Vascular tissue - boon or bane? How pathogens usurp long-distance transport in plants and the defence mechanisms deployed to counteract them.

Evolutionary emergence of specialised vascular tissues has enabled plants to coordinate their growth and adjust to unfavourable external conditions. Whilst holding a pivotal role in long-distance transport, both xylem and phloem can be encroached on by various biotic factors for systemic invasion and hijacking of nutrients. Therefore, a complete understanding of the strategies deployed by plants against such pathogens to restrict their entry and establishment within plant tissues, is of key importance for the future development of disease-tolerant crops. In this review, we aim to describe how microorganisms exploit the plant vascular system as a route for gaining access and control of different host tissues and metabolic pathways. Highlighting several biological examples, we detail the wide range of host responses triggered to prevent or hinder vascular colonisation and effectively minimise damage upon biotic invasions.

Phloem-specific overexpression of AtOPT6 alters glutathione, phytochelatin, and cadmium distribution in Arabidopsis thaliana.

The Arabidopsis oligopeptide transporter AtOPT6 is reportedly involved in the long-distance transport of thiol compounds into sink organs. In the present study, transgenic Arabidopsis lines overexpressing AtOPT6 under the control of a phloem-specific promoter, sucrose-proton symporter 2 (pSUC2), were analyzed for thiol and cadmium (Cd) distribution during the reproductive stage, both with and without Cd exposure. Phloem specific AtOPT6-overexpressing lines did not exhibit an evident impact on bolting time. In the absence of Cd exposure, these transgenic lines showed significantly enhanced transport of endogenous glutathione into siliques, accompanied by a reduction in the glutathione content of flowers and roots during the reproductive stage. Additionally, exposure of the roots of the phloem specific AtOPT6-overexpressing lines to Cd altered the distribution of thiol compounds, resulting in an increase in the content of phytochelatins in sink organs, contributing to a significant elevation of Cd contents in reproductive sink. Our findings confirm the crucial role of AtOPT6 in unloading phytochelatin-Cd conjugates from the phloem into sink organ.

OsHAK4 functions in retrieving sodium from the phloem at the reproductive stage of rice.

Soil salinity significantly limits rice productivity, but it is poorly understood how excess sodium (Na+) is delivered to the grains at the reproductive stage. Here, we functionally characterized OsHAK4, a member of the clade IV HAK/KUP/KT transporter subfamily in rice. OsHAK4 was localized to the plasma membrane and exhibited influx transport activity for Na+, but not for K+. Analysis of organ- and growth stage-dependent expression patterns showed that very low expression levels of OsHAK4 were detected at the vegetative growth stage, but its high expression in uppermost node I, peduncle, and rachis was found at the reproductive stage. Immunostaining indicated OsHAK4 localization in the phloem region of node I, peduncle, and rachis. Knockout of OsHAK4 did not affect the growth and Na+ accumulation at the vegetative stage. However, at the reproductive stage, the hak4 mutants accumulated higher Na+ in the peduncle, rachis, husk, and brown rice compared to the wild-type rice. Element imaging revealed higher Na+ accumulation at the phloem region of the peduncle in the mutants. These results indicate that OsHAK4 plays a crucial role in retrieving Na+ from the phloem in the upper nodes, peduncle, and rachis, thereby preventing Na+ distribution to the grains at the reproductive stage of rice.

Plant viruses exploit insect salivary GAPDH to modulate plant defenses.

Salivary proteins of insect herbivores can suppress plant defenses, but the roles of many remain elusive. One such protein is glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the saliva of the Recilia dorsalis (RdGAPDH) leafhopper, which is known to transmit rice gall dwarf virus (RGDV). Here we show that RdGAPDH was loaded into exosomes and released from salivary glands into the rice phloem through an exosomal pathway as R. dorsalis fed. In infected salivary glands of R. dorsalis, the virus upregulated the accumulation and subsequent release of exosomal RdGAPDH into the phloem. Once released, RdGAPDH consumed H2O2 in rice plants owing to its -SH groups reacting with H2O2. This reduction in H2O2 of rice plant facilitated R. dorsalis feeding and consequently promoted RGDV transmission. However, overoxidation of RdGAPDH could cause potential irreversible cytotoxicity to rice plants. In response, rice launched emergency defense by utilizing glutathione to S-glutathionylate the oxidization products of RdGAPDH. This process counteracts the potential cellular damage from RdGAPDH overoxidation, helping plant to maintain a normal phenotype. Additionally, salivary GAPDHs from other hemipterans vectors similarly suppressed H2O2 burst in plants. We propose a strategy by which plant viruses exploit insect salivary proteins to modulate plant defenses, thus enabling sustainable insect feeding and facilitating viral transmission.

