High outcrossing rates in a self-compatible and highly aggregated host-generalist mistletoe.

Plants have evolved various strategies to avoid inbreeding, but the mass flowering displayed by many plants predisposes them to within-plant pollen movements and self-pollination. Mistletoes often aggregate at multiple spatial scales. Their bird pollinators often visit several flowers of the same individual and of others on the same host tree. We hypothesized that hermaphroditic mistletoes have self-incompatibility mechanisms that reduce or prevent selfing. Whether their spatial distribution, affected by host specificity, host distribution, and the behaviour of seed dispersers, influences their mating system and population genetic structure remains unclear. We studied how mating system and spatial distribution affect genetic structure in four populations of the host-generalist mistletoe Dendrophthoe pentandra in southwestern China using microsatellite markers and progeny arrays. We also characterized the fine-scale spatial genetic structure among 166 mistletoes from four host trees in one population. Prevalence and intensity of infection both appeared to vary among host species, strongly affecting the degree of aggregation. Host tree size had a strong effect on infection intensity. Surprisingly, manual pollination experiments indicated that D. pentandra is self-compatible, but genetic analyses revealed that outcrossing rates were higher than expected in all four populations (MLTR tm 0.83-1.20, Bayesian tm 0.772-0.952). Spatial genetic structure was associated with distance between host trees but not at shorter scales (within hosts). Our results demonstrate that the combination of bird pollination, bird-mediated seed dispersal, and post-dispersal processes result in outcrossing and maintain relatively high diversity in the presence of biparental inbreeding, despite very high local densities and possible self-compatibility.

Geographical context outweighs habitat disturbance effects in explaining mistletoe population genetic differentiation at a regional scale.

Genetic differentiation depends on ecological and evolutionary processes that operate at different spatial and temporal scales. While the geographical context is likely to determine large-scale genetic variation patterns, habitat disturbance events will probably influence small-scale genetic diversity and gene flow patterns. Therefore, the genetic diversity patterns that we observe today result from the combination of both processes, but they are rarely assessed simultaneously. We determined the population structure and genetic diversity of a hemiparasitic mistletoe (Tristerix corymbosus) from the temperate rainforests of southern Chile to determine the effects of geographical context and habitat disturbance at a regional scale and if it is affected by the abundance and occurrence of its seed disperser mutualist (the arboreal marsupial Dromiciops gliroides). We genotyped 359 individuals from 12 populations using single nucleotide polymorphisms, across three different geographical contexts and four disturbance conditions. We also used camera traps to estimate the abundance and occurrence of the seed disperser. Our results suggest that genetic differences among populations are related more to geographical context than to habitat disturbance. However, as disturbance increased, D. gliroides abundance and occurrence decreased, and mistletoe inbreeding index (FIS ) increased. We also found highly uneven gene flow among study sites. Despite the high levels of disturbance that these temperate rainforests are facing, our results suggest that mistletoe genetic differentiation at a regional scale was more influenced by historical events. However, habitat disturbance can indirectly affect mistletoe population genetic differentiation via the seed dispersal process, which may increase levels of inbreeding.

Hybridization and differential introgression associated with environmental shifts in a mistletoe species complex.

Host specialization after host shifting is traditionally viewed as the pathway to speciation in parasitic plants. However, geographical and environmental changes can also influence parasite speciation, through hybridization processes. Here we investigated the impact of past climatic fluctuations, environment, and host shifts on the genetic structure and patterns of hybridization and gene flow between Psittacanthus calyculatus and P. schiedeanus, a Mesoamerican species complex. Using microsatellites (408 individuals), we document moderate genetic diversity but high genetic differentiation between widespread parental clusters, calyculatus in dry pine-oak forests and schiedeanus in cloud forests. Bayesian analyses identified a third cluster, with admixture between parental clusters in areas of xeric and tropical dry forests and high levels of migration rates following secondary contact. Coincidently host associations in these areas differ from those in areas of parental species, suggesting that past hybridization played a role in environmental and host shifts. Overall, the observed genetic and geographic patterns suggest that these Psittacanthus populations could have entered a distinct evolutionary pathway. The results provide evidence for highlights on the importance of the Pleistocene climate changes, habitat differences, and potential host shifts in the evolutionary history of Neotropical mistletoes.

The influence of climatic niche preferences on the population genetic structure of a mistletoe species complex.

