Developing type 1 diabetes resources: a qualitative study to identify resources needed to upskill and support community sport coaches.

Introduction: Community sport coaches in Western Australia lack an understanding, the confidence, and knowledge in supporting young people with Type 1 diabetes (T1D). This study aims to identify what T1D educational resources are required to upskill coaches in Western Australia. Methods: Semi-structured online interviews were conducted with i) young people living with T1D, ii) parents of young people living with T1D and iii) community sport coaches. The questions explored i) past experiences of T1D management in community sport ii) the T1D information coaches should be expected to know about and iii) the format of resources to be developed. Thematic analysis of interview transcripts was performed, and the themes identified were used to guide resource development. Results: Thirty-two participants (16 young people living with T1D, 8 parents, 8 coaches) were interviewed. From the interviews, young people wanted coaches to have a better understanding of what T1D is and the effect it has on their sporting performance, parents wanted a resource that explains T1D to coaches, and sports coaches wanted to know the actions to best support a player living with T1D. All groups identified that signs and symptoms of hypoglycaemia and hyperglycaemia needed to be a key component of the resource. Sports coaches wanted a resource that is simple, quick to read and available in a variety of different formats. Conclusion: The interviews resulted in valuable information gained from all groups and have reinforced the need for the development of specific resources to increase community knowledge and provide support for players with T1D, parents and sport coaches.

Experiences of Caregivers and At-Risk Children Enrolled in a Prospective Pregnancy-Birth Cohort Study into the Causes of Type 1 Diabetes: The ENDIA Study.

BACKGROUND: We sought research experiences of caregivers and their children were enrolled in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. METHODS: ENDIA is a pregnancy-birth cohort investigating early-life causes of type 1 diabetes (T1D). Surveys were sent to 1090 families between June 2021 and March 2022 with a median participation of >5 years. Caregivers completed a 12-item survey. Children ≥ 3 years completed a four-item survey. RESULTS: The surveys were completed by 550/1090 families (50.5%) and 324/847 children (38.3%). The research experience was rated as either "excellent" or "good" by 95% of caregivers, and 81% of children were either "ok", "happy" or "very happy". The caregivers were motivated by contributing to research and monitoring their children for T1D. Relationships with the research staff influenced the experience. The children most liked virtual reality headsets, toys, and "helping". Blood tests were least liked by the children and were the foremost reason that 23.4% of the caregivers considered withdrawing. The children valued gifts more than their caregivers. Only 5.9% of responses indicated dissatisfaction with some aspects of the protocol. The self-collection of samples in regional areas, or during the COVID-19 pandemic restrictions, were accepted. CONCLUSIONS: This evaluation identified modifiable protocol elements and was conducted to further improve satisfaction. What was important to the children was distinct from their caregivers.

Experiences and Attitudes of Parents Reducing Carbohydrate Intake in the Management of Their Child's Type 1 Diabetes: A Qualitative Study.

Reducing carbohydrate (CHO) intake is being used as an approach to manage type 1 diabetes (T1D) in children. This study aimed to investigate the experiences and attitudes of parents of children with T1D who are reducing CHO intake to help manage blood glucose levels (BGLs). Semi-structured interviews were conducted with the parents of children with T1D for >1 year who reported implementing a low CHO approach to manage BGLs. Data were analysed using a constant comparative analysis approach. Participants (n = 14) were parents of children (6.6 ± 2.0 years) with T1D in Western Australia. All parents reported different methods of CHO restriction and all perceived that benefits outweighed challenges. Parents reported feeling less worried, had improved sleep and felt their child was safer when using a low CHO approach due to more stable BGLs. Reported challenges included: increased cost and time spent preparing food; perceived judgement from others; and child dissatisfaction with restricted food choices. Parents reported accessing information and support through social media networks. Parents reported a desire for more research into this approach. Understanding the promoters and barriers for this dietary approach may inform strategies to better engage and support families with approaches that align with current evidence while considering their concerns around safety and hyperglycaemia.