The Increased aspartate levels in transgenic cotton (Gossypium hirsutum L.) lead to improved tolerance against whitefly (Bemisia tabaci, Gennadius).

The whitefly, a polyphagous insect pest feeding on nearly 1328 plant species, is a major threat to global cotton production and incurs up to 50% yield losses in cotton production in Pakistan. We investigated whether increased aspartate in phloem sap imparts whitefly toxicity and protects cotton plants from intense damage. The enzymatic step for aspartate production is carried through aspartate aminotransferase (AAT). In this study, we constitutively overexpressed the Oryza sativa cytoplasmic AAT (OsAAT2) under the CaMV35S promoter in Gossypium hirsutum cv. CIM-482. Real-time PCR analysis of the AAT transcripts revealed a 2.85- to 31.7-fold increase in mRNA levels between the different cotton lines. A substantial increase in the free-amino acid content of the major N-assimilation and transport amino acids (aspartate, glutamate, asparagine, and glutamine) was seen in the phloem sap of the transgenic cotton lines. The bioassay revealed that the two transgenic cotton lines with the highest free aspartate content in the phloem sap exhibited 97 and 94% mortality in the adult whitefly population and a 98 and 96% decline in subsequent nymph populations, respectively. There was also a significant change in the physiological behaviour of the transgenic cotton lines, with an increased net assimilation (A), gaseous exchange (Gs) and rate of transpiration (E). Improved morphological characteristics like plant height, total number of bolls and fiber yield were recorded in transgenic cotton lines. The AAT gene shows promise in mitigating whitefly infestations and enhancing the overall health and yield of cotton plants.

Participation of CWINV and SUS Genes in Sucrose Utilization in the Disruption of Cambium Derivatives Differentiation of Silver Birch.

BACKGROUND: The mechanisms that control the accumulation of woody biomass are of great interest to the study. Invertase and sucrose synthase are enzymes that are vital for distributing carbon in various biosynthetic pathways. Karelian birch (Betula pendula var. carelica) is a form of silver birch (B. pendula Roth) and is characterized by disruption of the differentiation of cambium derivatives towards both the xylem and phloem, which leads to a change in the proportion of the conducting tissues' structural elements and the figured wood formation. We researched the expression profiles of genes encoding sucrose-cleaving enzymes (CWINV and SUS gene families) and genes encoding CVIF protein, which is responsible for the post-translational regulation of the cell wall invertase activity. OBJECTIVE: In our study, 16-year-old common silver birch (Betula pendula var. pendula) and Karelian birch were used for sampling non-figured and figured trunk section tissues, respectively. Samples were selected for the research based on the radial vector: non-conductive, conductive phloem, cambial zone - differentiating xylem - mature xylem. METHODS: The enzyme's activity was investigated by biochemical methods. RT-PCR method was used to determine the level of gene expression. Anatomical and morphological methods were used to determine the stage of differentiation of xylem cambial derivatives. RESULTS: Our research revealed a shift in the composition of xylem components in figured Karelian birch, characterized by increased parenchymatization and reduced vessel quantity. In all studied trunk tissues of Karelian birch, compared with common silver birch, an increase in the expression of the CWINV gene family and the SUS3 gene and a decrease in the expression of SUS4 were shown. CONCLUSION: Therefore, the increase in parenchymatization in figured Karelian birch is linked to a shift in sucrose metabolism towards the apoplastic pathway, indicated by a higher cell wall invertase activity and gene expression. The expression of the SUS4 gene correlates with the decrease in xylem increments and vessel proportion. The research findings will enhance our understanding of how sucrose breaking enzymes regulate secondary growth in woody plants and aid in developing practical timber cultivation methods.

Metagenomic Analysis of Rhizospheric Bacterial Community of Citrus Trees Expressing Phloem-Directed Antimicrobials.

Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and ß-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.

The water and oridonin sources in the ice ribbons of Isodon rubescens.

To study the source and content change of oridonin in the ice ribbons, the contents of oridonin in the ice ribbons and bleeding sap of Isodon rubescens at different times were determined with RP-HPLC. The paraffin sectioning and electron microscopy imaging were performed to study the transport channel of oridonin in the stem. The results showed that there were abundant xylem rays and perfect pit pairs in the secondary xylem of I. rubescens stems. The oridonin content in the ice ribbons of I. rubescens stems was lower than that in the stem of I. rubescens and even decreased over time. The contents of oridonin in the bleeding sap of I. rubescens stems was equal to that in second-day ice ribbons and was lower than that in first-day ice ribbons. The water in the ice ribbons of I. rubescens stems originated from water absorbed by the roots from soil. This water was transported from the roots of I. rubescens to the stem and then transferred through efficient lateral conducting tissues to the surface of the stem. The oridonin in the phloem and cortex of I. rubescens stems dissolves in water originating from the soil and freezes in the form of ice ribbons below 0 °C.

Companion cell mediates wound-stimulated leaf-to-leaf electrical signaling.

Leaf wounding triggers rapid long-range electrical signaling that initiates systemic defense responses to protect the plants from further attack. In Arabidopsis, this process largely depends on clade three GLUTAMATE RECEPTOR-LIKE (GLR) genes GLR3.3 and GLR3.6. In the cellular context, phloem sieve elements and xylem contact cells where GLRs were mostly present are implicated in the signaling events. In spite of that, the spatial requirements of different leaf cell types for leaf-to-leaf signaling remain poorly investigated. In this study, we dissected cell-type-specific long-distance wound signaling mediated by GLR3s and showed that phloem companion cells are critical in shaping the functions of GLR3.3 and GLR3.6 in the signaling pathway. GLR3.3-mediated response is phloem-specific, during which, GLR3.3 has to be renewed from companion cells to allow its function in sieve elements. GLR3.6 functions dually in ectopic phloem companion cells, in addition to xylem contact cells. Furthermore, the action of GLR3.6 in phloem is independent of its paralog GLR3.3 and probably requires synthesis of GLR3.6 from xylem contact cells. Overall, our work highlights that the phloem companion cell is crucial for both GLRs in controlling leaf-to-leaf electrical signaling.

Brown planthoppers manipulate rice sugar transporters to benefit their own feeding.

The feeding of piercing-sucking insect herbivores often elicits changes in their host plants that benefit the insect.1 In addition to thwarting a host's defense responses, these phloem-feeding insects may manipulate source-sink signaling so as to increase resources consumed.2,3 To date, the molecular mechanisms underlying herbivore-induced resource reallocation remain less investigated. Brown planthopper (BPH), an important rice pest, feeds on the phloem and oviposits into leaf sheaths. BPH herbivory increases sugar accumulations 5-fold in the phloem sap of leaf sheaths and concurrently induces the expression of two clade III SWEET genes, SWEET13 and SWEET14, in leaf tissues, but not in leaf sheaths of attacked rice plants. Mutations of both genes by genome editing attenuate resistance to BPH without alterations of known chemical and physical defense responses. Moreover, BPH-elicited sugar levels in the phloem sap were significantly reduced in sweet13/14 mutants, which is likely to attenuate BPH feeding behavior on sweet13/14 mutants. In one of the two field seasons tested, the sweet13/14 mutants showed comparable yield to wild types, and in the other season, the mutants demonstrated stronger BPH resistance. These preliminary results suggested that the mutations in these SWEET transporters could enhance BPH resistance without yield penalties. Given that sweet13/14 mutants also exhibit resistance to bacterial blight pathogen, Xanthomonas oryzae pv. oryzae, these SWEET genes could serve as excellent molecular targets for the breeding of resistant rice cultivars.

WUSCHEL-RELATED HOMEOBOX genes are crucial for normal vascular organization and wood formation in poplar.

