Incorporating non-equilibrium dynamics into demographic history inferences of a migratory marine species.

Understanding how dispersal and gene flow link geographically separated the populations over evolutionary history is challenging, particularly in migratory marine species. In southern right whales (SRWs, Eubalaena australis), patterns of genetic diversity are likely influenced by the glacial climate cycle and recent history of whaling. Here we use a dataset of mitochondrial DNA (mtDNA) sequences (n = 1327) and nuclear markers (17 microsatellite loci, n = 222) from major wintering grounds to investigate circumpolar population structure, historical demography and effective population size. Analyses of nuclear genetic variation identify two population clusters that correspond to the South Atlantic and Indo-Pacific ocean basins that have similar effective breeder estimates. In contrast, all wintering grounds show significant differentiation for mtDNA, but no sex-biased dispersal was detected using the microsatellite genotypes. An approximate Bayesian computation (ABC) approach with microsatellite markers compared the scenarios with gene flow through time, or isolation and secondary contact between ocean basins, while modelling declines in abundance linked to whaling. Secondary-contact scenarios yield the highest posterior probabilities, implying that populations in different ocean basins were largely isolated and came into secondary contact within the last 25,000 years, but the role of whaling in changes in genetic diversity and gene flow over recent generations could not be resolved. We hypothesise that these findings are driven by factors that promote isolation, such as female philopatry, and factors that could promote dispersal, such as oceanographic changes. These findings highlight the application of ABC approaches to infer the connectivity in mobile species with complex population histories and, currently, low levels of differentiation.

A technique for improved focused ion beam milling of cryo-prepared life science specimens.

The combination of focused ion beam and scanning electron microscopy with a cryo-preparation/transfer system allows specimens to be milled at low temperatures. However, for biological specimens in particular, the quality of results is strongly dependent on correct preparation of the specimen surface. We demonstrate a method for deposition of a protective, planarizing surface layer onto a cryo-sample, enabling high-quality cross-sectioning using the ion beam and investigation of structures at the nanoscale.

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids.

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. leguminosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutant, or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis.

The biological reduction of atmospheric N2 to ammonium (nitrogen fixation) provides about 65% of the biosphere's available nitrogen. Most of this ammonium is contributed by legume-rhizobia symbioses, which are initiated by the infection of legume hosts by bacteria (rhizobia), resulting in formation of root nodules. Within the nodules, rhizobia are found as bacteroids, which perform the nitrogen fixation: to do this, they obtain sources of carbon and energy from the plant, in the form of dicarboxylic acids. It has been thought that, in return, bacteroids simply provide the plant with ammonium. But here we show that a more complex amino-acid cycle is essential for symbiotic nitrogen fixation by Rhizobium in pea nodules. The plant provides amino acids to the bacteroids, enabling them to shut down their ammonium assimilation. In return, bacteroids act like plant organelles to cycle amino acids back to the plant for asparagine synthesis. The mutual dependence of this exchange prevents the symbiosis being dominated by the plant, and provides a selective pressure for the evolution of mutualism.

A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity.

Epidemiological studies have documented a reduced prevalence of Alzheimer's disease among users of nonsteroidal anti-inflammatory drugs (NSAIDs). It has been proposed that NSAIDs exert their beneficial effects in part by reducing neurotoxic inflammatory responses in the brain, although this mechanism has not been proved. Here we report that the NSAIDs ibuprofen, indomethacin and sulindac sulphide preferentially decrease the highly amyloidogenic Abeta42 peptide (the 42-residue isoform of the amyloid-beta peptide) produced from a variety of cultured cells by as much as 80%. This effect was not seen in all NSAIDs and seems not to be mediated by inhibition of cyclooxygenase (COX) activity, the principal pharmacological target of NSAIDs. Furthermore, short-term administration of ibuprofen to mice that produce mutant beta-amyloid precursor protein (APP) lowered their brain levels of Abeta42. In cultured cells, the decrease in Abeta42 secretion was accompanied by an increase in the Abeta(1-38) isoform, indicating that NSAIDs subtly alter gamma-secretase activity without significantly perturbing other APP processing pathways or Notch cleavage. Our findings suggest that NSAIDs directly affect amyloid pathology in the brain by reducing Abeta42 peptide levels independently of COX activity and that this Abeta42-lowering activity could be optimized to selectively target the pathogenic Abeta42 species.

Cell wall architecture of the elongating maize coleoptile.