Protein Ingestion in Reducing the Risk of Late-Onset Post-Exercise Hypoglycemia: A Pilot Study in Adolescents and Youth with Type 1 Diabetes.

Dietary protein causes dose-dependent hyperglycemia in individuals with type 1 diabetes (T1D). This study investigated the effect of consuming 50 g of protein on overnight blood glucose levels (BGLs) following late-afternoon moderate-intensity exercise. Six participants (3M:3F) with T1D, HbA1c 7.5 ± 0.8% (58.0 ± 8.7 mmol/mol) and aged 20.2 ± 3.1 years exercised for 45 min at 1600 h and consumed a protein drink or water alone at 2000 h, on two separate days. A basal insulin euglycemic clamp was employed to measure the mean glucose infusion rates (m-GIR) required to maintain euglycemia on both nights. The m-GIR on the protein and water nights during the hypoglycemia risk period and overnight were 0.27 ± 043 vs. 1.60 ± 0.66 mg/kg/min (p = 0.028, r = 0.63) and 0.51 ± 0.16 vs. 1.34 ± 0.71 mg/kg/min (p = 0.028, r = 0.63), respectively. Despite ceasing intravenous glucose infusion on the protein night, the BGLs peaked at 9.6 ± 1.6 mmol/L, with a hypoglycemia risk period mean of 7.8 ± 1.5 mmol/L compared to 5.9 ± 0.4 mmol/L (p = 0.028) on the water night. The mean plasma glucagon levels were 51.5 ± 14.1 and 27.2 ± 10.1 ng/L (p = 0.028) on the protein and water night, respectively. This suggests that an intake of protein is effective at reducing the post-exercise hypoglycemia risk, potentially via a glucagon-mediated stimulation of glucose production. However, 50 g of protein may be excessive for maintaining euglycemia.

Phloem connectivity and transport are not involved in mature plant resistance (MPR) to Potato Virus Y in different potato cultivars, and MPR does not protect tubers from recombinant strains of the virus.

The aims of this study were: i) to investigate mature plant resistance (MPR) against four strains of Potato virus Y (PVYO, PVYN, PVYNTN and PVYN-Wi) in potato cultivars that differ in maturity (e.g. early or maincrop) at different developmental stages, and ii) to determine whether phloem translocation of photoassimilates at different stages including the source-sink transition influences MPR. The data showed that MPR was functional by the flowering stage in all cultivars, and that the host-pathogen interaction is highly complex, with all three variables (potato cultivar, virus strain and developmental stage of infection) having a significant effect on the outcome. However, virus strain was the most important factor, and MPR was less effective in protecting tubers from recombinant virus strains (PVYNTN and PVYN-Wi). Development of MPR was unrelated to foliar phloem connectivity, which was observed at all developmental stages, but a switch from symplastic to apoplastic phloem unloading early in tuber development may be involved in the prevention of tuber infections with PVYO. Recombinant virus strains were more infectious than parental strains and PVYNTN has a more effective silencing suppressor than PVYO, another factor that may contribute to the efficiency of MPR. The resistance conferred by MPR against PVYO or PVYN may be associated with or enhanced by the presence of the corresponding strain-specific HR resistance gene in the cultivar.

A Novel Mobile Health App to Educate and Empower Young People With Type 1 Diabetes to Exercise Safely: Prospective Single-Arm Mixed Methods Pilot Study.

