of Tengu-su include witches' broom and dwarfism. Infected plants produce large numbers of auxiliary or axillary shoots, and exhibit dwarfism (small flowers and leaves with shortened internodes), resulting in a broom-like appearance (Figs. S1 B–D and S2). Because this disease is accompanied by dramatic developmental abnormalities, identification of its virulence factor is important in terms of plant pathology, plant morphology, and plant physiology. However, the virulence factor involved in the production of these symptoms remains unknown.

Phytoplasmas (class Mollicutes, genus Phytoplasma) are bacterial plant pathogens that have had devastating effects on yields in diverse low- and high-value crops and plants worldwide (2). Coconut palm lethal yellowing disease, Paulownia witches' broom disease, and many other diseases caused by phytoplasmas induce fatal damage in plants and crops all over the world. Phytoplasmas infect more than 700 plant species and bring about dramatic changes in plant development, including witches' broom, dwarfism, proliferation (the growth of shoots from floral organs) (Fig. S2), phyllody, virescence, sterility of flowers, bolting, purple tops (the reddening of leaves and stems), generalized yellowing, and phloem necrosis (3, 4). Phytoplasmas reside within the sieve elements of plant phloem and within the cell in insects, and are transmitted from plant to plant by insect vectors. Phytoplasmas are pleiomorphic bacteria that lack cell walls, and their cell size (0.1–0.8 μm in diameter) and genome size (0.5–1.3 Mbp) are the smallest among bacteria (3, 4). Although the inability to culture phytoplasmas in vitro has hindered their characterization at the molecular level, the full genomic sequences of 4 phytoplasmas were recently determined (5–8). Analyses of these data indicate that phytoplasma has lost many metabolic genes, suggesting that reductive evolution is a consequence of being an intracellular parasite. An antigenic membrane protein (Amp) of Candidatus Phytoplasma asteris OY strain interacts with the microfilaments of its insect host Macrosteles striifrons, as well as with microfilament complexes in certain leafhopper species known to transmit OY, but not with those in leafhoppers that are unable to transmit OY, suggesting that the interaction between Amp and the insect microfilament complexes is involved in insect transmissibility (9). In addition to phytoplasma-insect interactions, some findings regarding phytoplasma-plant interactions have been reported. In particular, it has recently been reported that Ca. P. asteris AY-WB strain secretes a protein that targets plant cell nuclei, which is thus one of the candidate virulence factors of phytoplasma (10). However, the mechanism underlying its pathogenicity remains unknown.

Many Gram-negative bacterial pathogens that affect plants and animals use a type-III secretion system (TTSS) to deliver virulence factors into the host cell. These prokaryotic virulence factors often mimic eukaryotic proteins, allowing the pathogen to modulate the biological systems of the host to promote bacterial invasion, multiplication, and dispersal (11). Diverse enzymatic activities are associated with these TTSS virulence factors, including cysteine protease (12), SUMO protease (13), E3 ubiquitin ligase (14), protein phosphatase (15), and ADP-ribosyltransferase (16) activity. These virulence factors are generally involved in the suppression of plant immunity (1). The phytoplasma genome lacks genes that encode a TTSS (5). However, because phytoplasmas reside within the plant cell, they can secrete proteins into plant hosts via the bacterial Sec translocation system. The proteins secreted from phytoplasma may function in the host cytoplasm like TTSS virulence factors because phytoplasma is an intracellular parasite (4). In fact, it has been recently reported that phytoplasma produces a protein that targets the nuclei of plant host cells (10).

In this study, we identified a protein secreted from phytoplasma that induces symptoms of witches' broom and dwarfism in plants, causing abnormalities in plant morphology. These symptoms strongly resemble those of phytoplasma infection.

Results

Screening for a Virulence Factor of Phytoplasma Disease.

Because phytoplasmas reside within the cells of their hosts, we assumed that secreted proteins would be strong candidates for disease virulence factors. To investigate this hypothesis, we identified more than 30 putative secreted proteins from the OY phytoplasma genome (5) and expressed each of them in N. benthamiana using a plant virus vector-mediated expression system (17). OY phytoplasma-infected plants show distinctive symptoms such as witches' broom and dwarfism; therefore, we tested whether the expression of any of our predicted phytoplasma secreted proteins would induce these characteristic symptoms.

