<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> transformed roots of the parasitic plant <Emphasis Type="Italic">Triphysaria versicolor</Emphasis> retain parasitic competence

Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria versicolor Fischer and C. Meyer. Agrobacterium tumefaciens and Agrobacterium rhizogenes were both able to transiently express a GUS reporter in Triphysaria seedlings following vacuum infiltration. There was a correlation between the length of time seedlings were conditioned in the dark prior to infiltration and the tissue type transformed. In optimized experiments, nearly all of the vacuum infiltrated seedlings transiently expressed GUS activity in some tissue. Calluses that developed from transformed tissues were selected using non-destructive GUS staining and after several rounds of in vivo GUS selection, we recovered uniformly staining GUS calluses from which roots were subsequently induced. The presence and expression of the transgene in Triphysaria was verified using genomic PCR, RT PCR and Southern hybridizations. Transgenic roots were also obtained by inoculating A. rhizogenes into wounded Triphysaria seedlings. Stable transformed roots were identified using GUS staining or fluorescent microscopy following transformation with vectors containing GFP, dsRED or EYFP. Transgenic roots derived from both A. tumefaciens and A. rhizogenes transformations were morphologically normal and developed haustoria that attached to and invaded lettuce roots. Transgenic roots also remained competent to form haustoria in response to purified inducing factors. These transformation systems will allow an in planta assessment of genes predicted to function in plant parasitism. Alexey Tomilov and Natalya Tomilova made an equal contribution in the paper.     

Localized hormone fluxes and early haustorium development in the hemiparasitic plant Triphysaria versicolor

Perhaps the most obvious phenotypes associated with chemical signaling between plants are manifested by parasitic species of Orobanchaceae. The development of haustoria, invasive root structures that allow hemiparasitic plants to transition from autotrophic to heterotrophic growth, is rapid, highly synchronous, and readily observed in vitro. Haustorium development is initiated in aseptic roots of the facultative parasite Triphysaria versicolor when exposed to phenolic molecules associated with host root exudates and rhizosphere bioactivity. Morphological features of early haustorium ontogeny include rapid cessation of root elongation, expansion, and differentiation of epidermal cells into haustorial hairs, and cortical cell expansion. These developmental processes were stimulated in aseptic T. versicolor seedlings by the application of exogenous phytohormones and inhibited by the application of hormone antagonists. Surgically dissected root tips formed haustoria if the root was exposed to haustorial-inducing factors prior to dissection. In contrast, root tips that were dissected prior to inducing-factor treatment were unable to form haustoria unless supplemented with indole-3-acetic acid. A transient transformation assay demonstrated that auxin and ethylene-responsive promoters were up-regulated when T. versicolor was exposed to either exogenous hormones or purified haustoria-inducing factors. These experiments demonstrate that localized auxin and ethylene accumulation are early events in haustorium development and that parasitic plants recruit established plant developmental mechanisms to realize parasite-specific functions.     

Identification of the gene whose mutation leads to the morphological changes in the hypocotyl of Arabidopsis thaliana

The results of genetic and molecular genetic analysis of line 176 of Arabidopsis thaliana with reduced hypocotyls obtained from a previously obtained collection of insertion mutants, are presented. The examined mutation proved to be recessive and based on a single insertion of the T-DNA vector pLD3 into the A. thaliana genome. Computer-aided analysis of the DNA region adjacent to the left border of the insertion revealed a putative site of T-DNA insertion, the 2.5-kb At2g09920 gene located in the long arm of chromosome 2, near the centromere.     

Identification of a Mutant Gene Controlling Necrotic Cotyledons in Developing Seedlings of Arabidopsis thaliana

Genetic and molecular analyses of an Arabidopsis thalianamutant with necrotic cotyledons from the collection of insertion mutants obtained earlier were conducted. The mutation under study showed incomplete dominance and represented a single insertion of the T region of pLD3 vector used for transformation of germinating seeds to the plant genome during the creation of the collection. Using TAIL–PCR, a fragment of the mutant DNA adjacent to the left border of the T-DNA insertion was isolated and sequenced. Computer r-aided analysis showed that the insertion was located on the left arm of chromosome 1. The open reading frame containing the insertion has one exon and encodes a protein of 446 amino acids, whose function is unknown.     