Vascular cambium in tree species is a cylindrical domain of meristematic cells that are responsible for producing secondary xylem (also called wood) inward and secondary phloem outward. The poplar (Populus trichocarpa) WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family members, PtrWUSa and PtrWOX13b, were previously shown to be expressed in vascular cambium and differentiating xylem cells in poplar stems, but their functions remain unknown. Here, we investigated roles of PtrWUSa, PtrWOX13b and their close homologs in vascular organization and wood formation. Expression analysis showed that like PtrWUSa and PtrWOX13b, their close homologs, PtrWUSb, PtrWUS4a/b and PtrWOX13a/c, were also expressed in vascular cambium and differentiating xylem cells in poplar stems. PtrWUSa also exhibited a high level of expression in developing phloem fibers. Expression of PtrWUSa fused with the dominant EAR repression domain (PtrWUSa-DR) in transgenic poplar caused retarded growth of plants with twisted stems and curled leaves and a severe disruption of vascular organization. In PtrWUSa-DR stems, a drastic proliferation of cells occurred in the phloem region between vascular cambium and phloem fibers and they formed islands of ectopic vascular tissues or phloem fiber-like sclerenchyma cells. A similar proliferation of cells was also observed in PtrWUSa-DR leaf petioles and midveins. On the other hand, overexpression of PtrWOX4a-DR caused ectopic formation of vascular bundles in the cortical region, and overexpression of PtrWOX13a-DR and PtrWOX13b-DR led to a reduction in wood formation without affecting vascular organization in transgenic poplar plants. Together, these findings indicate crucial roles of PtrWUSa and PtrWOX13a/b in regulating vascular organization and wood formation, which furthers our understanding of the functions of WOX genes in regulating vascular cambium activity in tree species.

Potato spindle tuber viroid (PSTVd) loop 27 mutants promote cell-to-cell movement and phloem unloading of the wild type: Insights into RNA-based viroid interactions.

Variations in infection progression with concurrent or prior infections by different viruses, viroids, or their strains are evident, but detailed investigations into viroid variant interactions are lacking. We studied potato spindle tuber viroid intermediate strain (PSTVd-I) to explore variant interactions. Two mutants, U177A/A182U (AU, replication- and trafficking-competent) and U178G/U179G (GG, replication-competent but trafficking-defective) on loop 27 increased cell-to-cell movement of wild-type (WT) PSTVd without affecting replication. In mixed infection assays, both mutants accelerated WT phloem unloading, while only AU promoted it in separate leaf assays, suggesting that enhancement of WT infection is not due to systemic signals. The mutants likely enhance WT infection due to their loop-specific functions, as evidenced by the lack of impact on WT infection seen with the distantly located G347U (UU) mutant. This study provides the first comprehensive analysis of viroid variant interactions, highlighting the prolonged phloem unloading process as a significant barrier to systemic spread.

Genome-Wide Identification of TLP Gene Family in Populus trichocarpa and Functional Characterization of PtTLP6, Preferentially Expressed in Phloem.

Thaumatin-like proteins (TLPs) in plants are involved in diverse biotic and abiotic stresses, including antifungal activity, low temperature, drought, and high salinity. However, the roles of the TLP genes are rarely reported in early flowering. Here, the TLP gene family was identified in P. trichocarpa. The 49 PtTLP genes were classified into 10 clusters, and gene structures, conserved motifs, and expression patterns were analyzed in these PtTLP genes. Among 49 PtTLP genes, the PtTLP6 transcription level is preferentially high in stems, and GUS staining signals were mainly detected in the phloem tissues of the PtTLP6pro::GUS transgenic poplars. We generated transgenic Arabidopsis plants overexpressing the PtTLP6 gene, and its overexpression lines showed early flowering phenotypes. However, the expression levels of main flowering regulating genes were not significantly altered in these PtTLP6-overexpressing plants. Our data further showed that overexpression of the PtTLP6 gene led to a reactive oxygen species (ROS) burst in Arabidopsis, which might advance the development process of transgenic plants. In addition, subcellular localization of PtTLP6-fused green fluorescent protein (GFP) was in peroxisome, as suggested by tobacco leaf transient transformation. Overall, this work provides a comprehensive analysis of the TLP gene family in Populus and an insight into the role of TLPs in woody plants.

Leafhopper salivary carboxylesterase suppresses JA-Ile synthesis to facilitate initial arbovirus transmission in rice phloem.