We found that N. benthamiana plants expressing PAM765, 1 of the 30 secreted proteins, developed clear symptoms of phytoplasma infection, including witches' broom and dwarfism. In particular, the number of shoots and leaves that emanated from the apical meristem was dramatically increased while plant height was reduced (Fig. 1A and B). These phenotypes resembled typical phytoplasma disease symptoms. In contrast, the phenotype of N. benthamiana plants expressing the other secreted protein, PAM486, was similar to the phenotype of the control plant inoculated with empty viral vector (pCAMV) (Fig. 1A). These results strongly suggest that PAM765 is the virulence factor responsible for witches' broom and dwarfism in plants. Surprisingly, PAM765 encodes a very small protein of 4.5 kDa. The mature protein, after the cleavage of its N-terminal signal peptide, is only 38 amino acids in length (Fig. S3).

Fig. 1.

Identification of a virulence factor inducing phytoplasma disease symptoms. (A) N. benthamiana plants inoculated with A. tumefaciens harboring empty vector (pCAMV) (Left), pCAMV-PAM765 (tengu) (Center), or pCAMV-PAM486 (Right). (Lower) Stems are highlighted with red lines; leaf petioles are highlighted with black lines. The center plant showed witches' broom disease symptoms (a dramatically increased shoot system). (B) The number of leaves per plant following inoculation with the viral vector. The error bars indicate the SD. An asterisk indicates a significant difference (P < 0.05). 1, pCAMV; 2, pCAMV-PAM765 (tengu).

Transgenic A. thaliana-expressing PAM765 Exhibited Witches' Broom and Dwarfism.

To further analyze the effect of PAM765 on plant organ formation and development, we produced transgenic A. thaliana plants (ecotype Col-0) that constitutively expressed PAM765. Wild-type plants inoculated with OY phytoplasma via sap-feeding of OY-infected insect vectors exhibited severe developmental abnormalities, including symptoms of witches' broom and dwarfism (Fig. 2A, center and right). Interestingly, similar symptoms were observed in the PAM765-transgenic plants (Fig. 2B, center and right, and Table S1). The severity of the symptoms varied across the transgenic lines. Among 87 transgenic lines, 6 lines (6.9%) exhibited severe dwarfism (i.e., short internodes) and produced sterile flowers (Fig. 2B, center); 18 lines (20.7%) did not develop dwarfism, but instead exhibited witches' broom (i.e., increased shoot branching) (Fig. 2B, right, Table S1). In contrast, 25 transgenic lines that expressed GUS alone showed no symptoms (0%) and were similar to plants inoculated with healthy insects (Fig. 2A, left, and B, left). There are significant differences between the number of abnormal plants of PAM765-transgenic lines and that of GUS-transgenic lines (Fisher's exact probability test, P < 0.01, Table S1).

Fig. 2.

Comparison of 35S∷PAM765 (tengu) transgenic plants and phytoplasma (OY strain)-infected plants. (A) Phenotypes of the OY-infected plants. (Left) uninfected plant (control). (Center and Right) OY-infected plants. The center and right plants show severe dwarfism and witches' broom symptoms, respectively. (B) Phenotypes of the 35S∷PAM765 transgenic A. thaliana lines. (Left) 35S∷GUS transgenic line (control). (Center and Right) 35S∷PAM765 transgenic lines. The center and right transgenic lines show severe dwarfism with short internodes and witches' broom symptoms, respectively.

In terms of their reproductive organs, the transgenic lines with witches' broom also had defects in phyllotaxis (leaf arrangement) such that 2 or more flowers grew from a single point on the stem (Fig. 3B–D), and some of these lines produced sterile flowers (Fig. 3E). All of the abnormal phenotypes observed in the PAM765-transgenic plants were the same as those observed in OY phytoplasma-infected A. thaliana (Fig. 3G–I). In contrast, transgenic plants that expressed the GUS gene alone were similar to the plants inoculated with healthy insects in that they had normal reproductive organs (Fig. 3 A and F). These results suggest that PAM765 is the virulence factor that induces phytoplasma-related witches' broom, dwarfism and abnormal reproductive organogenesis (i.e., Tengu-su disease). Thus, we designated PAM765 as phytoplasma tengu-su inducer (tengu).