The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans (GAXs), and mixed-linkage beta-glucans, together with smaller amounts of xyloglucans, glucomannans, pectins, and a network of polyphenolic substances. Chemical imaging by Fourier transform infrared microspectroscopy revealed large differences in the distributions of many chemical species between different tissues of the maize (Zea mays) coleoptile. This was confirmed by chemical analyses of isolated outer epidermal tissues compared with mesophyll-enriched preparations. Glucomannans and esterified uronic acids were more abundant in the epidermis, whereas beta-glucans were more abundant in the mesophyll cells. The localization of beta-glucan was confirmed by immunocytochemistry in the electron microscope and quantitative biochemical assays. We used field emission scanning electron microscopy, infrared microspectroscopy, and biochemical characterization of sequentially extracted polymers to further characterize the cell wall architecture of the epidermis. Oxidation of the phenolic network followed by dilute NaOH extraction widened the pores of the wall substantially and permitted observation by scanning electron microscopy of up to six distinct microfibrillar lamellae. Sequential chemical extraction of specific polysaccharides together with enzymic digestion of beta-glucans allowed us to distinguish two distinct domains in the grass primary wall. First, a beta-glucan-enriched domain, coextensive with GAXs of low degrees of arabinosyl substitution and glucomannans, is tightly associated around microfibrils. Second, a GAX that is more highly substituted with arabinosyl residues and additional glucomannan provides an interstitial domain that interconnects the beta-glucan-coated microfibrils. Implications for current models that attempt to explain the biochemical and biophysical mechanism of wall loosening during cell growth are discussed.

Evidence for significant differences in microsomal drug glucuronidation by canine and human liver and kidney.

The in vitro glucuronidation of a range of structurally diverse chemicals has been studied in hepatic and renal microsomes from human donors and the beagle dog. These studies were undertaken to improve on the limited knowledge of glucuronidation by the dog and to assess its suitability as a model species for pharmacokinetic studies. In general, the compounds studied were glucuronidated severalfold more rapidly (based on intrinsic clearance estimates) by DLM than by HLM. Intrinsic clearance values for human UGT1A1 and UGT2B7 substrates were an order of magnitude higher in DLM than in HLM (e.g., gemfibrozil: 31 microl/min/mg versus 3.0 microl/min/mg; ketoprofen: 2.4 microl/min/mg versus 0.2 microl/min/mg). There were also drug-specific differences. HLM readily glucuronidated propofol (2.4 microl/min/mg) whereas DLM appeared unable to glucuronidate this drug directly. Regioselective differences in morphine glucuronidation were also apparent. Human kidney microsomes catalyzed the glucuronidation of many xenobiotics, although glucuronidation of the endobiotic bilirubin was not detectable in this tissue. In direct contrast, dog kidney microsomes glucuronidated bilirubin only (no glucuronidation of all other xenobiotics was detected). These preliminary studies indicated significant differences in the glucuronidation of xenobiotics by microsomes from the livers and kidneys of human and dog and should be confirmed using a larger panel of tissues from individual dogs. Early knowledge of the relative rates of in vitro glucuronidation, the UGTs responsible for drug glucuronidation, and their tissue distribution in different species could assist the design and analysis of preclinical pharmacokinetic and safety evaluation studies.

Escherichia coli strains blocked in Tat-dependent protein export exhibit pleiotropic defects in the cell envelope.

The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect. Both phenotypes are similar to those displayed by strains carrying point mutations in the lpxC (envA) gene. The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates.

Cell-free assays for gamma-secretase activity.

The amyloid b-protein (Ab) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid b-protein precursor (APP). Generation of Ab from the APP requires two sequential proteolytic events: an initial b-secretase cleavage at the amino terminus of the Ab sequence followed by g-secretase cleavage at the carboxyl terminus of Ab. We describe the development of a robust in vitro assay for g-secretase cleavage by showing de novo Ab production in vitro and establish that this assay monitors authentic gamma-secretase activity by documenting the production of a cognate g-CTF, confirming the size of the Ab produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter g-secretase activity in living cells. Using this assay, we demonstrate that the g-secretase activity 1) is tightly associated with the membrane, 2) can be solubilized, 3) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4) ascertain that activities of the g-40 and g-42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.

WhiA, a protein of unknown function conserved among gram-positive bacteria, is essential for sporulation in Streptomyces coelicolor A3(2).

