The CLE33 peptide represses phloem differentiation via autocrine and paracrine signaling in Arabidopsis.

Plant meristems require a constant supply of photoassimilates and hormones to the dividing meristematic cells. In the growing root, such supply is delivered by protophloem sieve elements. Due to its preeminent function for the root apical meristem, protophloem is the first tissue to differentiate. This process is regulated by a genetic circuit involving in one side the positive regulators DOF transcription factors, OCTOPUS (OPS) and BREVIX RADIX (BRX), and in the other side the negative regulators CLAVATA3/EMBRYO SURROUNDING REGION RELATED (CLE) peptides and their cognate receptors BARELY ANY MERISTEM (BAM) receptor-like kinases. brx and ops mutants harbor a discontinuous protophloem that can be fully rescued by mutation in BAM3, but is only partially rescued when all three known phloem-specific CLE genes, CLE25/26/45 are simultaneously mutated. Here we identify a CLE gene closely related to CLE45, named CLE33. We show that double mutant cle33cle45 fully suppresses brx and ops protophloem phenotype. CLE33 orthologs are found in basal angiosperms, monocots, and eudicots, and the gene duplication which gave rise to CLE45 in Arabidopsis and other Brassicaceae appears to be a recent event. We thus discovered previously unidentified Arabidopsis CLE gene that is an essential player in protophloem formation.

Long-distance transport of the pear HMGR1 mRNA via the phloem is associated with enhanced salt tolerance.

Grafting is the main asexual propagation method for horticultural crops and can enhance their resistance to biotic or abiotic stress. Many mRNAs can be transported over long distances through the graft union, however, the function of mobile mRNAs remains poorly understood. Here, we exploited lists of candidate mobile mRNAs harboring potential 5-methylcytosine (m5C) modification in pear (Pyrus betulaefolia). dCAPS RT-PCR and RT-PCR were employed to demonstrate the mobility of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase1 (PbHMGR1) mRNA in grafted plants of both pear and tobacco (Nicotiana tabacum). Overexpressing PbHMGR1 in tobacco plants enhanced salt tolerance during seed germination. In addition, both histochemical staining and GUS expression analysis showed that PbHMGR1 could directly respond to salt stress. Furthermore, it was found that the relative abundance of PbHMGR1 increased in heterografted scion, which avoided serious damage under salt stress. Collectively, these findings established that PbHMGR1 mRNA could act as a salt-responsive signal and move through the graft union to enhance salt tolerance of scion, which might be used as a new plant breeding technique to improve resistance of scion through a stress-tolerant rootstock.

Analysis of companion cell and phloem metabolism using a transcriptome-guided model of Arabidopsis metabolism.

Companion cells and sieve elements play an essential role in vascular plants, and yet the details of the metabolism that underpins their function remain largely unknown. Here, we construct a tissue-scale flux balance analysis (FBA) model to describe the metabolism of phloem loading in a mature Arabidopsis (Arabidopsis thaliana) leaf. We explore the potential metabolic interactions between mesophyll cells, companion cells, and sieve elements based on the current understanding of the physiology of phloem tissue and through the use of cell type-specific transcriptome data as a weighting in our model. We find that companion cell chloroplasts likely play a very different role to mesophyll chloroplasts. Our model suggests that, rather than carbon capture, the most crucial function of companion cell chloroplasts is to provide photosynthetically generated ATP to the cytosol. Additionally, our model predicts that the metabolites imported into the companion cell are not necessarily the same metabolites that are exported in phloem sap; phloem loading is more efficient if certain amino acids are synthesized in the phloem tissue. Surprisingly, in our model predictions, the proton-pumping pyrophosphatase (H+-PPiase) is a more efficient contributor to the energization of the companion cell plasma membrane than the H+-ATPase.

Loss of OPT3 function decreases phloem copper levels and impairs crosstalk between copper and iron homeostasis and shoot-to-root signaling in Arabidopsis thaliana.