The prevalent view on genetic structuring in parasitic plants is that host-race formation is caused by varying degrees of host specificity. However, the relative importance of ecological niche divergence and host specificity to population differentiation remains poorly understood. We evaluated the factors associated with population differentiation in mistletoes of the Psittacanthus schiedeanus complex (Loranthaceae) in Mexico. We used genetic data from chloroplast sequences and nuclear microsatellites to study population genetic structure and tested its association with host preferences and climatic niche variables. Pairwise genetic differentiation was associated with environmental and host preferences, independent of geography. However, environmental predictors appeared to be more important than host preferences to explain genetic structure, supporting the hypothesis that the occurrence of the parasite is largely determined by its own climatic niche and, to a lesser degree, by host specificity. Genetic structure is significant within this mistletoe species complex, but the processes associated with this structure appear to be more complex than previously thought. Although host specificity was not supported as the major determinant of population differentiation, we consider this to be part of a more comprehensive ecological model of mistletoe host-race formation that incorporates the effects of climatic niche evolution.

De novo assembly and transcriptome characterization of spruce dwarf mistletoe Arceuthobium sichuanense uncovers gene expression profiling associated with plant development.

BACKGROUND: The parasitic flowering plant dwarf mistletoe (Arceuthobium spp., Viscaceae) is one of the most destructive forest pests, posing a major threat to numerous conifer species worldwide. Arceuthobium sichuanense (spruce dwarf mistletoe, SDM) infects Qinghai spruce (Picea crassifolia) and causes severe damage to spruce forests in Northwest China. SDM is a Chinese native parasitic plant and acquires carbohydrates and mineral nutrition from its hosts. However, underlying molecular basis of the physiological development is largely unknown. Investigations of these physiological traits have been hampered by the lack of genomic resources for this species. RESULTS: In this study, to investigate the transcriptomic processes underlying physiological traits and development in SDM, we used RNA from four major tissues (i.e., shoots, flowers, fruits, and seeds) for de novo assembly and to annotate the transcriptome of this species. We uncovered the annotated transcriptome and performed whole genome expression profiling to uncover transcriptional dynamics during physiological development, and we identified key gene categories involved in the process of sexual development. The assembled SDM transcriptome reported in this work contains 331,347 assembled transcripts; 226,687 unigenes were functionally annotated by Gene Ontology analysis. RNA-Seq analysis using this reference transcriptome identified 22,641 differentially expressed genes from shoots, flowers, fruits, and seeds. These genes are enriched in processes including organic substance metabolism, cellular metabolism, biosynthesis, and cellular component. In addition, genes related to transport, transcription, hormone biosynthesis and signaling, carbohydrate metabolism, and photosynthesis were differentially expressed between tissues. CONCLUSION: This work reveals tissue-specific gene expression patterns and pathways of SDM and implied to a difference between photosynthetic and non-photosynthetic tissues in plants. The data can potentially be used for future investigations on endophytic parasitism and SDM-spruce interaction, and it dramatically increases the available genomic resources for Arceuthobium and dwarf mistletoe communities. This preliminary study of the Arceuthobium transcriptome provides excellent opportunities for characterizing plant parasitic genes with unknown functions.

Development of ten microsatellite markers from the keystone mistletoe Tristerix corymbosus (Loranthaceae) using 454 next generation sequencing and their applicability to population genetic structure studies.

Tristerix corymbosus (Loranthaceae) is a keystone mistletoe from the South American temperate rainforests. As most mistletoes, T. corymbosus relies on biotic pollination and seed dispersal, which may cause population structure. For a better understanding of its ecology, we isolated and characterized ten polymorphic microsatellite loci for this species. We used 454 Next Generation Sequencing to build a microsatellite library from a high quality DNA sample. We tested 90 sequences from which we obtained ten polymorphic markers. In order to assess their variability, the novel markers were tested in 48 individuals from three locations of the Valdivian Coastal Reserve in Chile. We also estimated genetic differences between pairs of populations using the FST statistic. The mean number of alleles per locus in the 48 individuals studied was 7.1 (2-17 alleles per locus). The observed and expected heterozygosity ranged from 0.298 to 0.634 and from 0.310 to 0.881, respectively. There were genetic differences among the three populations assessed, according to the FST values (ranging from 0.048 to 0.100, all significant) and, the number of alleles per locus ranged from 3.9 to 5.1. These are the first microsatellite markers developed for T. corymbosus, and they arise as a powerful tool for studying population structure, genetic diversity and gene flow at the landscape scale, along its distribution.

Massive gene loss in mistletoe (Viscum, Viscaceae) mitochondria.