Plant jasmonoyl-L-isoleucine (JA-Ile) is a major defense signal against insect feeding, but whether or how insect salivary effectors suppress JA-Ile synthesis and thus facilitate viral transmission in the plant phloem remains elusive. Insect carboxylesterases (CarEs) are the third major family of detoxification enzymes. Here, we identify a new leafhopper CarE, CarE10, that is specifically expressed in salivary glands and is secreted into the rice phloem as a saliva component. Leafhopper CarE10 directly binds to rice jasmonate resistant 1 (JAR1) and promotes its degradation by the proteasome system. Moreover, the direct association of CarE10 with JAR1 clearly impairs JAR1 enzyme activity for conversion of JA to JA-Ile in an in vitro JA-Ile synthesis system. A devastating rice reovirus activates and promotes the co-secretion of virions and CarE10 via virus-induced vesicles into the saliva-storing salivary cavities of the leafhopper vector and ultimately into the rice phloem to establish initial infection. Furthermore, a virus-mediated increase in CarE10 secretion or overexpression of CarE10 in transgenic rice plants causes reduced levels of JAR1 and thus suppresses JA-Ile synthesis, promoting host attractiveness to insect vectors and facilitating initial viral transmission. Our findings provide insight into how the insect salivary protein CarE10 suppresses host JA-Ile synthesis to promote initial virus transmission in the rice phloem.

Understanding phloem's role in long-distance transport and accumulation of arsenic (As) in rice: toward low-As-accumulating grain development.

MAIN CONCLUSION: This review highlights the roles of phloem in the long-distance transport and accumulation of As in rice plants, facilitating the formulation of new strategies to reduce the grain As content. Rice is a staple diet for a significant proportion of the global population. As toxicity is a major issue affecting the rice productivity and quality worldwide. Phloem tissues of rice plants play vital roles in As speciation, long-distance transport, and unloading, thereby controlling the As accumulation in rice grains. Phloem transport accounts for a significant proportion of As transport to grains, ranging from 54 to 100% depending on the species [inorganic arsenate (As(V)), arsenite (As(III)), or organic dimethylarsinic acid (DMA(V)]. However, the specific mechanism of As transport through phloem leading to its accumulation in grains remains unknown. Therefore, understanding the molecular mechanism of phloem-mediated As transport is necessary to determine the roles of phloem in long-distance As transport and subsequently reduce the grain As content via biotechnological interventions. This review discusses the roles of phloem tissues in the long-distance transport and accumulation of As in rice grains. This review also highlights the biotechnological approaches using critical genetic factors involved in nodal accumulation, vacuolar sequestration, and cellular efflux of As in phloem- or phloem-associated tissues. Furthermore, the limitations of existing transgenic techniques are outlined to facilitate the formulation of novel strategies for the development of rice with reduced grain As content.

Histochemical and ultrastructural localization of triterpene saponins in Medicago truncatula.

In the Medicago genus, saponins are complex mixtures of triterpene pentacyclic glycosides extensively studied for their different and economically relevant biological and pharmaceutical properties. This research is aimed at determining for the first time the tissue and cellular localization of triterpene saponins in vegetative organs of Medicago truncatula, a model plant species for legumes, by histochemistry and transmission electron microscopy. The results showed that saponins are present mainly in the palisade mesophyll layer of leaves, whereas in stems they are mostly located in the primary phloem and the subepidermal cells of cortical parenchyma. In root tissue, saponins occur in the secondary phloem region. Transmission electron microscopy revealed prominent saponin accumulation within the leaf and stem chloroplasts, while in the roots the saponins are found in the vesicular structures. Our results demonstrate the feasibility of using histochemistry and transmission electron microscopy to localize M. truncatula saponins at tissue and cellular levels and provide important information for further studies on biosynthesis and regulation of valuable bioactive saponins on agronomic relevant Medicago spp., such as alfalfa (Medicago sativa L.). RESEARCH HIGHLIGHTS: The Medicago genus represents a valuable rich source of saponins, one of the most interesting groups of secondary plant metabolites, which possess relevant biological and pharmacological properties. Plant tissue and cellular localization of saponins is of great importance to better understand their biological functions, biosynthetic pathway, and regulatory mechanisms. We elucidate the localization of saponins in Medicago truncatula with histochemical and transmission electron microscopy studies.

Evaluation of novel promoters for vascular tissue-specific gene expression in Populus.