Copper (Cu) and iron (Fe) are essential micronutrients that are toxic when accumulating in excess in cells. Thus, their uptake by roots is tightly regulated. While plants sense and respond to local Cu availability, the systemic regulation of Cu uptake has not been documented in contrast to local and systemic control of Fe uptake. Fe abundance in the phloem has been suggested to act systemically, regulating the expression of Fe uptake genes in roots. Consistently, shoot-to-root Fe signaling is disrupted in Arabidopsis thaliana mutants lacking the phloem companion cell-localized Fe transporter, OLIGOPEPTIDE TRANSPORTER 3 (AtOPT3). We report that AtOPT3 also transports Cu in heterologous systems and contributes to its delivery from sources to sinks in planta. The opt3 mutant contained less Cu in the phloem, was sensitive to Cu deficiency and mounted a transcriptional Cu deficiency response in roots and young leaves. Feeding the opt3 mutant and Cu- or Fe-deficient wild-type seedlings with Cu or Fe via the phloem in leaves downregulated the expression of both Cu- and Fe-deficiency marker genes in roots. These data suggest the existence of shoot-to-root Cu signaling, highlight the complexity of Cu/Fe interactions, and the role of AtOPT3 in fine-tuning root transcriptional responses to the plant Cu and Fe needs.

Tobacco mosaic virus movement protein complements a Potato spindle tuber viroid RNA mutant impaired for mesophyll entry but not mutants unable to enter the phloem.

Tobacco mosaic virus movement protein (TMV MP) is essential for virus spread between cells. To accomplish its task, TMV MP binds viral RNA, interacts with components of the cytoskeleton, and increases the size exclusion limit (SEL) of plasmodesmata. Plasmodesmata are gated intercellular channels that allow passage of small molecules and macromolecules, including RNA and protein, between plant cells. Moreover, plasmodesmata are diverse and those connecting different cell types appear to have unique mechanisms to regulate macromolecular trafficking, which likely contributes to the establishment of distinct cell boundaries. Consequently, TMV MP might be competent to mediate RNA transport through some but not all plasmodesmal gates. Due to a lack of viral mutants defective for movement between specific cell types, the ability of TMV MP in this regard is incompletely understood. In contrast, a number of trafficking impaired Potato spindle tuber viroid (PSTVd) mutants have been identified. PSTVd is a systemically infectious non-coding RNA that nevertheless can perform all functions required for replication as well as cell-to-cell and systemic spread. Previous studies have shown that PSTVd employs different structure and sequence elements to move between diverse cell types in host plants, and mutants defective for transport between specific cell types have been identified. Therefore, PSTVd may serve as a tool to analyze the functions of MPs of viral and cellular origin. To probe the RNA transport activity of TMV MP, transgenic plants expressing the protein were inoculated with PSTVd mutants. Remarkably, TMV MP complemented a PSTVd mutant defective for mesophyll entry but could not support two mutants impaired for phloem entry, suggesting it fails to productively interface with plasmodesmata at the phloem boundary and that additional viral and host factors may be required. Consistent with this idea, TMV co-infection, but not the combination of MP and coat protein (CP) expression, was able to complement one of the phloem entry mutants. These observations suggest that phloem loading is a critical impediment to establishing systemic infection that could involve the entire ensemble of TMV proteins. They also demonstrate a novel strategy for analysis of MPs.

NST- and SND-subgroup NAC proteins coordinately act to regulate secondary cell wall formation in cotton.