Fig. 3.

Analysis of branching in 35S∷PAM765 (tengu) transgenic plants and phytoplasma-infected plants. (A) A 35S∷GUS transgenic plant (control). (B–E) Phenotypes of the 35S∷PAM765 transgenic A. thaliana lines. (B–D) The 35S∷PAM765 transgenic plants exhibited defects in phyllotaxis (2 or more flowers growing from a single point on the stem). (E) A 35S∷PAM765 transgenic plant with sterile flowers. (F) An uninfected plant (control). (G–I) Phenotypes of the OY-infected plants. (G and H) The OY-infected plants exhibited defects in phyllotaxis, similar to (B–D). (I) An OY-infected plant with sterile flowers, as in (E). (Scale bars, 50 mm.) (J) The transcription of tengu was examined by quantitative real-time RT-PCR and the results were normalized against the expression of tufB. The error bars indicate the SD. An asterisk indicates a significant difference (P < 0.05). 1, Phytoplasma-infected plants (C. coronarium). 2, Phytoplasma-infected insects (M. striifrons).

TENGU Is Not a Silencing Suppressor.

It has been reported that the constitutive expression of a post-transcriptional gene-silencing (PTGS) suppressor of some plant viruses can induce developmental defects in plants (18). To investigate the possibility that TENGU functions as a PTGS suppressor, we tested whether TENGU could suppress PTGS of the GFP gene using a transient expression assay (SI Text). Leaves of non-transgenic N. benthamiana infiltrated with A. tumefaciens carrying GFP (35S∷GFP) exhibited strong fluorescence at 2–4 days post-inoculation (dpi); however, the level of fluorescence gradually decreased after 4 dpi and finally disappeared by 5 dpi because of the induction of PTGS (Fig. S4). When both GFP and the PTGS suppressor P19 of tomato bushy stunt virus (TBSV) were co-expressed by agroinfiltration, strong fluorescence was observed at 7 dpi (Fig. S4), suggesting that the silencing of GFP was suppressed by P19. On the other hand, because GUS does not have PTGS suppressor activity, no GFP fluorescence was observed at 5 dpi when GFP and GUS were co-expressed. Likewise, when GFP and TENGU were co-expressed, no fluorescence was observed at 5 dpi (Fig. S4), suggesting that TENGU does not function as a PTGS suppressor.

TENGU Is More Highly Expressed in Plant Hosts than in Insect Hosts.

Because phytoplasmas can infect both plant and insect hosts, we compared the expression of tengu in a phytoplasma-infected plant to that in a phytoplasma-infected insect by real-time PCR to gauge the importance of the tengu gene in each host. Total RNA was extracted from OY-infected insects (M. striifrons) and plants (Chrysanthemum coronarium). Subsequently, real-time PCR was performed with the elongation factor Tu gene of OY (tufB) as an internal standard. The level of expression of tengu in the plant host was approximately 5 times that in the insect host (Fig. 3J).

TENGU Is Transported from Phloem Tissue into Adjacent Tissues.

Because TENGU is quite a small protein (≈4.5 kDa), we hypothesized that it could be transported from cell to cell through symplasm. To examine this hypothesis, we used immunohistochemical analysis to investigate the localization of TENGU protein in tissue. It has been reported that the intracellular localization of phytoplasma can be specifically detected by immunohistochemical analysis using an antibody against Amp protein, which is a phytoplasmal membrane protein (19). Similarly, the blue signal of Amp protein was specifically detected in the phloem of the OY-infected plant by immunohistochemical analysis using anti-Amp antibody (Fig. 4 B and E), suggesting that the localization of phytoplasma was restricted within the phloem. However, in an immunohistochemical analysis with an anti-TENGU antibody, the blue signal of TENGU protein was observed not only in phloem tissues but also in parenchyma and meristem tissues (Fig. 4A). Surprisingly, quite a strong signal was extensively detected in the tip region of the stem and the branching region of axillary buds (Fig. 4 D and F). In addition, the TENGU signal was also detected in the apical meristem (Fig. 4H). We confirmed that TENGU and Amp proteins were not detected in healthy plants (Fig. 4C). These results indicate that TENGU is transported from phloem tissues into parenchyma and meristem tissues, especially into the tip region of the stem and axillary buds.

