Josef Vlasák

Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli β-glucuronidase

Human papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity. In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb‐binding site of the HPV16 E7 protein.

Global diversity of the Ganoderma lucidum complex (Ganodermataceae, Polyporales) inferred from morphology and multilocus phylogeny.

Species of the Ganoderma lucidum complex are used in many types of health products. However, the taxonomy of this complex has long been chaotic, thus limiting its uses. In the present study, 32 collections of the complex from Asia, Europe and North America were analyzed from both morphological and molecular phylogenetic perspectives. The combined dataset, including an outgroup, comprised 33 ITS, 24 tef1α, 24 rpb1 and 21 rpb2 sequences, of which 19 ITS, 20 tef1α, 20 rpb1 and 17 rpb2 sequences were newly generated. A total of 13 species of the complex were recovered in the multilocus phylogeny. These 13 species were not strongly supported as a single monophyletic lineage, and were further grouped into three lineages that cannot be defined by their geographic distributions. Clade A comprised Ganoderma curtisii, Ganoderma flexipes, Ganoderma lingzhi, Ganoderma multipileum, Ganoderma resinaceum, Ganoderma sessile, Ganoderma sichuanense and Ganoderma tropicum, Clade B comprised G. lucidum, Ganoderma oregonense and Ganoderma tsugae, and Clade C comprised Ganoderma boninense and Ganoderma zonatum. A dichotomous key to the 13 species is provided, and their key morphological characters from context, pores, cuticle cells and basidiospores are presented in a table. The taxonomic positions of these species are briefly discussed. Noteworthy, the epitypification of G. sichuanense is rejected.

Use of phosphomannose isomerase-based selection system for Agrobacterium -mediated transformation of tomato and potato

Two selection systems for Agrobacterium tumefaciens mediated transformation of tomato and potato were compared. In the tomato (Lycopersicon esculentum cv. Moneymaker), the highest transformation rate, 4.2 %, of cotyledon explants on mannose-selection medium was obtained when mannose/sucrose concentration in the regeneration medium was 5/15 g dm−3. The best transformation efficacy with the commonly used concentration of 100 mg dm−3 kanamycin as a selection agent was 9 %. In the potato (Solanum tuberosum cv. Bintje), the highest transformation frequency was 53.3 % when mannose concentration in the regeneration medium was 5 g dm−3 during the first 3 weeks after transformation and 10 g dm−3 afterwards. The optimum concentration of sucrose was 20 g dm−3. The transformation efficiency using kanamycin as a selection agent at a concentration 100 mg dm−3 was 33.3 % with potato. Our results demonstrate that the transformation efficiency using mannose selection is 1.6-fold higher for potato and about 2 times lower for tomato comparing with the ordinary protocol using kanamycin.

Taxonomy and phylogeny of the brown-rot fungi: Fomitopsis and its related genera

Taxonomic and phylogenetic studies on the brown-rot fungi, Fomitopsis and its related genera, are carried out. On the basis of morphological characters and phylogenetic evidence of DNA sequences of multiple loci including the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit nuclear ribosomal RNA gene (nSSU), the small subunit mitochondrial rRNA gene sequences (mtSSU), the translation elongation factor 1-α gene (tef1) and the second subunit of RNA polymerase II (rpb2), six new genera, Fragifomes, Niveoporofomes, Piptoporellus, Rhodofomitopsis, Rubellofomes and Ungulidaedalea are established. Four new species, Buglossoporus eucalypticola, Daedalea allantoidea, Piptoporellus hainanensis and P. triqueter are descibed from China. Illustrated descriptions of the novel species are provided. Identification keys to Fomitopsis and its related genera, as well as keys to the species of each genus are provided.

Global diversity and taxonomy of the Inonotus linteus complex (Hymenochaetales, Basidiomycota): Sanghuangporus gen. nov.,Tropicoporus excentrodendri and T. guanacastensis gen. et spp. nov., and 17 new combinations

Although Inonotus linteus complex is placed in the genus Inonotus, its perennial basidiocarps with a dimitic hyphal system, at least in the trama, distinguish the complex from other species in the genus, which have an annual habit and a monomitic hyphal system. The species number of Inonotus linteus complex has been increased in China and tropical America in recent publications. However, whether species in this complex belong to Inonotus has not specifically been addressed. To explore the phylogenetic placement of species of Inonotus linteus complex, we performed a comprehensive study using morphological and phylogenetic data based on global samples. nLSU (53 with 13 new) and ITS (70 with 18 new) datasets were used to produce the phylogenetic results. Taking into consideration the phylogenies inferred from the nLSU and ITS datasets, Inonotus is a polyphyletic genus comprising at least three clades. Clade A, the core Inonotus clade, contains the generic type, Inonotus hispidus, while Clades B and C comprise species from the Inonotus linteus complex. Morphological and phylogenetic evidence indicates that Clades B and C are new genera, and Sanghuangporus and Tropicoporus are introduced in this study. Ten species are transferred to Sanghuangporus and seven to Tropicoporus. Tropicoporus excentrodendri and T. guanacastensis spp. nov. are described, and their distinctive characters are discussed. Keys to the two new genera and the Inonotus sensu stricto, and to species of each new genus are provided.