Parasitism is a successful survival strategy across all kingdoms and has evolved repeatedly in angiosperms. Parasitic plants obtain nutrients from other plants and some are agricultural pests. Obligate parasites, which cannot complete their lifecycle without a host, may lack functional photosystems (holoparasites), or have retained photosynthesis (hemiparasites). Plastid genomes are often reduced in parasites, but complete mitochondrial genomes have not been sequenced and their mitochondrial respiratory capacities are largely unknown. The hemiparasitic European mistletoe (Viscum album), known from folklore and postulated therapeutic properties, is a pest in plantations and forestry. We compare the mitochondrial genomes of three Viscum species based on the complete mitochondrial genome of V. album, the first from a parasitic plant. We show that mitochondrial genes encoding proteins of all respiratory complexes are lacking or pseudogenized raising several questions relevant to all parasitic plants: Are any mitochondrial gene functions essential? Do any genes need to be located in the mitochondrial genome or can they all be transferred to the nucleus? Can parasitic plants survive without oxidative phosphorylation by using alternative respiratory pathways? More generally, our study is a step towards understanding how host- and self-perception, host integration and nucleic acid transfer has modified ancestral mitochondrial genomes.

Plastome Evolution in Hemiparasitic Mistletoes.

Santalales is an order of plants consisting almost entirely of parasites. Some, such as Osyris, are facultative root parasites whereas others, such as Viscum, are obligate stem parasitic mistletoes. Here, we report the complete plastome sequences of one species of Osyris and three species of Viscum, and we investigate the evolutionary aspects of structural changes and changes in gene content in relation to parasitism. Compared with typical angiosperms plastomes, the four Santalales plastomes are all reduced in size (10-22% compared with Vitis), and they have experienced rearrangements, mostly but not exclusively in the border areas of the inverted repeats. Additionally, a number of protein-coding genes (matK, infA, ccsA, rpl33, and all 11 ndh genes) as well as two transfer RNA genes (trnG-UCC and trnV-UAC) have been pseudogenized or completely lost. Most of the remaining plastid genes have a significantly changed selection pattern compared with other dicots, and the relaxed selection of photosynthesis genes is noteworthy. Although gene loss obviously reduces plastome size, intergenic regions were also shortened. As plastome modifications are generally most prominent in Viscum, they are most likely correlated with the increased nutritional dependence on the host compared with Osyris.

The first mistletoes: origins of aerial parasitism in Santalales.

Past molecular phylogenetic work has shown that aerial parasites have evolved five times independently in the sandalwood order (Santalales), but the absolute timing of these diversifications was not addressed. DNA sequences from nuclear SSU and LSU rDNA, and chloroplast rbcL, matK and trnL-F from 39 santalalean taxa were obtained. Separate and combined data partitions were analyzed with maximum parsimony and Bayesian inference. Time estimates were performed with Bayesian relaxed molecular clock and penalized likelihood methods using published fossil data. Both methods gave comparable divergence dates for the major clades. These data confirm five origins of aerial parasitism, first in Misodendraceae ca. 80 Mya and subsequently in Viscaceae (72 Mya), "Eremolepidaceae" (53 Mya), tribe Amphorogyneae in Santalaceae (46 Mya), and Loranthaceae (28 Mya). The rapid adaptive radiation and speciation in Loranthaceae coincides with the appearance of savanna biomes during the Oligocene. In all clades except Misodendraceae, it appears that aerial parasites evolved from ancestors that were polymorphic for either root or stem parasitism-a condition here termed amphiphagous. Convergences in morphological features associated with the mistletoe habit have occurred such as the squamate habit, seed attachment structures, unisexual flowers, and loss of chlorophyll.

[Obtaining and characterization of the B-chains of mistletoe toxic lectins].

Mistletoe toxic lectins consist of two polypeptide chains: an enzymatic A chain, the toxic component, is joined by disulfide bond to a B chain conferring the lectin properties to the complete molecules. Mistletoe leaves contain three of toxic lectins encoded by three genes. The three B chains were produced in Escherichia coli in a soluble form. The recombinant proteins were found to bind to asialofetuin but in contrast to native proteins could be competed to a less extent by simple sugars D-galactose and N-acetyl-D-galactosamine. The functional properties of the proteins were strongly influenced by storage conditions such as salt concentration and simple sugar presence thus indicating an unstable folding. The lectin activity of one of the recombinant B chains was most close to the native protein that possibly results from the absence of N-glycosylation.

A new way of describing meiosis that uses fractal dimension to predict metaphase I.

Meiosis, the reductive nuclear division, is a continuum, but for purposes of communication, is described in stages. In sexually-reproducing organisms, including the dwarf mistletoe Arceuthobium americanum, prophase I of meiosis is prolonged (8 months for female A. americanum). Conversely, metaphase I, where chromosome pairs line up along a dividing cell's "equator", is relatively brief, difficult to predict, but critical regarding the random distribution of the paternal and maternal chromosomes in sexual organisms. However, descriptions of meiosis as either a continuum or stages are limited to qualitative observations. A quantification of meiosis can provide mathematical descriptors and allow for the prediction of when chromosomes reach the equator; this will not only be useful to researchers of cell division, but also to those requiring a large sample of metaphase I materials. Here, the probability-density function was used to calculate the fractal dimension of A. americanum nuclei undergoing early meiosis, and it predicted the onset of metaphase I by 2 days.