Fig. 4.

Immunohistochemical detection of TENGU and Amp proteins in apical meristem tissue. (A and B) Apical meristem tissue sections from OY-infected plants were reacted with the anti-TENGU antibody (A) or the anti-Amp antibody (B). Bars, 1 mm. (C) An apical meristem tissue section from healthy plant was reacted with the anti-TENGU antibody. (Scale bar, 1 mm.) (D, F, and H) Enlarged section of (A). (E, G, and I) Enlarged section of (B). (D and E) Branching region of axillary buds. (Scale bar, 200 μm.) (F and G) Tip region of stem. Bar, 200 μm. (H and I) Apical meristem. (Scale bar, 100 μm.) ph; phloem, pa; parenchyma. (J) Analysis of the subcellular localization of TENGU. Chimeric constructs (35S∷GFP [left], 35S∷GFP-tengu [center], and 35S∷tengu-GFP [right]) were transiently expressed in onion epidermal cells.

To investigate the intracellular localization of TENGU, we transiently expressed a TENGU-GFP fusion protein in onion epidermal cells (Fig. 4J). The fusion protein was localized in the cytoplasm regardless of whether GFP was fused to the N- or C-terminus of TENGU (Fig. 4J, center and right), suggesting that TENGU would function in the cytoplasm of a plant cell. The cytoplasmic localization of TENGU was also supported by protein localization prediction using PSORT (20). In addition, subcellular localization signal sequences, such as a nuclear localization signal, were not identified in the secreted region of TENGU by InterProScan (21).

TENGU Affects Plant Auxin Responses.

To investigate the influence of TENGU expression on the transcription profile of a host plant, we identified differences in the gene expression profiles between tengu-transgenic and GUS-transgenic Arabidopsis plants using microarray analysis. A total of 373 genes was significantly up-regulated, with ratios of more than 2.0 (P < 0.05), and 575 genes were significantly down-regulated, with ratios of less than 0.5 (P < 0.05) in the tengu-transgenic plants compared with the GUS-transgenic plants. Among these genes, the number of auxin-related genes was significantly high according to classification analysis based on gene ontology (24 genes, P < 0.05), most of which were down-regulated by the expression of TENGU (Table 1). Auxin-related genes that were down-regulated in microarray analysis contained early auxin-responsive genes, that is, AUX/IAA family genes (IAA29, IAA7/AUX2), small auxin-induced RNA (SAUR) family genes (SAUR_AC1 and other 14 genes), and GH3 family genes (GH3.5/WES1), all of which are known to be induced after exposure to auxins (22). Additionally, the expression level of dormancy-associated protein 1 (DRM1) was approximately 5.7-fold lower in the tengu-transgenic plants than in the GUS-transgenic plants. It has been reported that the DRM1 gene is up-regulated in non-growing axillary buds (dormant state) but down-regulated in growing axillary buds (23). Moreover, the expression levels of 2 pin-formed (PIN) genes (PIN7 and At5g01990) were approximately 2–3-fold lower in tengu-transgenic plants than in GUS-transgenic plants. Auxin is mainly synthesized in young meristems and leaves, and is transported to the root. PIN genes encoding components of the auxin efflux machinery are involved in this polar auxin transport (24). Taken together, these results suggest that TENGU disrupts auxin signaling or biosynthesis pathways.

Table 1.

Auxin-related genes down-regulated in tengu-transgenic plants as identified by microarray analysis

AGI number	Fold change	AGI number	Fold change
	AUX/IAA genes
At4 g32280	28.6	At3 g23050	2.5
	Auxin responsive SAUR genes
At3 g53250	10.8	At4 g38850	9.1
At1 g75590	8.3	At5 g18010	7.8
At5 g18080	6.8	At5 g18060	6.5
At5 g18030	6.5	At1 g29460	6.2
At5 g18050	6.0	At5 g18020	5.5
At5 g03310	5.4	At1 g29500	3.8
At1 g29450	3.6	At1 g29440	2.9
At1 g29510	2.8	At3 g03850	2.4
	GH3 genes
At4 g27260	3.3
	Other auxin responsive protein
At5 g19140	3.9
	Dormancy-associated proteins
At1 g28330	5.7	At2 g33830	5.6
	Auxin efflux carrier family proteins
At1 g23080	3.1	At5 g01990	2.0

Genes with a ratio (GUS-transgenic plants/tengu-transgenic plants) of > 2 and P < 0.05 are listed.