The whiA sporulation gene of Streptomyces coelicolor A3(2), which plays a key role in switching aerial hyphae away from continued extension growth and toward sporulation septation, was cloned by complementation of whiA mutants. DNA sequencing of the wild-type allele and five whiA mutations verified that whiA is a gene encoding a protein with homologues in all gram-positive bacteria whose genome sequence is known, whether of high or low G+C content. No function has been attributed to any of these WhiA-like proteins. In most cases, as in S. coelicolor, the whiA-like gene is downstream of other conserved genes in an operon-like cluster. Phenotypic analysis of a constructed disruption mutant confirmed that whiA is essential for sporulation. whiA is transcribed from at least two promoters, the most downstream of which is located within the preceding gene and is strongly up-regulated when colonies are undergoing sporulation. The up-regulation depends on a functional whiA gene, suggesting positive autoregulation, although it is not known whether this is direct or indirect. Unlike the promoters of some other sporulation-regulatory genes, the whiA promoter does not depend on the sporulation-specific sigma factor encoded by whiG.

Characterization of the uridine diphosphate-glucuronosyltransferase-catalyzing thyroid hormone glucuronidation in man.

Increased thyroid hormone glucuronidation in rats caused by exposure to xenobiotics has stimulated a search for the individual uridine diphosphate-glucuronosyltransferases (UGTs) catalyzing this reaction in rats and man. Microsomal preparations from Crigler-Najjar liver, normal human liver, and kidney have been used to try to identify the UGT isoforms responsible for glucuronidation of the thyroid hormones. The predominant thyroid hormone released from the thyroid gland, T4, and the inactive rT3 are glucuronidated by cloned expressed bilirubin UGT1A1 and also phenol UGT1A9. Results from Crigler-Najjar microsomal samples indicate that UGT1A1 is the main contributor to thyroid hormone glucuronidation in the liver, with rT3 being the preferential substrate. In kidney microsomes thyroid hormone glucuronidation is more complex, suggesting that more than just the UGT1A9 isoform may be involved. Bioactive T3 is not significantly glucuronidated by these isoforms and other UGTs, and sulfotransferases may be involved.

Presenilin 1 regulates pharmacologically distinct gamma -secretase activities. Implications for the role of presenilin in gamma -secretase cleavage.

Presenilins (PSs) are polytopic membrane proteins that have been implicated as potential therapeutic targets in Alzheimer's disease because of their role in regulating the gamma-secretase cleavage that generates the amyloid beta protein (Abeta). It is not clear how PSs regulate gamma-secretase cleavage, but there is evidence that PSs could be either essential cofactors in the gamma-secretase cleavage, gamma-secretase themselves, or regulators of intracellular trafficking that indirectly influence gamma-secretase cleavage. Using presenilin 1 (PS1) mutants that inhibit Abeta production in conjunction with transmembrane domain mutants of the amyloid protein precursor that are cleaved by pharmacologically distinct gamma-secretases, we show that PS1 regulates multiple pharmacologically distinct gamma-secretase activities as well as inducible alpha-secretase activity. It is likely that PS1 acts indirectly to regulate these activities (as in a trafficking or chaperone role), because these data indicate that for PS1 to be gamma-secretase it must either have multiple active sites or exist in a variety of catalytically active forms that are altered to an equivalent extent by the mutations we have studied.

Virus-induced silencing of a plant cellulose synthase gene.

Specific cDNA fragments corresponding to putative cellulose synthase genes (CesA) were inserted into potato virus X vectors for functional analysis in Nicotiana benthamiana by using virus-induced gene silencing. Plants infected with one group of cDNAs had much shorter internode lengths, small leaves, and a "dwarf" phenotype. Consistent with a loss of cell wall cellulose, abnormally large and in many cases spherical cells ballooned from the undersurfaces of leaves, particularly in regions adjacent to vascular tissues. Linkage analyses of wall polysaccharides prepared from infected leaves revealed a 25% decrease in cellulose content. Transcript levels for at least one member of the CesA cellulose synthase gene family were lower in infected plants. The decrease in cellulose content in cell walls was offset by an increase in homogalacturonan, in which the degree of esterification of carboxyl groups decreased from approximately 50 to approximately 33%. The results suggest that feedback loops interconnect the cellular machinery controlling cellulose and pectin biosynthesis. On the basis of the phenotypic features of the infected plants, changes in wall composition, and the reduced abundance of CesA mRNA, we concluded that the cDNA fragments silenced one or more cellulose synthase genes.