Secondary cell wall (SCW) has a strong impact on plant growth and adaptation to the environments. Previous studies have shown that NAC (NAM, ATAF1/2, and CUC2) transcription factors act as key regulators of SCW biosynthesis. However, the regulatory network triggered by NAC proteins is largely unknown, especially in cotton, a model plant for SCW development studies. Here, we show that several cotton NAC transcription factors are clustered in the same group with Arabidopsis secondary wall NACs (SWNs), including secondary wall-associated NAC domain protein1 (SND1) and NAC secondary wall thickening promoting factor1/2 (NST1/2), so we name these cotton orthologs as SND1s and NST1s. We found that simultaneous silencing of SND1s and NST1s led to severe xylem and phloem developmental defect in cotton stems, however silencing either SND1s or NST1s alone had no visible phenotype. Silencing both SND1s and NST1s but not one subgroup caused decreased expression of a set of SCW-associated genes, while over-expression of cotton SWNs in tobacco leaves resulted in SCW deposition. SWNs could bind the promoter of MYB46 and MYB83, which are highly expressed in SCW-rich tissues of cotton. In total, our data provide evidence that cotton SWNs positively and coordinately regulate SCW formation.

Utilizing Potato Virus X to Monitor RNA Movement.

Mobility assays coupled with RNA profiling have revealed the presence of hundreds of full-length non-cell-autonomous messenger RNAs that move through the whole plant via the phloem cell system. Monitoring the movement of these RNA signals can be difficult and time consuming. Here we describe a simple, virus-based system for surveying RNA movement by replacing specific sequences within the viral RNA genome of potato virus X (PVX) that are critical for movement with other sequences that facilitate movement. PVX is a RNA virus dependent on three small proteins that facilitate cell-to-cell transport and a coat protein (CP) required for long-distance spread of PVX. Deletion of the CP blocks movement, whereas replacing the CP with phloem-mobile RNA sequences reinstates mobility. Two experimental models validating this assay system are discussed. One involves the movement of the flowering locus T RNA that regulates floral induction and the second involves movement of StBEL5, a long-distance RNA signal that regulates tuber formation in potato.

Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana.

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

Engineering Plant Resistance to Tomato Yellow Leaf Curl Thailand Virus Using a Phloem-Specific Promoter Expressing Hairpin RNA.

Transgenic approaches employing RNA interference (RNAi) strategies have been successfully applied to generate desired traits in plants; however, variations between RNAi transgenic siblings and the ability to quickly apply RNAi resistance to diverse cultivars remain challenging. In this study, we assessed the promoter activity of a cauliflower mosaic virus 35S promoter (35S) and a phloem-specific promoter derived from rice tungro bacilliform virus (RTBV) and their efficacy to drive RNAi against the endogenous glutamate-1-semialdehyde aminotransferase gene (GSA) that acts as a RNAi marker, through chlorophyll synthesis inhibition, and against tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family Geminiviridae) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic Nicotiana benthamiana expressing hairpin RNA of GSA driven by either the 35S or RTBV promoter revealed that RTBV::hpGSA induced stronger silencing along the vein and more uniformed silencing phenotype among its siblings than 35S::hpGSA. Analysis of transgenic N. benthamiana, 35S::hpTYLCTHV, and RTBV::hpTYLCTHV revealed that, although 35S::hpTYLCTHV generated a higher abundance of small RNA than RTBV::hpTYLCTHV, RTBV::hpTYLCTHV transgenic plants conferred better TYLCTHV resistance than 35S::hpTYLCTHV. Grafting of wild-type (WT) scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. A TYLCTHV-inoculation assay showed that noninfected WT scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor WT rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.

Identification of Phloem Mobile mRNAs Using the Solanaceae Heterograft System.

Large numbers of mRNAs move in the phloem and some may function as signals to exert important physiological functions in the distal recipient organs. Generating an authentic list of phloem mobile mRNA is a prerequisite for elucidating their physiological functions. Nicotiana benthamiana can be used as a scion to graft on a tomato (Solanum lycopersicum) rootstock. Thereby, shoot-to-root mobile N. benthamiana mRNAs transported via the phloem can be identified from the root of the tomato rootstock. Due to the close relationship and similar genome sequences of the two species, stringent informatics procedures should be applied to avoid false identification. This heterograft system can be used to study physiological processes associated with mRNAs that are mobile under either normal or adverse growth condition.

PbTTG1 forms a ribonucleoprotein complex with polypyrimidine tract-binding protein PbPTB3 to facilitate the long-distance trafficking of PbWoxT1 mRNA.