Discussion

Identification of an Inducer of Witches' Broom and Dwarfism.

Several phytopathogenic fungi and bacteria are known to cause witches' broom and/or dwarfism (25, 26), for example, the bacterium Rhodococcus fascians, which affects carnation plants (25), and the fungus Taphrina wiesneri, which affects cherry trees (26). It has been demonstrated that these phytopathogens have biosynthetic genes for auxin or cytokinin, and produce these phytohormones to cause disease symptoms (25–27). Here, we showed that a single protein from a plant-pathogenic bacterium induces witches' broom and dwarfism in plants (Figs. 1A and 2B). Although some bacteria are known to induce histological symptom such as canker or gall (1), this is the first report of a bacterial virulence factor that causes morphological abnormalities at the whole plant level.

TENGU Induces Symptoms by Changing Auxin Responses.

Microarray analysis indicated that the expression levels of many auxin-related genes were down-regulated in the tengu-transgenic plant compared with the GUS- transgenic plant (Table 1), suggesting that TENGU may suppress the auxin signaling and biosynthesis pathways. Auxin is known to be involved in apical dominance, which is where an apical bud inhibits development of an axillary bud growth. Auxin biosynthesized in the apical bud is transported to the root, and inhibits the growth of axillary buds (28). This growth inhibition of axillary buds is released by loss of or damage to the apical bud (29). In this study, the expression levels of early auxin-responsive genes, auxin efflux-related genes, and dormancy-associated genes were reduced in the tengu-transgenic plant (Table 1). It has been reported that the A. thaliana mutant of IAA/AXR2, which is an early auxin-responsive gene, exhibited severe dwarfism (30). Interestingly, this symptom greatly resembles that in the tengu-transgenic plant (Fig. 2). Taken together, these results suggest that TENGU suppresses plant auxin responses, resulting in the growth inhibition of apical buds in tengu-transgenic or OY-infected plants. Similarly, apical dormancy would be released by TENGU in tengu-transgenic or OY-infected plants, thus promoting the growth of axillary buds. It has been recently reported that auxin treatment induced recovery of phytoplasma-infected periwinkles (31). These findings imply that the symptoms caused by phytoplasma may be involved in an auxin-related pathway.

Some phytohormones, for example, auxin and cytokinin, are thought to be involved in morphological changes caused by phytoplasmas, such as witches' broom or dwarf symptoms (3). However, this has not been experimentally demonstrated so far, and these disease symptoms were thought to be caused by indirect effects, for example, consumption of metabolites of infected plants by phytoplasmas. Our findings suggest that TENGU could be a missing link between phytoplasmal symptoms and phytohormones. However, many additional genes besides the auxin-related genes were deregulated in the microarray analysis. It is unclear whether their deregulation was due to auxin imbalance or rather the deregulation of some auxin metabolism related genes as a downstream effect of other molecular event(s). Therefore, further analyses are needed to elucidate a direct connection between TENGU and auxin.

As stated above, some phytopathogenic fungi and bacteria are known to produce phytohormones that cause disease symptoms (25–27). However, phytoplasmas do not have genes for the biosynthesis of phytohormones (5). Instead, through secretion of TENGU, phytoplasmas are thought to perturb the auxin pathway of the host plant. This represents a mechanism by which pathogenic bacteria affect plant development.

TENGU Is Transported from Phloem Tissue and Localizes in Non-phloem Cells.