Due to the extended generation cycle of trees, the breeding process for forest trees tends to be time-consuming. Genetic engineering has emerged as a viable approach to expedite the genetic breeding of forest trees. However, current genetic engineering techniques employed in forest trees often utilize continuous expression promoters such as CaMV 35S, which may result in unintended consequences by introducing genes into non-target tissues. Therefore, it is imperative to develop specific promoters for forest trees to facilitate targeted and precise design and breeding. In this study, we utilized single-cell RNA-Seq data and co-expression network analysis during wood formation to identify three vascular tissue-specific genes in poplar, PP2-A10, PXY, and VNS07, which are expressed in the phloem, cambium/expanding xylem, and mature xylem, respectively. Subsequently, we cloned the promoters of these three genes from '84K' poplar and constructed them into a vector containing the eyGFPuv visual selection marker, along with the 35S mini enhancer to drive GUS gene expression. Transgenic poplars expressing the ProPagPP2-A10::GUS, ProPagPXY::GUS, and ProPagVNS07::GUS constructs were obtained. To further elucidate the tissue specificity of these promoters, we employed qPCR, histochemical staining, and GUS enzyme activity. Our findings not only establish a solid foundation for the future utilization of these promoters to precisely express of specific functional genes in stems but also provide a novel perspective for the modular breeding of forest trees.

Study on the Design, Synthesis, Bioactivity and Translocation of the Conjugates of Phenazine-1-carboxylic Acid and N-Phenyl Alanine Ester.

The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by 1H NMR, 13C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC50 value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1-F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 µmol/L, which was six times higher than Metalaxyl (3.56 µmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the "ion trap" effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds.

Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins.

The signature feature of all plant viruses is the encoding of movement proteins (MPs) that supports the movement of the viral genome into adjacent cells and through the vascular system. The recent discovery of umbravirus-like viruses (ULVs), some of which only encode replication-associated proteins, suggested that they, as with umbraviruses that lack encoded capsid proteins (CPs) and silencing suppressors, would require association with a helper virus to complete an infection cycle. We examined the infection properties of 2 ULVs: citrus yellow vein associated virus 1 (CY1), which only encodes replication proteins, and closely related CY2 from hemp, which encodes an additional protein (ORF5CY2) that was assumed to be an MP. We report that both CY1 and CY2 can independently infect the model plant Nicotiana benthamiana in a phloem-limited fashion when delivered by agroinfiltration. Unlike encoded MPs, ORF5CY2 was dispensable for infection of CY2, but was associated with faster symptom development. Examination of ORF5CY2 revealed features more similar to luteoviruses/poleroviruses/sobemovirus CPs than to 30K class MPs, which all share a similar single jelly-roll domain. In addition, only CY2-infected plants contained virus-like particles (VLPs) associated with CY2 RNA and ORF5CY2. CY1 RNA and a defective (D)-RNA that arises during infection interacted with host protein phloem protein 2 (PP2) in vitro and in vivo, and formed a high molecular weight complex with sap proteins in vitro that was partially resistant to RNase treatment. When CY1 was used as a virus-induced gene silencing (VIGS) vector to target PP2 transcripts, CY1 accumulation was reduced in systemic leaves, supporting the usage of PP2 for systemic movement. ULVs are therefore the first plant viruses encoding replication and CPs but no MPs, and whose systemic movement relies on a host MP. This explains the lack of discernable helper viruses in many ULV-infected plants and evokes comparisons with the initial viruses transferred into plants that must have similarly required host proteins for movement.

Studying plant vascular development using single-cell approaches.

Vascular cells form a highly complex and heterogeneous tissue. Its composition, function, shape, and arrangement vary with the developmental stage and between organs and species. Understanding the transcriptional regulation underpinning this complexity thus requires a high-resolution technique that is capable of capturing rapid events during vascular cell formation. Single-cell and single-nucleus RNA sequencing (sc/snRNA-seq) approaches provide powerful tools to extract transcriptional information from these lowly abundant and dynamically changing cell types, which allows the reconstruction of developmental trajectories. Here, we summarize and reflect on recent studies using single-cell transcriptomics to study vascular cell types and discuss current and future implementations of sc/snRNA-seq approaches in the field of vascular development.