The effect of an elevated cytokinin level using the **ipt** gene and N^{6} -benzyladenine on single node and intact potato plant tuberization **in vitro**

Two models of potato (Solanum tuberosum L.) tuberization in vitro (intact plants and single nodes) were used to study the role of cytokinins in this process. We applied hormone in two different ways. The exogenous addition of 10 mg · L-1N6-benzyladenine (BA) into the tuberization medium resulted in advanced tuber formation in intact plants, and microtubers appeared 10–20 days earlier than in the experiments in which no cytokinin was supplied. Transformation with the Agrobacterium tumefaciens ipt gene provided potato clones with endogenously elevated cytokinin levels (3–20 times higher zeatin riboside content in different clones). The onset of tuberization in intact ipt-transformed plants with low transgene expression was advanced in comparison with control material, and exogenously applied BA further promoted the tuberization process. On the contrary, tuberization was strongly inhibited in ipt-transformed nodes, and an external increase of the cytokinin level caused complete inhibition of expiant growth. In untransformed (control) nodes cytokinin application resulted in primary and secondary tuber formation, which depended on the BA concentration in cultivation media.

Phylogeny and global diversity of Polyporus group Melanopus (Polyporales, Basidiomycota)

Polyporus accommodates species with stipitate basidiocarps, a dimitic hyphal system with skeleto-binding hyphae, and hyaline, thin-walled, more or less cylindrical basidiospores. The species of Polyporus are divided into six morphological groups, of which Melanopus is characterized by coriaceous basidiocarps, thin context and a black cuticle on the stipe. In this study, we explore the phylogeny and species diversity of Polyporus sensu lato focusing on the group Melanopus based on global samples. Our combined ITS and nLSU dataset included 117 collections represented by 117 ITS and 94 nLSU sequences, of which 43 ITS and 37 nLSU sequences were newly generated. The resulting phylogenetic analysis recovered four clades, Favolus, Neofavolus, Melanopus and Polyporellus clades, while several species in the morphological groups Melanopus and Polyporus were outside these clades. Combined with morphological evidence, three new species in the Melanopus clade, Polyporus americanus, P. austroandinus and P. conifericola, are newly described and illustrated, and their distinctive characters are discussed. The illegitimate name Polyporus fraxineus is renamed as Polyporus fraxinicola. The phylogeny and taxonomy of each species in group Melanopus (morphology) or Melanopus clade (phylogeny) and related species are briefly discussed.

What is Antrodia sensu stricto

The polypore genus Antrodia (Polyporales, Basidiomycota) in the strict sense consists of a small number of species grouped around the type species A. serpens in phylogenetic analyses. This distinct clade (Antrodia sensu stricto in our view) contains species of the Antrodia heteromorpha complex, A. macra coll. and Antrodia mappa (formerly Postia mappa). Nuclear rDNA ITS and tef1 data show that the Antrodia heteromorpha species complex includes four species: A. heteromorpha sensu stricto (mostly on gymnosperms, large pores and spores), A. serpens (on angiosperms in Europe, resupinate, smaller pores but large spores), A. favescens (smaller pores and spores, pileate species in North America, formerly known as Trametes sepium), and A. tanakai (a close kin of A. favescens in Eurasia). Antrodia albida is a synonym of A. heteromorpha sensu stricto. We combine A. mappa, A. favescens and A. tanakai in Antrodia and designate neotypes for A. albida and A. heteromorpha, and an epitype for A. serpens. We also compare the morphologically similar but distantly related A. albidoides and A. mellita, and conclude that A. macrospora and A. subalbidoides are synonyms of A. albidoides.

The Agrobacterium tumefaciens C58-6b gene confers resistance to N 6 -benzyladenine without modifying cytokinin metabolism in tobacco seedlings

Abstract. The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin. To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L. cv. Samsun) plants were transformed with the A. tumefaciens C58-6b gene. Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N6-benzyladenine (BA). The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited. Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment. Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth. Intracellular levels of the major BA metabolite, N6-benzyladenine-7-glucoside (80–92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings. Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

Pileate Fomitiporia species in the USA. New combinations Fomitiporia calkinsii and F. bakeri

Sequencing of the ribosomal ITS region was used to resolve the relationship among USA collections of the morphological species Fomitiporia robusta. F. robusta corresponding to European collections was not found and its occurrence in the USA is regarded as questionable. Birch-growing fungus from mid-western and eastern United States known as Phellinus bakeri is a closely related polypore, but because of the absence of typical F. robusta here, it should be considered a separate species. Oak-growing pileate fungus from the south-eastern USA, also known as Fomes calkinsii, is a distinct species, rather distant from the European F. robusta. New combinations Fomitiporia bakeri and F. calkinsii are proposed. Notes on other similar species are provided.