WhiD and WhiB, homologous proteins required for different stages of sporulation in Streptomyces coelicolor A3(2).

The whiD locus, which is required for the differentiation of Streptomyces coelicolor aerial hyphae into mature spore chains, was localized by map-based cloning to the overlap between cosmids 6G4 and D63 of the minimal ordered library of Redenbach et al. (M. Redenbach et al., Mol. Microbiol. 21:77-96, 1996). Subcloning and sequencing showed that whiD encodes a homologue of WhiB, a protein required for the initiation of sporulation septation in S. coelicolor. WhiD and WhiB belong to a growing family of small (76- to 112-residue) proteins of unknown biochemical function in which four cysteines are absolutely conserved; all known members of this family are found in the actinomycetes. A constructed whiD null mutant showed reduced levels of sporulation, and those spores that did form were heat sensitive, lysed extensively, and were highly irregular in size, arising at least in part from irregularity in septum placement. The whiD null mutant showed extreme variation in spore cell wall deposition; most spores had uniformly thin (20- to 30-nm) walls, but spore chains were frequently observed in which there was irregular but very pronounced (up to 170 nm) cell wall thickening at the junctions between spores. whiD null mutant spores were frequently partitioned into irregular smaller units through the deposition of additional septa, which were often laid down in several different planes, very close to the spore poles. These "minicompartments" appeared to be devoid of chromosomal DNA. Two whiD promoters, whiDp1 and whiDp2, were identified, and their activities were analyzed during development of wild-type S. coelicolor on solid medium. Both promoters were developmentally regulated; whiDp1 and whiDp2 transcripts were detected transiently, approximately at the time when sporulation septa were observed in the aerial hyphae.

Tissue distribution and interindividual variation in human UDP-glucuronosyltransferase activity: relationship between UGT1A1 promoter genotype and variability in a liver bank.

The variability in a liver bank and tissue distribution of three probe UDP-glucuronosyltransferase (UGT) activities were determined as a means to predict interindividual differences in expression and the contribution of extrahepatic metabolism to presystemic and systemic clearance. Formation rates of acetaminophen-O-glucuronide (APAPG), morphine-3-glucuronide (M3G), and oestradiol-3-glucuronide (E3G) as probes for UGT1A6, 2B7, and 1A1, respectively, were determined in human kidney, liver, and lung microsomes, and in microsomes from intestinal mucosa corresponding to duodenum, jejunum and ileum. While formation of E3G and APAPG were detectable in human kidney microsomes, M3G formation rates from kidney microsomes approached the levels seen in liver, indicating significant expression of UGT2B7. Interestingly, rates of E3G formation in human intestine exceeded the hepatic rates by several fold, while APAPG and M3G formation rates were low. The intestinal apparent Km value for E3G formation was essentially identical to that seen in liver, consistent with intestinal UGT1A1 expression. No UGT activities were observed in lung. Variability in APAPG and M3G activity across a bank of 20 human livers was modest (< or = 7-fold), compared to E3G formation, which varied approximately 30-fold. The E3G formation rates were found to segregate by UGT1A1 promoter genotype, with wild-type (TA)6 rates significantly greater than homozygous mutant (TA)7 individuals. Kinetic analyses were performed to demonstrate that the promoter mutation altered apparent Vmax without significantly affecting apparent Km. These results suggest that glucuronidation, and specifically UGT1A1 activity, can profoundly contribute to intestinal first pass metabolism and interindividual variability due to the expression of common allelic variants.

The SERRATE locus controls the formation of the early juvenile leaves and phase length in Arabidopsis.

The development of the shoot can be divided into a series of distinct developmental phases based on leaf character-istics and inflorescence architecture. The relationship between phase length, defined by the number of organs produced, and the timing of the floral induction (V3-I1 transition) is relatively ill defined. Characterization of the serrate mutant (CS3257; Arabidopsis Biological Research Center) revealed defects in both vegetative and inflores-cence phase lengths, the timing of phase transitions, leaf number, the leaf initiation rate, and phyllotaxy. The timing of floral induction, however, is the same as in wild-type in extended short days as well as in short days, whereas the flowering time response to photoperiod is unaffected. SERRATE is shown to be required for the development of early juvenile leaves (V1) and to promote late juvenile leaf development (V2), while suppressing adult leaf (V3) and inflorescence development (I1 and I2). The se mutation supports the hypothesis that the timing of floral induction is independent of vegetative and inflorescence phase lengths. The role of SERRATE in the regulation of phase length and leaf identity is discussed.