Combined action of the entomopathogenic fungus <Emphasis Type="Italic">Metarhizium robertsii</Emphasis> and avermectins on the larvae of the colorado potato beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> (Say) (Coleoptera,Chrysomelidae)

Synergy between the entomopathogenic fungus Metarhizium robertsii J.F. Bisch., S.A. Rehner et Humber and a complex of natural avermectins was observed after combined treatment of the Leptinotarsa decemlineata (Say) larvae. This effect was also recorded at the larval stage most resistant to fungi (5 days after molting to the IV instar). Intoxication with avermectins led to a 2.7-fold decrease in food consumption and arrest of larval development. Synergy between the fungus and avermectins was revealed in field experiments conducted in the steppe zone of Western Siberia under strong fluctuations of temperature and humidity. In particular, the median lethal time (LT₅₀) decreased 2.8-fold after combined treatment as compared to treatment with M. robertsii alone. The mechanisms of synergy and the prospects of using the combined treatment for Colorado potato beetle control are discussed.     

Thermostable DNA Polymerase from Thermus thermophilus B35: Preparation and Study of a Modified Form of the Enzyme with High Affinity to ddNTP

The hybrid protein consisting of Tte DNA polymerase fragment and mutant Taq DNA polymerase (F667Y) fragment in the ratio 20 : 1 was constructed. Affinity of the modified enzyme (substitutions F669Y, V667I, and S692Q) to ddNTP was two orders higher than that of the wild type enzyme. The modified enzyme was used for sequencing DNA fragment with total deoxyguanosine and deoxycytidine content of 68%. In the polymerase chain reaction, the modified enzyme exhibits properties typical of the wild type Tte DNA polymerase.     

Effect of the Entomopathogenic Fungus Beauveria bassiana on the Development of Faba Bean (Vicia faba) Diseases in the Field Conditions

Doklady Biochemistry and Biophysics - Several ascomycetous entomopathogenic fungi, including species in the genera Beauveria, are plant symbionts/endophytes and are termed as endophytic...     

Hormone-Dependent Insertional Mutants of Arabodopsis thalianawith Reduced Viability and Fertility

We present data on the phenotype identification and genetic analysis of offspring in three lines of dominant morphological mutants of Arabidopsis thalianahaving drastically reduced fertility (a sterile calluslike mutant, a flower mutant, and a dwarf mutant) and in five lines of recessive morphological mutants (four mutants with lethal seedlings and one pigmentation mutant). The mutants were selected from a collection of transgenic plants that had genomes carrying a T-DNA insertion of plasmid vectors pLD3 and pPCVRN4; the collection was created earlier via agrobacterial transformation of germinating seeds. The results presented here were obtained using compensation of hormonal imbalance in the insertional morphological mutants of A. thalianaby exogenous hormones.     

Identification of a Gene Involved in the Control of the Root System Development in Arabidopsis thaliana

Genetic and molecular genetic analysis of a lethal root mutant of Arabidopsis thaliana was carried out. The mutant was obtained from a collection created earlier by means of insertion mutagenesis. The mutation was found to be recessive. It was caused by an insertion of the T region of vector pLD3 used for transformation of germinating seeds when creating the collection of insertion mutants. A 118-bp DNA fragment flanking the left border of the insertion was isolated using the TAIL PCR technique, and its nucleotide sequence was determined. Computer analysis of this DNA region demonstrated that it was located in exon 32 of the YUP8H12R.44 gene in chromosome 1.     

The structure of muscle spindles in the intercostal muscles and rectus abdominis of the human fetus]

The structure and innervation of muscle fibres were studied in external intercostal muscles and the streight muscle of the abdomen in 24--26-week human fetuses. The diameter of most spindles was shown to be within the range of 50--70 mkm, while in the streight muscle of the abdomen it could reach 100 mkm. In the external intercostal muscles the spindles, as a rule, were longer (300-500 mkm) than in the streight muscle of the abdomen (100--300 mkm) which was likely due to special functioning of the spindles in rhythmically working muscles. According to sensory innervation all the spindles may be divided into 3 main types: simple, intermediate and complex. In the external intercostal muscles there occur 3 types of spindles, while in the streight muscle of the abdomen there are spindles of a complex type.