Differences in amino acid sequences of mistletoe lectin I and III B-subunits determining carbohydrate binding specificity.

Toxic lectins of European mistletoe Viscum album L.--MLI (viscumin), MLII and MLIII--are present in water extracts of this plant. Earlier we have cloned the full-length gene of MLIII precursor [A.G. Tonevitsky, I.I. Agapov, I.B. Pevzner, N.V. Maluchenko, M.M. Mojsenovich, U. Pfueller, M.P. Kirpichnikov, (2004) Biochemistry (Mosc.), 69 (6), 790-800, in press]. Here for the first time we report the cloning and expression in Escherichia coli cells of MLIII gene fragment encoding the carbohydrate-binding subunit. We have proved with our panel of monoclonal antibodies against ML toxins that the cloned fragment encoded MLIII B-subunit. The immunochemical and sugar-binding activities of renatured recombinant MLIII B-subunit were demonstrated in ELISA and ELLA, respectively. The comparative analysis of amino acid sequences of the cloned rMLIIIB and the B-subunits of other type II RIPs--MLI, ricin, abrin and nigrin b--was performed, revealing the main differences in primary structure of MLI and MLIII B-chains, which could determine their sugar specificity. The antigenicity analysis of MLI and MLIII B-subunits showed one epitope 25RDDDFRDGNQ34 in MLIB that is absent in MLIIIB sequence. The role of the toxic lectins and their subunits in immunological properties of mistletoe extracts is discussed.

Cloning and characterization of the genes encoding toxic lectins in mistletoe (Viscum album L).

Leaves of mistletoe (Viscum album L) contain three toxic lectins (type 2 ribosome-inactivating proteins) MLI, MLII, and MLIII, differing in molecular mass and carbohydrate specificity. Clones, containing sequences of three gene variants designated ml1p, ml2p, and ml3p, were obtained using PCR amplification from cDNA and from mistletoe genomic DNA. The quantitative ratio of the ml1p, ml2p, and ml3p genes in genomic DNA was found to be 1.5 : 1 : 4, respectively, whereas the ratio of their mRNA was 50 : 10 : 1. The quantitative prevalence of the ml1p transcript correlates well with the observation that MLI is quantitatively dominant over MLII and MLIII in the mistletoe extract. The sequences of the proteins encoded by the ml1p, ml2p, and ml3p genes are identical to MLI by 98, 88, and 77%, respectively. The similarity to MLI of the amino acid sequence encoded by the gene ml1p, the quantitative prevalent of its mRNA, as well as structural properties of the B-chain indicate that the gene, ml1p, corresponds to MLI. Western blot analysis of recombinant A-chains encoded by the three variants of mlp genes with the monoclonal antibody MNA4 having differential affinity to MLI, MLII and MLIII A-chains suggests that the ml2p and ml3p genes correspond to MLII and MLIII, respectively. Structural differences in the carbohydrate-binding sites of the B-subunits of ML1p, ML2p, and ML3p probably explain the difference in sugar specificity of MLI, MLII and MLIII.

A new gene encoding the ribosome-inactivating protein from mistletoe extracts.

Extracts from mistletoe (Viscum album L.) contain three main toxic proteins--the lectins MLI (also known as viscumin), MLII and MLIII. A catalytic subunit of the mistletoe plant toxic lectin MLIII has been cloned and expressed in Escherichia coli cells. The structure and immunochemical properties of recombinant MLIII A-subunit were investigated using a panel of monoclonal antibodies against ML-toxins. Ribosome-inactivating activity of the recombinant MLIII A-subunit was determined in a cell-free system exhibiting inhibition of endogenous protein synthesis. The comparative analysis of nucleotide and deduced amino acid sequences of the cloned MLIII A and the native MLI A-subunits was performed, revealing the main differences in the primary structure of these proteins. Antigenicity analysis of the MLIII A-subunit has revealed a new epitope D179-E184 that is not present in viscumin. The role of toxic lectins with respect to the immunological properties of mistletoe extracts is discussed.

Primary structure, isoforms, and molecular modeling of a chitin-binding mistletoe lectin.