The grafting of horticultural crops enables breeders to induce phenotypic changes in rootstocks and scions. A number of signaling molecules, including RNAs and proteins, were recently shown to underlie these changes; however, little is known about the composition of ribonucleoprotein (RNP) complexes or how these macromolecules are transported. Here, we used a polypyrimidine tract-binding protein, PbPTB3, as a bait to screen a library of phloem cDNA from a pear variety 'Du Li' (Pyrus betulaefolia). We identified a new protein constituent of the RNP complex, TRANSPARENT TESTA GLABRA1 (PbTTG1), a WD40 protein that interacts with PbPTB3 to facilitate its transport with PbWoxT1 mRNA through the phloem. Overexpression experiments indicated that PbTTG1 binds to PbPTB3, facilitating its transmission from the leaf through the petiole, while silencing of PbTTG1 expression prevented their translocation. Heterografting experiments also showed that silencing of PbTTG1 prevented the transport of PbPTB3 from the rootstock to the scion. Collectively, these findings established that PbTTG1 binds to PbPTB3 and PbWoxT1 to form an RNP complex, which facilitates their long-distance movement.

Elucidation of the Mechanisms of Long-Distance mRNA Movement in a Nicotiana benthamiana/Tomato Heterograft System.

Recent heterograft analyses showed that large-scale messenger RNA (mRNA) movement takes place in the phloem, but the number of mobile transcripts reported varies widely. However, our knowledge of the mechanisms underlying large-scale mRNA movement remains limited. In this study, using a Nicotiana benthamiana/tomato (Solanum lycopersicum) heterograft system and a transgenic approach involving potato (Solanum tuberosum), we found that: (1) the overall mRNA abundance in the leaf is not a good indicator of transcript mobility to the root; (2) increasing the expression levels of nonmobile mRNAs in the companion cells does not promote their mobility; (3) mobile mRNAs undergo degradation during their movement; and (4) some mRNAs arriving in roots move back to shoots. These results indicate that mRNA movement has both regulated and unregulated components. The cellular origins of mobile mRNAs may differ between herbaceous and woody species. Taken together, these findings suggest that the long-distance movement of mRNAs is a complex process and that elucidating the physiological roles associated with this movement is challenging but remains an important task for future research.

AtOPT6 Protein Functions in Long-Distance Transport of Glutathione in Arabidopsis thaliana.

The involvement of the Arabidopsis oligopeptide transporter AtOPT6, which was previously shown to take up glutathione (GSH) when expressed in yeast cells or in Xenopus laevis oocytes, in GSH transport was analyzed using opt6 knockout mutant lines. The concentration of GSH in flowers or siliques was lower in opt6 mutants relative to wild-type plants, suggesting involvement of AtOPT6 in long-distance transport of GSH. The GSH concentration in phloem sap was similar between opt6 mutants and wild-type plants. These results, combined with earlier reports showing expression of AtOPT6 in the vascular bundle, especially in the cambial zone, suggest that AtOPT6 functions to transport GSH into cells surrounding the phloem in sink organs. The opt6 mutant plants showed delayed bolting, implying the importance of AtOPT6 for regulation of the transition from vegetative to reproductive growth. After cadmium (Cd) treatment, the concentration of the major phytochelatin PC2 was lower in flowers in the opt6 mutants and Cd was accumulated in roots of opt6 mutant plants compared with wild-type plants. These results suggest that AtOPT6 is likely to be involved in transporting GSH, PCs and Cd complexed with these thiols into sink organs.

Small RNA Isolation from Tissues of Grapevine and Woody Plants.

A protocol is described to purify small (s)RNA molecules from tissues of grapevine and other woody plants. The protocol has been specifically developed to analyze sRNA populations by high-throughput sequencing. It has been widely used on species of the genera Prunus and Vitis particularly rich in polyphenols and other enzyme-inhibiting compounds. The high quality of the sRNAs extracted from leaf or phloem tissues makes them suitable for all molecular biology reactions, in particular for next-generation sequencing library preparation.