The DIF1 gene of Arabidopsis is required for meiotic chromosome segregation and belongs to the REC8/RAD21 cohesin gene family.

Cohesins are a group of conserved proteins responsible for cohesion between replicated sister chromatids during mitosis and meiosis and which are implicated in double-strand break repair and meiotic recombination. We describe here the identification and characterisation of an Arabidopsis gene - DETERMINATE, INFERTILE1 (DIF1), which is a homolog of the Schizosaccharomyces pombe REC8/RAD21 cohesin genes, and is essential for meiotic chromosome segregation. Five independent alleles of the DIF1 gene were isolated by transposon mutagenesis, and the mutants show complete male and female sterility. Pollen mother cells (PMCs) of dif1 mutants show multiple meiotic defects which are represented by univalent chromosomes and chromosome fragmentation at metaphase I, and acentric fragments and chromatin bridges in meiosis I and II. Consequently, chromosome segregation is strongly affected, resulting in meiotic products of uneven size, shape and of variable ploidy. The similarities in phenotype, and the sequence homology between DIF1 and the REC8/RAD21 cohesins suggests that cohesin function is largely conserved between eukaryotes and highlights the essential role cohesins play in plant meiosis.

New sporulation loci in Streptomyces coelicolor A3(2).

Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci, whiA, whiB, whiD, whiE, whiG, whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9-28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB, whiG, whiH, or whiJ (but not whiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated whiK, whiL, whiM, whiN, and whiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM, whiN, and whiO occupied positions distinct from previously cloned whi genes; whiK was located on the same cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.

Interaction of BP180 (type XVII collagen) and alpha6 integrin is necessary for stabilization of hemidesmosome structure.

The hemidesmosome is a multimolecular complex that integrates the extracellular matrix with the keratin cytoskeleton and that stabilizes epithelial attachment to connective tissue. A 180 kDa protein (BP180, type XVII collagen), first identified by its reactivity with autoantibodies in the serum of patients with a blistering skin disease called bullous pemphigoid (BP), is a transmembrane component of the hemidesmosome with a collagen-like extracellular domain. Here, using recombinantly expressed molecules and the yeast two-hybrid assay, we have identified alpha6 integrin as a BP180-binding partner. The association between specific domains of the BP180 and alpha6 integrin molecules is inhibited by a 14 mer peptide, whose sequence is identical to amino acid residues 506-519 in the noncollagenous region of the ectodomain of the BP180 molecule, as well as by antibodies raised against this peptide. The 14 mer peptide sequence is part of an epitope recognized by autoantibodies that are pathogenic in BP. In vivo, when 804G cells are plated into medium containing the same peptide, they fail to assemble hemidesmosomes. Furthermore, although BP180 and certain cytoplasmic components of the hemidesmosome colocalize in the peptide-treated cells, they are aberrantly distributed and fail to show extensive association with (alpha6beta4 integrin. Taken together, our results indicate that BP180 is a novel transmembrane ligand of the alpha6beta4 integrin heterodimer. In addition, our data provide support for the possibility that BP180 and alpha6 integrin interaction is not only mediated by the BP epitope but is necessary for hemidesmosome formation.

Ultrastructural and temporal observations of the potyvirus cylindrical inclusions (Cls) show that the Cl protein acts transiently in aiding virus movement.

A systematic ultrastructural study across the edge of an advancing infection in pea seed-borne mosaic potyvirus-infected pea cotyledons showed the cylindrical inclusion (CI) protein to exist in transient functional states. Initially, the characteristic CI pinwheel inclusion bodies were positioned centrally over the plasmodesmal apertures (including those of plasmodesmata connected to the previously infected cell), in agreement with a proposed role in virus movement (Carrington et al., 1998, Plant J., 13, in press). The viral coat protein was associated with these structures and was seen within the modified plasmodesma, most notably in a continuous channel that passed along the axis of the pinwheel and through the plasmodesma. The CI protein was not detected within the plasmodesmal cavities. Later in the infection (i.e., behind the zone of active virus replication) the CI was no longer associated with cell walls, or with coat protein, and showed signs of structural degeneration. In contrast, the coat protein remained within plasmodesmal cavities. The role of the CI in assisting virus movement is not known but the presence of the CI was linked with an apparent transient reduction in callose in the vicinity of the plasmodesmata.