BACKGROUND: Empowering young people with type 1 diabetes (T1D) to manage their blood glucose levels during exercise is a complex challenge faced by health care professionals due to the unpredictable nature of exercise and its effect on blood glucose levels. Mobile health (mHealth) apps would be useful as a decision-support aid to effectively contextualize a blood glucose result and take appropriate action to optimize glucose levels during and after exercise. A novel mHealth app acT1ve was recently developed, based on expert consensus exercise guidelines, to provide real-time support for young people with T1D during exercise. OBJECTIVE: Our aim was to pilot acT1ve in a free-living setting to assess its acceptability and functionality, and gather feedback on the user experience before testing it in a larger clinical trial. METHODS: A prospective single-arm mixed method design was used. Ten participants with T1D (mean age 17.7 years, SD 4.2 years; mean HbA1c, 54 mmol/mol, SD 5.5 mmol/mol [7.1%, SD 0.5%]) had acT1ve installed on their phones, and were asked to use the app to guide their exercise management for 6 weeks. At the end of 6 weeks, participants completed both a semistructured interview and the user Mobile Application Rating Scale (uMARS). All semistructured interviews were transcribed. Thematic analysis was conducted whereby interview transcripts were independently analyzed by 2 researchers to uncover important and relevant themes. The uMARS was scored for 4 quality subscales (engagement, functionality, esthetics, and information), and a total quality score was obtained from the weighted average of the 4 subscales. Scores for the 4 objective subscales were determined by the mean score of each of its individual questions. The perceived impact and subjective quality of acT1ve for each participant were calculated by averaging the scores of their related questions, but were not considered in the total quality score. All scores have a maximal possible value of 5, and they are presented as medians, IQRs, and ranges. RESULTS: The main themes arising from the interview analysis were "increased knowledge," "increased confidence to exercise," and "suitability" for people who were less engaged in exercise. The uMARS scores for acT1ve were high (out of 5) for its total quality (median 4.3, IQR 4.2-4.6), engagement (median 3.9, IQR 3.6-4.2), functionality (median 4.8, IQR 4.5-4.8), information (median 4.6, IQR 4.5-4.8), esthetics (median 4.3, IQR 4.0-4.7), subjective quality (median 4.0, IQR 3.8-4.2), and perceived impact (median 4.3, IQR 3.6-4.5). CONCLUSIONS: The acT1ve app is functional and acceptable, with a high user satisfaction. The efficacy and safety of this app will be tested in a randomized controlled trial in the next phase of this study. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12619001414101; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=378373.

TERMINAL FLOWER-1/CENTRORADIALIS inhibits tuberisation via protein interaction with the tuberigen activation complex.

Potato tuber formation is a secondary developmental programme by which cells in the subapical stolon region divide and radially expand to further differentiate into starch-accumulating parenchyma. Although some details of the molecular pathway that signals tuberisation are known, important gaps in our knowledge persist. Here, the role of a member of the TERMINAL FLOWER 1/CENTRORADIALIS gene family (termed StCEN) in the negative control of tuberisation is demonstrated for what is thought to be the first time. It is shown that reduced expression of StCEN accelerates tuber formation whereas transgenic lines overexpressing this gene display delayed tuberisation and reduced tuber yield. Protein-protein interaction studies (yeast two-hybrid and bimolecular fluorescence complementation) demonstrate that StCEN binds components of the recently described tuberigen activation complex. Using transient transactivation assays, we show that the StSP6A tuberisation signal is an activation target of the tuberigen activation complex, and that co-expression of StCEN blocks activation of the StSP6A gene by StFD-Like-1. Transcriptomic analysis of transgenic lines misexpressing StCEN identifies early transcriptional events in tuber formation. These results demonstrate that StCEN suppresses tuberisation by directly antagonising the function of StSP6A in stolons, identifying StCEN as a breeding marker to improve tuber initiation and yield through the selection of genotypes with reduced StCEN expression.

Potato Mop-Top Virus Co-Opts the Stress Sensor HIPP26 for Long-Distance Movement.