FLOWERING LOCUS T mRNA is synthesized in specialized companion cells in Arabidopsis and Maryland Mammoth tobacco leaf veins.

Flowering is triggered by the transmission of a mobile protein, FLOWERING LOCUS T (FT), from leaves to the shoot apex. FT originates in the phloem of leaf veins. However, the identity of the FT-synthesizing cells in the phloem is not known. As a result, it has not been possible to determine whether the complex regulatory networks that control FT synthesis involve intercellular communication, as is the case in many aspects of plant development. We demonstrate here that FT in Arabidopsis thaliana and FT orthologs in Maryland Mammoth tobacco (Nicotiana tabacum) are produced in two unique files of phloem companion cells. These FT-activating cells, visualized by fluorescent proteins, also activate the GALACTINOL SYNTHASE (CmGAS1) promoter from melon (Cucumis melo). Ablating the cells by expression of the diphtheria toxin gene driven by the CmGAS1 promoter delays flowering in both Arabidopsis and Maryland Mammoth tobacco. In Arabidopsis, toxin expression reduces expression of FT and flowering-associated genes downstream, but not upstream, of FT Our results indicate that specific companion cells mediate the essential flowering function. Since the identified cells are present in the minor veins of two unrelated dicotyledonous species, this may be a widespread phenomenon.

Perception of root-active CLE peptides requires CORYNE function in the phloem vasculature.

Arabidopsis root development is orchestrated by signaling pathways that consist of different CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide ligands and their cognate CLAVATA (CLV) and BARELY ANY MERISTEM (BAM) receptors. How and where different CLE peptides trigger specific morphological or physiological changes in the root is poorly understood. Here, we report that the receptor-like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) are necessary to fully sense root-active CLE peptides. We uncover BAM3 as the CLE45 receptor in the root and biochemically map its peptide binding surface. In contrast to other plant peptide receptors, we found no evidence that SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) proteins act as co-receptor kinases in CLE45 perception. CRN stabilizes BAM3 expression and thus is required for BAM3-mediated CLE45 signaling. Moreover, protophloem-specific CRN expression complements resistance of the crn mutant to root-active CLE peptides, suggesting that protophloem is their principal site of action. Our work defines a genetic framework for dissecting CLE peptide signaling and CLV/BAM receptor activation in the root.

Bioinformatic and expression analysis of the Brassica napus L. cyclophilins.

Cyclophilins (CYPs) are a group of ubiquitous proteins characterized by their ability to bind to the immunosuppressive drug cyclosporin A. The CYP family occurs in a wide range of organisms and contains a conserved peptidyl-prolyl cis/trans isomerase domain. In addition to fulfilling a basic role in protein folding, CYPs may also play diverse important roles, e.g. in protein degradation, mRNA processing, development, and stress responses. We performed a genome-wide database survey and identified a total of 94 CYP genes encoding 91 distinct proteins. Sequence alignment analysis of the putative BnCYP cyclophilin-like domains revealed highly conserved motifs. By using RNA-Seq, we could verify the presence of 77 BnCYP genes under control conditions. To identify phloem-specific BnCYP proteins in a complementary approach, we used LC-MS/MS to determine protein abundances in leaf and phloem extracts. We detected 26 BnCYPs in total with 12 being unique to phloem sap. Our analysis provides the basis for future studies concentrating on the functional characterization of individual members of this gene family in a plant of dual importance: as a crop and a model system for polyploidization and long-distance signalling.

Lack of Cytosolic Glutamine Synthetase1;2 Activity Reduces Nitrogen-Dependent Biosynthesis of Cytokinin Required for Axillary Bud Outgrowth in Rice Seedlings.

