Dye- and fluorescence-based assay to characterize symplastic and apoplastic trafficking in soybean (Glycime max L.) endosperm.

BACKGROUND: Endosperm is a triploid tissue in seed resulting from a sperm nucleus fused with the binucleate central cell after double fertilization. Endosperm may be involved in metabolite production, solute transport, nutrient storage, and germination. In the legume family (Fabaceae), with the greatest number of domesticated crops, approximately 60% of genera contain well-differentiated endosperm in mature seeds. Soybean seeds, the most important legume crop in the worlds, have endosperm surrounding embryos during all stages of seed development. However, the function of soybean endosperm is still unknown. RESULTS: Flow cytometry assay confirmed that soybean endosperm was triploid. Cytobiological observation showed that soybean endosperm cells were alive with zigzag-shape cell wall. Soybean endosperm cells allowed fusion proteins (42 kDa) to move from bombarded cells to adjacent unbombarded-cells. Such movement is not simple diffusion because the fusion proteins failed to move into dead cells. We used symplastic tracers to test the transport potential of soybean endosperm. Small organic dye and low-molecular-weight symplastic tracers revealed fast symplastic transport. After a treatment of an inhibitor of ATPase, N,N'-dicyclohexylcarbodiimide (DCCD), symplastic transport was blocked, but all tracers still showed fast apolopastic transport. The transport speed of 8-hydroxypyrene-1,3,6-trisulfonic acid in endosperm was 1.5 to 3 times faster than in cotyledon cells or Arabidopsis embryos. CONCLUSIONS: Soybean endosperm is a membrane-like, semi-transparent, and fully active tissue located between the seed coat and cotyledon. Soybean endosperm cells allowed macromolecules to move fast via plasmodesmata transport. The size exclusion limit is larger for soybean endosperm cells than its cotyledon or even Arabidopsis embryo cells. Soybean endosperm may be involved in fast and horizontal transport during the mid-developmental stage of seeds.

Differentiations in Gene Content and Expression Response to Virulence Induction Between Two Agrobacterium Strains.

Agrobacterium tumefaciens is important in biotechnology due to its ability to transform eukaryotic cells. Although the molecular mechanisms have been studied extensively, previous studies were focused on the model strain C58. Consequently, nearly all of the commonly used strains for biotechnology application were derived from C58 and share similar host ranges. To overcome this limitation, better understanding of the natural genetic variation could provide valuable insights. In this study, we conducted comparative analysis between C58 and 1D1609. These two strains belong to different genomospecies within the species complex and have distinct infectivity profiles. Genome comparisons revealed that each strain has >1,000 unique genes in addition to the 4,115 shared genes. Furthermore, the divergence in gene content and sequences vary among replicons. The circular chromosome is much more conserved compared to the linear chromosome. To identify the genes that may contribute to their differentiation in virulence, we compared the transcriptomes to screen for genes differentially expressed in response to the inducer acetosyringone. Based on the RNA-Seq results with three biological replicates, ∼100 differentially expressed genes were identified in each strain. Intriguingly, homologous genes with the same expression pattern account for <50% of these differentially expressed genes. This finding indicated that phenotypic variation may be partially explained by divergence in expression regulation. In summary, this study characterized the genomic and transcriptomic differences between two representative Agrobacterium strains. Moreover, the short list of differentially expressed genes are promising candidates for future characterization, which could improve our understanding of the genetic mechanisms for phenotypic divergence.

A termite symbiotic mushroom maximizing sexual activity at growing tips of vegetative hyphae.

BACKGROUND: Termitomyces mushrooms are mutualistically associated with fungus-growing termites, which are widely considered to cultivate a monogenotypic Termitomyces symbiont within a colony. Termitomyces cultures isolated directly from termite colonies are heterokaryotic, likely through mating between compatible homokaryons. RESULTS: After pairing homokaryons carrying different haplotypes at marker gene loci MIP and RCB from a Termitomyces fruiting body associated with Odontotermes formosanus, we observed nuclear fusion and division, which greatly resembled meiosis, during each hyphal cell division and conidial formation in the resulting heterokaryons. Surprisingly, nuclei in homokaryons also behaved similarly. To confirm if meiotic-like recombination occurred within mycelia, we constructed whole-genome sequencing libraries from mycelia of two homokaryons and a heterokaryon resulting from mating of the two homokaryons. Obtained reads were aligned to the reference genome of Termitomyces sp. J132 for haplotype reconstruction. After removal of the recombinant haplotypes shared between the heterokaryon and either homokaryons, we inferred that 5.04% of the haplotypes from the heterokaryon were the recombinants resulting from homologous recombination distributed genome-wide. With RNA transcripts of four meiosis-specific genes, including SPO11, DMC1, MSH4, and MLH1, detected from a mycelial sample by real-time quantitative PCR, the nuclear behavior in mycelia was reconfirmed meiotic-like. CONCLUSION: Unlike other basidiomycetes where sex is largely restricted to basidia, Termitomyces maximizes sexuality at somatic stage, resulting in an ever-changing genotype composed of a myriad of coexisting heterogeneous nuclei in a heterokaryon. Somatic meiotic-like recombination may endow Termitomyces with agility to cope with termite consumption by maximized genetic variability.