It was previously shown that the size exclusion limit (SEL) of plasmodesmata between a sieve element and a companion cell ranges from 10 to 40 kDa (32), which is much higher than the SEL of plasmodesmata between non-phloem cells such as mesophyll cells (≈1 kDa) (33). However, Imlau et al. showed that GFP proteins (27 kDa), which are specifically produced in phloem cells under control of the phloem-specific AtSUC2 promoter in A. thaliana and tobacco, could be transported through plasmodesmata from the phloem into developing (sink) tissues, that is, young rosette leaves, petals, root tips, etc. (34). Therefore, because the molecular weight of TENGU is approximately 4.5 kDa, which is smaller than GFP, we assume that TENGU can be transported into non-phloem cells through symplasm. In fact, TENGU proteins were detected not only in phloem tissues but also in parenchyma and meristem tissues via immunohistochemical analysis using an anti-TENGU antibody (Fig. 4A). This suggests that TENGU has the ability to be transported from phloem to non-phloem cells. In addition, TENGU proteins were strongly detected in the tip region of stems and the branching region of axillary buds (Fig. 4D and F), and even in the apical meristem region (Fig. 4H), suggesting that TENGU can be transported into apical buds. Because auxin is biosynthesized in apical buds, TENGU may directly suppress the auxin biosynthetic pathway in the cells of apical buds. It has been demonstrated that phytoplasmas selectively localize to the phloem, and are not observed in younger tissues of the apical meristem (19). Thus, although phytoplasmas cannot invade apical buds, phytoplasmas may secrete TENGU to perturb plant metabolism in apical buds by remote manipulation. Alternatively, TENGU may down-regulate the expression of auxin efflux-related genes (Table 1) or indirectly suppress the auxin signaling pathway by inhibition of auxin transport, resulting in an impact on plant development.

TENGU Is Not a Silencing Suppressor.

Previous studies have demonstrated that some PTGS suppressors might cause the disturbance of miRNA function (18). Such PTGS suppressors may affect the expression of plant genes that are normally regulated by miRNA and may also result in the induction of developmental defects (18). However, we demonstrated that TENGU does not possess PTGS suppressor activity. Moreover, a viral vector containing TENGU did not induce necrosis (Fig. 1A), whereas viral vectors that contain PTGS suppressors induce severe necrosis in plants (35). These results strongly suggest that TENGU is not a PTGS suppressor. Therefore, the developmental changes induced by TENGU are probably not caused by interference with the host's miRNA pathways.

TENGU May Increase the Evolutionary Fitness of Phytoplasma.

The reason that phytoplasmas possess TENGU and induce morphological changes in plants may be to increase their own evolutionary fitness by modifying plants. Disease symptoms of phytoplasma-infected plants, including witches' broom, phyllody, and virescence, have common characteristics: i.e., the aggressive production of young and green organs. These characteristic symptoms may be related to the life cycle of phytoplasmas. Because phytoplasmas are transmitted by insect vectors, sap-feeding by insects is one of the most important steps in the phytoplasmal life cycle (3, 4). Leafhoppers, which are the main insect vector of phytoplasmas, prefer young and green/yellow tissues for feeding, as well as for laying eggs (4). Therefore, phytoplasmas that are able to increase the production of young and green/yellow leaves in plants would increase their own transmission efficiency by making the infected plants appear more attractive to insects, in turn increasing their own survival (4). Thus, although speculative, witches' broom, a characteristic symptom of Ca. P. asteris-infected plants, may be the result of the phytoplasma manipulating the host to increase its own fitness and extend its ecological niche. We demonstrated that tengu is highly expressed in plant hosts (Fig. 3J), implying that it plays an important role in supporting the existence of the phytoplasma in the plant host. It is a very intriguing phenomenon that these host controls are governed by a single protein (TENGU). However, our hypothesis may not apply to other phytoplasmas since many of them do not induce witches' broom. Further analysis of this protein will provide additional insight into the interactions among insects, plants, and phytoplasmas.

The discovery of virulence factors that induce cell death has greatly contributed to study of the mechanisms and signaling cascades involved in plant immunity. Likewise, the discovery of TENGU will certainly contribute expanding progress in the fields of plant pathology, plant physiology, and plant development.

Materials and Methods

Identification of Secreted Proteins from OY Phytoplasma.

Secreted proteins generally have both a transmembrane region and a signal sequence at the N terminus (4). We identified putative secreted proteins encoded in the OY genome using the SOSUI (36) and Signal P (37) programs. Using this approach, more than 200 open-reading frames (ORFs) were predicted to have at least 1 transmembrane region, of which more than 30 ORFs had a predicted signal sequence.

Transient Expression Assays.