A mutation abolishing cytosolic glutamine synthetase1;2 (GS1;2) activity impairs assimilation of ammonium into glutamine in both roots and basal portions of shoots, and severely decreases axillary bud outgrowth (tillering) in mutant rice seedlings. Although the gs1;2 mutant phenotype is independent of strigolactone, which inhibits tillering, it also demonstrates glutamine- or related metabolite-responsive biosynthesis of cytokinin (CK), which promotes tillering. Here, we examined the connection between GS1;2 and CK biosynthesis during tillering, focusing on basal portions of the shoots as well as apical and axillary bud meristems in the gs1;2 mutant. Despite a sufficient ammonium supply, decreases in precursor CK contents and a decrease in ammonium assimilation into glutamine were observed in basal portions of mutant shoots. Reintroducing expression of OsGS1;2 cDNA driven by its own promoter restored precursor CK contents and ammonium assimilation to wild-type levels. In basal portions of the shoots, glutamine-responsive adenosine phosphate-isopentenyltransferase4 (OsIPT4), which is also predominant in rice roots, was the predominant isogene for IPT, which synthesizes CK. Cell-specific expression of OsIPT4 in phloem companion cells in nodal vascular anastomoses connected to the axillary bud vasculature also decreased in the gs1;2 mutant. Expression of CK-responsive type-A response regulator genes as local indicators of active CKs was also abolished in the axillary bud meristem of the mutant. These results suggest that the lack of GS1;2 activity decreased levels of glutamine or a related metabolite required for CK biosynthesis, causing a deficiency in active CK in the axillary bud meristem necessary for tillering.

Chilli leaf curl virus-based vector for phloem-specific silencing of endogenous genes and overexpression of foreign genes.

Geminiviruses are the largest and most devastating group of plant viruses which contain ssDNA as a genetic material. Geminivirus-derived virus-induced gene silencing (VIGS) vectors have emerged as an efficient and simple tool to study functional genomics in various plants. However, previously developed VIGS vectors have certain limitations, owing to their inability to be used in tissue-specific functional study. In the present study, we developed a Chilli leaf curl virus (ChiLCV)-based VIGS vector for its tissue-specific utilization by replacing the coat protein gene (open reading frame (ORF) AV1) with the gene of interest for phytoene desaturase (PDS) of Nicotiana benthamiana. Functional validation of ChiLCV-based VIGS in N. benthamiana resulted in systemic silencing of PDS exclusively in the phloem region of inoculated plants. Furthermore, expression of enhanced green fluorescence protein (EGFP) using the same ChiLCV vector was verified in the phloem region of the inoculated plants. Our results also suggested that, during the early phase of infection, ChiLCV was associated with the phloem region, but at later stage of pathogenesis, it can spread into the adjoining non-vascular tissues. Taken together, the newly developed ChiLCV-based vector provides an efficient and versatile tool, which can be exploited to unveil the unknown functions of several phloem-specific genes.

A phloem-limited fijivirus induces the formation of neoplastic phloem tissues that house virus multiplication in the host plant.

A number of phloem-limited viruses induce the development of tumours (enations) in the veins of host plants, but the relevance of tumour induction to the life cycle of those viruses is unclear. In this study, we performed molecular and structural analyses of tumours induced by rice black-streaked dwarf virus (RBSDV, genus Fijivirus) infection in maize plants. The transcript level of the maize cdc2 gene, which regulates the cell cycle, was highly elevated in tumour tissues. Two-dimensional electrophoresis identified 25 cellular proteins with altered accumulation in the tumour tissues. These proteins are involved in various metabolic pathways, including photosynthesis, redox, energy pathways and amino acid synthesis. Histological analysis indicated that the tumours predominantly originated from hyperplastic growth of phloem, but those neoplastic tissues have irregular structures and cell arrangements. Immunodetection assays and electron microscopy observations indicated that in the shoots, RBSDV is confined to phloem and tumour regions and that virus multiplication actively occurs in the tumour tissue, as indicated by the high accumulation of non-structural proteins and formation of viroplasms in the tumour cells. Thus, the induction of tumours by RBSDV infection provides a larger environment that is favourable for virus propagation in the host plant.