Virus movement proteins facilitate virus entry into the vascular system to initiate systemic infection. The potato mop-top virus (PMTV) movement protein, TGB1, is involved in long-distance movement of both viral ribonucleoprotein complexes and virions. Here, our analysis of TGB1 interactions with host Nicotiana benthamiana proteins revealed an interaction with a member of the heavy metal-associated isoprenylated plant protein family, HIPP26, which acts as a plasma membrane-to-nucleus signal during abiotic stress. We found that knockdown of NbHIPP26 expression inhibited virus long-distance movement but did not affect cell-to-cell movement. Drought and PMTV infection up-regulated NbHIPP26 gene expression, and PMTV infection protected plants from drought. In addition, NbHIPP26 promoter-reporter fusions revealed vascular tissue-specific expression. Mutational and biochemical analyses indicated that NbHIPP26 subcellular localization at the plasma membrane and plasmodesmata was mediated by lipidation (S-acylation and prenylation), as nonlipidated NbHIPP26 was predominantly in the nucleus. Notably, coexpression of NbHIPP26 with TGB1 resulted in a similar nuclear accumulation of NbHIPP26. TGB1 interacted with the carboxyl-terminal CVVM (prenyl) domain of NbHIPP26, and bimolecular fluorescence complementation revealed that the TGB1-HIPP26 complex localized to microtubules and accumulated in the nucleolus, with little signal at the plasma membrane or plasmodesmata. These data support a mechanism where interaction with TGB1 negates or reverses NbHIPP26 lipidation, thus releasing membrane-associated NbHIPP26 and redirecting it via microtubules to the nucleus, thereby activating the drought stress response and facilitating virus long-distance movement.

Femtosecond optoinjection of intact tobacco BY-2 cells using a reconfigurable photoporation platform.

A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant ("diffraction-free") Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.

The role of the potato (Solanum tuberosum) CCD8 gene in stolon and tuber development.

· Strigolactones (SLs) are a class of phytohormones controlling shoot branching. In potato (Solanum tuberosum), tubers develop from underground stolons, diageotropic stems which originate from basal stem nodes. As the degree of stolon branching influences the number and size distribution of tubers, it was considered timely to investigate the effects of SL production on potato development and tuber life cycle. · Transgenic potato plants were generated in which the CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) gene, key in the SL biosynthetic pathway, was silenced by RNA interference (RNAi). · The resulting CCD8-RNAi potato plants showed significantly more lateral and main branches than control plants, reduced stolon formation, together with a dwarfing phenotype and a lack of flowering in the most severely affected lines. New tubers were formed from sessile buds of the mother tubers. The apical buds of newly formed transgenic tubers grew out as shoots when exposed to light. In addition, we found that CCD8 transcript levels were rapidly downregulated in tuber buds by the application of sprout-inducing treatments. · These results suggest that SLs could have an effect, solely or in combination with other phytohormones, in the morphology of potato plants and also in controlling stolon development and maintaining tuber dormancy.

The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids.

The potato mop-top virus (PMTV) triple gene block 2 (TGB2) movement proteins fused to monomeric red fluorescent protein (mRFP-TGB2) was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localizations and interactions of mRFP-TGB2 were investigated using confocal imaging [confocal laser-scanning microscope, (CLSM)] and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum (ER), mobile granules, small round structures (1-2 µm in diameter), and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labeled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein (CP), genomic RNA and fluorescently-labeled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localized to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts.

The TGB1 movement protein of Potato virus X reorganizes actin and endomembranes into the X-body, a viral replication factory.

Potato virus X (PVX) requires three virally encoded proteins, the triple gene block (TGB), for movement between cells. TGB1 is a multifunctional protein that suppresses host gene silencing and moves from cell to cell through plasmodesmata, while TGB2 and TGB3 are membrane-spanning proteins associated with endoplasmic reticulum-derived granular vesicles. Here, we show that TGB1 organizes the PVX "X-body," a virally induced inclusion structure, by remodeling host actin and endomembranes (endoplasmic reticulum and Golgi). Within the X-body, TGB1 forms helically arranged aggregates surrounded by a reservoir of the recruited host endomembranes. The TGB2/3 proteins reside in granular vesicles within this reservoir, in the same region as nonencapsidated viral RNA, while encapsidated virions accumulate at the outer (cytoplasmic) face of the X-body, which comprises a highly organized virus "factory." TGB1 is both necessary and sufficient to remodel host actin and endomembranes and to recruit TGB2/3 to the X-body, thus emerging as the central orchestrator of the X-body. Our results indicate that the actin/endomembrane-reorganizing properties of TGB1 function to compartmentalize the viral gene products of PVX infection.

Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture.

Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties.

The N-terminal domain of PMTV TGB1 movement protein is required for nucleolar localization, microtubule association, and long-distance movement.