For Agrobacterium-mediated viral vector assays, the PAM765 (tengu) gene was amplified by PCR with KOD DNA polymerase (Toyobo) (Table S2), and the product was cloned into the binary potato virus X (PVX) vector pCAMV to produce pCAMV-tengu. The pCAMV is a derivative of the binary vector pCAMBIA1301, which contains the PVX cDNA from pP2C2S (17) that was kindly provided by Dr. David Baulcombe (Sainsbury Laboratory, Norwich, U.K.). A. tumefaciens strain EHA105 was transformed with the vector to generate an agroinfiltration-ready clone. PVX agroinfection assays were performed as described previously (38).

Inoculation of A. thaliana with Phytoplasma Strain OY.

A. thaliana plants (ecotype Col-0) were grown and maintained in a growth chamber at 25 °C. Plants at the stage of 4 to 5 rosette leaves were covered with clear tubes, and 5 OY-infected leafhoppers (M. striifrons) were released into the tube and then removed after 5 days.

Transgenic Plants.

To create transgenic A. thaliana plants, the tengu gene was cloned into the binary plasmid vector pBI121 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. A. tumefaciens strain EHA105 was then transformed with the pBI121-tengu construct (35S∷tengu).

A. thaliana plants (ecotype Col-0) were transformed using the floral dip method. To select transformed plants, sterilized T1 seeds were plated on kanamycin selection plates [Murashige and Skoog salt (Wako), MS vitamin (Sigma), 1% sucrose, 0.7% agar, and 50 μg/ml kanamycin]. The expression of tengu was confirmed by RT-PCR.

Quantitative Real-time RT-PCR.

Total RNA from OY-infected insects (M. striifrons) and plants (C. coronarium) was extracted with ISOGEN (Nippon Gene) and reverse-transcribed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative real-time RT-PCR assays were performed by the 7300 Real-Time PCR System (Applied Biosystems) with tufB as an internal standard (Table S2).

Determination of the Subcellular Localization of TENGU.

The subcellular localization of TENGU was investigated using the following constructs: CaMV 35S∷tengu-GFP, CaMV 35S∷GFP-tengu, and CaMV 35S∷GFP. The tengu gene was cloned downstream of the CaMV 35S promoter to produce CaMV 35S∷tengu. The GFP gene was cloned downstream or upstream of tengu in the CaMV 35S∷tengu construct to produce CaMV 35S∷tengu-GFP (C-terminal fusion) and CaMV 35S∷ GFP-tengu (N-terminal fusion), respectively. Each vector was bombarded into onion epidermal cells using the Helios Gene Gun System (Bio-Rad). Following the incubation of the cells overnight in the dark, the level of GFP fluorescence was examined by confocal laser scanning microscopy (LSM5 PASCAL; Carl Zeiss).

Immunohistochemical Analysis.

Immunohistochemical analysis was performed according to a previously described method with some modifications (19). Tissues including the apical meristem were excised from OY-infected and healthy plants. These tissues were fixed, embedded in Paraplast Plus (Sherwood Medical), and sectioned to 10-μm thickness with a microtome. Anti-TENGU and anti-Amp IgG were used with an alkaline phosphatase-mediated reporter system to detect TENGU and Amp proteins in each tissue. These tissues were observed by Axio Imager microscopy (Carl Zeiss).

Microarray Analysis.

For microarray analysis, 3 independent transgenic lines were harvested from 2-week-old tengu- or GUS-transgenic plants. Total RNA was isolated from each plant with ISOGEN (Nippon Gene), and used for the preparation of Cy3-labeled cRNA probes. Samples were subjected to microarray experiments using an Arabidopsis 3 (4 × 44K) Oligo Microarray (Agilent Technologies). All microarray experiments were performed according to the supplier's manual. The slides were scanned with a Microarray Scanner (Agilent Technologies) at a 5-μm resolution, and extraction and image analysis software (Feature Extraction version 9.5.3.1; Agilent Technologies) was used to integrate each spot's intensity. We used the GeneSpring GX 9.0 (Agilent Technologies) for filtering, normalization, and statistical and gene ontology analyses. Statistical significance of gene expression was tested using a t test combined with a Benjamini and Hochberg false discovery rate multiple correction algorithm. We selected statistically significant genes (P < 0.05) only if their fold change was >2.0.