From mistletoe Viscum album L. extracts three chitin-binding lectin isoforms, cbML1, cbML2, and cbML3, were isolated and their primary structure determined. All three cbML isoforms are composed of two protein chains of 48 or 49 amino acid residues, linked by an intermolecular disulfide bond. The sequence of each single cbML chain is characterized by a relatively high number of cysteine and glycine residues, 9 and 6, respectively, and contains four intramolecular disulfide bridges. On the basis of the combined interpretation of sequencing and MALDI MS data, the following results for the three cbML isoforms were obtained: the first one consists of two identical truncated polypeptide chains (1--48), the second is a heterodimer, containing one truncated (1--48) and one full-length chain (1--49), and the third is composed of two full length chains (1--49). The cbML sequence shows 55% identity to hevein, a single-chain chitin-binding protein of 43 amino acids, one of the most predominant proteins in natural rubber latex. On the basis of the NMR data on hevein from Hevea brasiliensis the three-dimensional structure of cbML3 was modelled. The 26 sequence changes between cbML3 and hevein were accommodated with only little perturbation in the main chain folding. A comparison of the primary structures of cbML3 and hevein is shown and the effects of the sequence changes are discussed. Differences have been identified in the loop region of the molecule and the potential interface region of cbML3 supporting the dimer formation. The high-affinity chitin-binding site seems to be highly conserved.

Genetic evidence for host specificity in the hemi-parasitic Viscum album L. (Viscaceae).

Nuclear ribosomal DNA (nrDNA) ITS sequences and partial sequences of three non-coding chloroplast DNA (cpDNA) introns and spacers were used to assess genetic variation within and among three presumed host races of the hemi-parasite Viscum album L. Currently, identification of host races occurs via the host trees, and morphological differences are minute at best. cpDNA and nrDNA ITS sequences revealed little sequence variation, but the variation found consistently supported the distinction of three host races. cpDNA and ITS sequences were not incongruent, as assessed by the incongruence length difference test. A combined analysis supported the sister group relationship between mistletoes from deciduous trees and fir.

Cloning of the mistletoe lectin gene and characterization of the recombinant A-chain.

Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in the same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A-chains.

Primary structure and molecular modeling of mistletoe lectin I from Viscum album.

The first three-dimensional structure of the ribosome inactivating protein mistletoe lectin I (ML-I) from Viscum album has been modeled on the basis of the X-ray structure of castor bean ricin from Ricinus communis. The relative high sequence homology and conserved secondary structure enabled accurate modeling. The 196 sequence changes between ML-I and ricin could be accomodated with only little pertubation in the main chain folding. A close comparison of the primary structures of ML-I and ricin is given and the effects of the sequence changes are elucidated on the basis of the modeled three-dimensional structure. Differences have been identified in the vicinity of the active site, in the high affinity galactose binding site and in the interface between the A and B chains, which might account for the reduced cytotoxicity of ML-I.

High-level expression of a viscotoxin in Arabidopsis thaliana gives enhanced resistance against Plasmodiophora brassicae.

Viscotoxins are a group of toxic thionins found in several mistletoe species. The constitutive CaMV-omega promoter was used to drive the expression of the viscotoxin A3 cDNA from Viscum album in transgenic Arabidopsis thaliana C24. Lines with high viscotoxin A3 levels in all parts of the plant were selected and tested for resistance against the clubroot pathogen Plasmodiophora brassicae. The transgenic lines were more resistant to infection by this pathogen than the parental line.

Amino acid sequence, S-S bridge arrangement and distribution in plant tissues of thionins from Viscum album.

The complete primary structure of a cytotoxic 5 kDa polypeptide, viscotoxin A1, isolated from Viscum album L., has been determined by combining classical Edman degradation methodology with advanced mass spectrometric procedures. The same integrated approach allowed correction of the sequence of viscotoxin A2 and definition of the pattern of the disulfide bridges. The arrangement of the cysteine pairing was determined as Cys3-Cys40, Cys4-Cys32 and Cys16-Cys26. The primary structure of viscotoxin A1 shares a high degree of similarity with the known viscotoxins and more generally with the plant alpha- and beta-thionins. The pattern of S-S bridges determined for viscotoxin A2 and A1 is similar to that inferred by X-ray and NMR analysis in crambin and related to that present in alpha-purothionin and beta-hordothionin, thus indicating a highly conserved organization of the S-S pairings within the entire family. This arrangement of S-S bridges describes a peculiar structural motif, indicated as 'concentric motif', which is suggested to stabilize a common structure occurring in various small proteins able to interact with cell membranes. The distribution of the new variant toxin in different mistletoe subspecies was investigated. Viscotoxin A1 is abundant in the seeds of the three European subspecies of V. album whereas it represents a minor component in the shoots.