The triple-gene-block (TGB)1 protein of Potato mop-top virus (PMTV) was fused to fluorescent proteins and expressed in epidermal cells of Nicotiana benthamiana under the control of the 35S promoter. TGB1 fluorescence was observed in the cytoplasm, nucleus, and nucleolus and occasionally associated with microtubules. When expressed from a modified virus (PMTV.YFP-TGB1) which formed local lesions but was not competent for systemic movement, yellow fluorescent protein (YFP)-TGB1 labeled plasmodesmata in cells at the leading edge of the lesion and plasmodesmata, microtubules, nuclei, and nucleoli in cells immediately behind the leading edge. Deletion of 84 amino acids from the N-terminus of unlabeled TGB1 within the PMTV genome abolished movement of viral RNA to noninoculated leaves. When the same deletion was introduced into PMTV.YFP-TGB1, labeling of microtubules and nucleoli was abolished. The N-terminal 84 amino acids of TGB1 were fused to green fluorescent protein (GFP) and expressed in epidermal cells where GFP localized strongly to the nucleolus (not seen with unfused GFP), indicating that these amino acids contain a nucleolar localization signal; the fusion protein did not label microtubules. This is the first report of nucleolar and microtubule association of a TGB movement protein. The results suggest that PMTV TGB1 requires interaction with nuclear components and, possibly, microtubules for long-distance movement of viral RNA.

The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection.

Fluorescent proteins (FPs) based on green fluorescent protein (GFP) are widely used throughout cell biology to study protein dynamics, and have extensive use as reporters of virus infection and spread. However, FP-tagging of viruses is limited by the constraints of viral genome size resulting in FP loss through recombination events. To overcome this, we have engineered a smaller ( approximately 10 kDa) flavin-based alternative to GFP ( approximately 25 kDa) derived from the light, oxygen or voltage-sensing (LOV) domain of the plant blue light receptor, phototropin. Molecular evolution and Tobacco mosaic virus (TMV)-based expression screening produced LOV variants with improved fluorescence and photostability in planta. One variant in particular, designated iLOV, possessed photophysical properties that made it ideally suited as a reporter of subcellular protein localization in both plant and mammalian cells. Moreover, iLOV fluorescence was found to recover spontaneously after photobleaching and displayed an intrinsic photochemistry conferring advantages over GFP-based FPs. When expressed either as a cytosolic protein or as a viral protein fusion, iLOV functioned as a superior reporter to GFP for monitoring local and systemic infections of plant RNA viruses. iLOV, therefore, offers greater utility in FP-tagging of viral gene products and represents a viable alternative where functional protein expression is limited by steric constraints or genome size.

A role for symplastic gating in the control of the potato tuber life cycle.

The control of the potato tuber life cycle has been the subject of significant interest over many years. A number of different approaches have been adopted and data is available regarding hormonal, metabolic and gene expression changes that occur over the tuber life cycle. Despite this intense effort, no unifying model for the control of the potato tuber life cycle has emerged. We have undertaken a detailed analysis of the tuber life cycle utilising physiological, biochemical and cell-biological techniques. It has emerged that a major factor contributing to both tuber induction and dormancy break is symplastic gating which controls the allocation of resources to meristematic or vegetative tissues. Future challenges include the determination of factors regulating symplastic gating at the molecular level and the extrapolation of these findings to other systems.

Symplastic connection is required for bud outgrowth following dormancy in potato (Solanum tuberosum L.) tubers.

To gain greater insight into the mechanism of dormancy release in the potato tuber, an investigation into physiological and biochemical changes in tuber and bud tissues during the transition from bud dormancy (immediately after harvest) to active bud growth was undertaken. Within the tuber, a rapid shift from storage metabolism (starch synthesis) to reserve mobilization within days of detachment from the mother plant suggested transition from sink to source. Over the same period, a shift in the pattern of [U-(14)C]sucrose uptake by tuber discs from diffuse to punctate accumulation was consistent with a transition from phloem unloading to phloem loading within the tuber parenchyma. There were no gross differences in metabolic capacity between resting and actively growing tuber buds as determined by [U-(14)C]glucose labelling. However, marked differences in metabolite pools were observed with large increases in starch and sucrose, and the accumulation of several organic acids in growing buds. Carboxyfluorescein labelling of tubers clearly demonstrated strong symplastic connection in actively growing buds and symplastic isolation in resting buds. It is proposed that potato tubers rapidly undergo metabolic transitions consistent with bud outgrowth; however, growth is initially prevented by substrate limitation mediated via symplastic isolation.

