Asparate transcarbamylase activity in etiolated cowpea hypocotyls treated with 2,4-dichlorophenoxyacetic Acid

Treating etiolated cowpea (Vigna unguiculata) seedlings with 2,4-dichlorophenoxyacetic acid resulted in 2.5-, 3.9-, and 6.5-fold increases in DNA, soluble protein, and RNA, respectively, over untreated controls 84 hours after treatment. Aspartate transcarbamylase activity increased within 12 hours after treatment, and by 84 hours it was almost 12-fold greater than that in the untreated controls. During that time, activity in untreated controls dropped 60%. The assay used ¹⁴C-aspartate, which was then separated from the ¹⁴C-ureidosuccinate product by Dowex 50 (H⁺ form) column chromatography. Thin layer chromatography of the reaction product indicated that most of the carbamyl-phosphate-dependent radioactivity co-chromatographed with ureidosuccinate. The reaction has a pH optimum near 10.0 and is inhibited by uridine 5′-phosphate and succinate. The data suggest that aspartate transcarbamylase is important in pyrimidine biosynthesis in 2,4-dichlorophenoxyacetic acid-treated seedlings.     

Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA

The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Pythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual cultures or from mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi.     

Application of recombinant DNA technology to plant protection: molecular approaches to engineering virus resistance in crop plants

Developments in plant tissue culture, plant transformation and regeneration, and improvements in techniques to isolate and manipulate viral genes have led to the exploitation of the concept of cross protection: turning the virus onto itself and controlling it with its own genes. By introducing and expressing genes of viral origin in crop plants, scientists have engineered resistance to several plant viruses. Some of the approaches, used singly or in combination, include expression of viral-coat protein, untranslatable sense or antisense RNA, satellite RNA, virusspecific neutralizing antibody genes, plant viral replicase, protease or movement proteins and defective, interfering RNA. All of these approaches have resulted in manifestation of virus resistance to varying degrees in several commercially important crop plants. This review summarizes the recent advances in engineering virus resistance using the above approaches, and lists specific examples of their use in cultivated crop plants of economic importance.H.R. Pappu and C.L. Niblett are with the Plant Pathology Department, University of Florida. Gainesville, FL 32611-0680, USA; R.F. Lee is with the Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, USA. Florida Agricultural Experiment Station Journal Series No. R-04558.     

Regeneration of fertile Arachis paraguariensis plants from callus and suspension cultures

Highly morphogenic callus cultures were isolated from stamens of a wild peanut species, Arachis paraguariensis. These cultures were initiated on modified N6 medium containing 0.2 mg1l^-1 4amino-3,5,6-trichloro-picolinic acid (picloram) and 0.5 mg l^-1 6-benzylaminopurine (BAP) and were maintained on modified N6 medium with 0.008 mg l^-1 picloram and 0.25 mg l^-1 BAP. Buds formed on the calli growing on the maintenance medium developed into shoots when they were transferred to a MS salts based medium with no hormones. The cultures could also be maintained as a suspension culture in N6 liquid medium. When cell clumps larger tham 840 m were collected from the suspension culture and transferred to MS medium without hormones, they formed shoots in liquid culture. Root formation rarely occurred in agar or liquid cultures. Therefore, grafting to stems of rooted seedlings was used to obtain plants from regenerated shoots. Eight out of 50 field grown plants produced viable seed.     

Characterization of grapefruit plants (<Emphasis Type="Italic">Citrus paradisi</Emphasis> Macf.) transformed with citrus tristeza closterovirus genes

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.     

Hematologic and urinary excretion anomalies in patients with chronic fatigue syndrome

Patients with chronic fatigue syndrome (CFS) have a broad and variable spectrum of signs and symptoms with variable onsets. This report outlines the results of a single-blind, cross-sectional research project that extensively investigated a large cohort of 100 CFS patients and 82 non fatigued control subjects with the aim of performing a case-control evaluation of alterations in standard blood parameters and urinary amino and organic acid excretion profiles. Blood biochemistry and full blood counts were unremarkable and fell within normal laboratory ranges. However, the case-control comparison of the blood cell data revealed that CFS patients had a significant decrease in red cell distribution width and increases in mean platelet volume, neutrophil counts, and the neutrophil-lymphocyte ratio. Evaluation of the urine excretion parameters also revealed a number of anomalies. The overnight urine output and rate of amino acid excretion were both reduced in the CFS group (P < 0.01). Significant decreases in the urinary excretion of asparagine (P < 0.0001), phenylalanine (P < 0.003), the branch chain amino acids (P < 0.005), and succinic acid (P < 0.0001), as well as increases in 3-methylhistidine (P < 0.05) and tyrosine (P < 0.05) were observed. It was concluded that the urinary excretion and blood parameters data supported the hypothesis that alterations in physiologic homeostasis exist in CFS patients.     

Control of brown patch (<Emphasis Type="Italic">Rhizoctonia solani</Emphasis>) in tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) by host induced gene silencing

Key message

Transgenic tall fescue plants expressing RNAi constructs of essential genes of Rhizoctonia solani were resistant to R. solani.

Abstract

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species widely used for home lawns and on golf courses in North Carolina and other transition zone states in the US. The most serious and frequently occurring disease of tall fescue is brown patch, caused by a basidiomycete fungus, Rhizoctonia solani. This research demonstrates resistance to brown patch disease achieved by the application of host induced gene silencing. We transformed tall fescue with RNAi constructs of four experimentally determined “essential” genes from R. solani (including genes encoding RNA polymerase, importin beta-1 subunit, Cohesin complex subunit Psm1, and a ubiquitin E3 ligase) to suppress expression of those genes inside the fungus and thus inhibit fungal infection. Four gene constructs were tested, and 19 transgenic plants were obtained, among which 12 plants had detectable accumulation of siRNAs of the target genes. In inoculation tests, six plants displayed significantly improved resistance against R. solani. Lesion size was reduced by as much as 90 %. Plants without RNAi accumulation did not show resistance. To our knowledge, this is the first case that RNAi constructs of pathogen genes introduced into a host plant can confer resistance against a necrotrophic fungus.

     

Effects of inhibitors of RNA and protein synthesis on aspartate transcarbamylase activity in etiolated plant tissue

Aspartate transcarbamylase (ATCase) activity declines in etiolated cowpea (Vigna unguiculata L. Walp.) and soybean (Glycine max L. Merr.) hypocotyls between 3 and 11 days after planting. Treating cow-pea hypocotyls with cycloheximide (CH), actinomycin D (AMD), 6-methyl purine (6-MP), or cordycepin increases ATCase activity up to 740, 350, 465, and 305%, respectively, over water-treated controls 48 to 72 hours after treatment. In contrast erythromycin had no effect, and d-threo-chloramphenicol (CHL) reduced ATCase activity nearly 40%. CH, AMD, and CHL, whose effects were further characterized, each markedly reduced total RNA synthesis and protein synthesis. Respiration was stimulated by CH and AMD and reduced by CHL. In soybean, CHL-treated tissues and water-treated controls had comparable ATCase activities 48 hours after treatment, while AMD, 6-MP, and CH treatments reduced activities 29, 37, and 78%, respectively. The results suggest that the level of ATCase activity in etiolated cowpea hypocotyls is regulated by a mechanism or mechanisms that are interfered with by inhibition of RNA and protein synthesis. Possibly the mechanism is absent from etiolated soybean hypocotyls.