Screening a cDNA library for protein-protein interactions directly in planta.

Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein- or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ~2 × 10(5) cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.

Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily.

To infer the phylogenetic relationships of Xylaria species associated with termite nests within the genus Xylaria and among genera of the subfamily Xylarioideae, beta-tubulin, RPB2, and alpha-actin sequences of 131 cultures of 114 species from Xylaria and 11 other genera of the subfamily were analyzed. These 11 genera included Astrocystis, Amphirosellinia, Discoxylaria, Entoleuca, Euepixylon, Kretzschmaria, Nemania, Podosordaria, Poronia, Rosellinia, and Stilbohypoxylon. We showed that Xylaria species were distributed among three major clades, TE, HY, and PO, with clade TE-an equivalent of the subgenus Pseudoxylaria-encompassing exclusively those species associated with termite nests and the other two clades containing those associated with substrates other than termite nests. Xylaria appears to be a paraphyletic genus, with most of the 11 genera submerged within it. Podosordaria and Poronia, which formed a distinct clade, apparently diverged from Xylaria and the other genera early. Species of Entoleuca, Euepixylon, Nemania, and Rosellinia constituted clade NR, a major clade sister to clade PO, while those of Kretzschmaria were inserted within clade HY and those of Astrocystis, Amphirosellinia, Discoxylaria, and Stilbohypoxylon were within clade PO.

Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells.

BACKGROUND: The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed. RESULTS: We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP), and prey proteins are tagged with the N-terminal portions of either Venus (nVenus) or Cerulean (nCerulean). Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus. CONCLUSION: Multi-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

Theissenia rogersii sp. nov. and phylogenetic position of Theissenia.

Theissenia rogersii deviates from known Theissenia species primarily in having large ascospores with a thick wall layer and a unique configuration of two stromatal tissue types, one carbonaceous and the other fibrous. The carbonaceous tissue forms palisades on and beneath the perithecial layer as well as encasing individual perithecia, whereas the fibrous tissue fills the spaces between columns of the palisades as well as between encased perithecia. Phylogenetic analyses based on DNA sequences of beta-tubulin and alpha-actin genes placed Theissenia in the subfamily Hypoxyloideae among the genera that are characterized by having bipartite stromata (i.e. with the stromata differentiated into an outer dehiscing layer and an inner perithecium-bearing layer).

Gene expression of Na+-K+-ATPase alpha 1 and alpha 3 subunits in gills of the teleost Oreochromis mossambicus, adapted to different environmental salinities.

The objective of the present study was to test the hypothesis that fish gills can express more than one isoform of the Na+-K+-ATPase a subunit responsible for ion regulation in seawater and freshwater environments. Using rapid amplification of complementary DNA ends (RACE), we cloned and sequenced full-length cDNAs encoding Na+-K+-ATPase alpha 1 and alpha 3 subunits of tilapia (Oreochromis mossambicus). Clone TG33 is 3390 bp in length and encodes a polypeptide of 1023 amino acids, while clone TH3 is 3581 bp in length and encodes a protein of 1010 amino acids. Clones TG33 and TH3 showed 91% and 88% identities at the amino acid level with previously described animal Na+-K+-ATPase alpha 1 and alpha 3 subunits, respectively. Northern blot and reverse transcriptase polymerase chain reaction analyses indicated that the alpha 1 subunit is expressed predominantly in kidney and intestine, while the alpha 3 subunit is expressed mainly in brain and heart. However, lower levels of expression of both genes were detected in other tissues such as gill, spleen, and testis. The amounts of both alpha 1 and alpha 3 subunit messenger RNA in gill tissue increased with the level of environmental salinity. This provides direct evidence of enhanced transcription of N+-K+-ATPase alpha 1 and alpha 3 subunit genes upon salinity challenge.