Targeting of TMV movement protein to plasmodesmata requires the actin/ER network: evidence from FRAP.

Fluorescence recovery after photobleaching (FRAP) was used to study the mechanism by which fluorescent-protein-tagged movement protein (MP) of tobacco mosaic virus (TMV) is targeted to plasmodesmata (PD). The data show that fluorescence recovery in PD at the leading edge of an infection requires elements of the cortical actin/endoplasmic reticulum (ER) network and can occur in the absence of an intact microtubule (MT) cytoskeleton. Inhibitors of the actin cytoskeleton (latrunculin and cytochalasin) significantly inhibited MP targeting, while MT inhibitors (colchicine and oryzalin) did not. Application of sodium azide to infected cells implicated an active component of MP transfer to PD. Treatment of cells with Brefeldin A (BFA) at a concentration that caused reabsorption of the Golgi bodies into the ER (precluding secretion of viral MP) had no effect on MP targeting, while disruption of the cortical ER with higher concentrations of BFA caused significant inhibition. Our results support a model of TMV MP function in which targeting of MP to PD during infection is mediated by the actin/ER network.

Plasmodesmal Targeting and Accumulation of TMV Movement Protein.

The interaction between tobacco mosaic virus and its host plant cells has been intensively studied as a model for macromolecular trafficking. The observation that GFP-labelled TMV movement protein localises to microtubules led to the suggestion that microtubules are required for the cell to cell movement of the virus. In a recent paper we have demonstrated that the targeting of TMV movement protein to plasmodesmata requires the actin and ER networks, which supports previous evidence from our laboratory that showed that disruption of microtubules did not prevent cell to cell movement of TMV virus, and that a mutated movement protein, which did not localise to micro-tubules, showed enhanced viral movement. In this addendum we speculate where the TMV movement protein accumulates within plasmodesmata, and the relationship of this accumulation to the cell to cell movement of the virus.

Quantification of plasmodesmatal endoplasmic reticulum coupling between sieve elements and companion cells using fluorescence redistribution after photobleaching.

Transgenic tobacco (Nicotiana tabacum) was studied to localize the activity of phloem loading during development and to establish whether the endoplasmic reticulum (ER) of the companion cell (CC) and the sieve element (SE) reticulum is continuous by using a SUC2 promoter-green fluorescent protein (GFP) construct targeted to the CC-ER. Expression of GFP marked the collection phloem in source leaves and cotyledons as expected, but also the transport phloem in stems, petioles, midveins of sink leaves, nonphotosynthetic flower parts, roots, and newly germinated seedlings, suggesting that sucrose retrieval along the pathway is an integral component of phloem function. GFP fluorescence was limited to CCs where it was visualized as a well-developed ER network in close proximity to the plasma membrane. ER coupling between CC and SEs was tested in wild-type tobacco using an ER-specific fluorochrome and fluorescence redistribution after photobleaching (FRAP), and showed that the ER is continuous via pore-plasmodesma units. ER coupling between CC and SE was quantified by determining the mobile fraction and half-life of fluorescence redistribution and compared with that of other cell types. In all tissues, fluorescence recovered slowly when it was rate limited by plasmodesmata, contrasting with fast intracellular FRAP. FRAP was unaffected by treatment with cytochalasin D. The highest degree of ER coupling was measured between CC and SE. Intimate ER coupling is consistent with a possible role for ER in membrane protein and signal exchange between CC and SE. However, a complete lack of GFP transfer between CC and SE indicated that the intraluminal pore-plasmodesma contact has a size exclusion limit below 27 kD.