Two Resistance Modes to Clover yellow vein virus in Pea Characterized by a Green Fluorescent Protein-Tagged Virus

ABSTRACT This study characterized resistance in pea lines PI 347295 and PI 378159 to Clover yellow vein virus (ClYVV). Genetic cross experiments showed that a single recessive gene controls resistance in both lines. Conventional mechanical inoculation did not result in infection; however, particle bombardment with infectious plasmid or mechanical inoculation with concentrated viral inocula did cause infection. When ClYVV No. 30 isolate was tagged with a green fluorescent protein (GFP) and used to monitor infection, viral cell-to-cell movement differed in the two pea lines. In PI 347595, ClYVV replicated at a single-cell level, but did not move to neighboring cells, indicating that resistance operated at a cell-to-cell step. In PI 378159, the virus moved to cells around the infection site and reached the leaf veins, but viral movement was slower than that in the susceptible line. The viruses observed around the infection sites and in the veins were then recovered and inoculated again by a conventional mechanical inoculation method onto PI 378159 demonstrating that ClYVV probably had mutated and newly emerged mutant viruses can move to neighboring cells and systemically infect the plants. Tagging the virus with GFP was an efficient tool for characterizing resistance modes. Implications of the two resistance modes are discussed.     

Temporal and topographic profiles of tissue hypoxia following transient focal cerebral ischemia in rats

Intravascular accumulation of blood cells after brain ischemia-reperfusion can cause obstruction of cerebral blood flow and tissue hypoxia/ischemia as a consequence. In the present study, we examined temporal and topographic changes of tissue hypoxia/ischemia after occlusion of the middle cerebral artery (MCA) for 60 min in rats with immunohistochemical staining for hypoxia (2-nitroimidazole hypoxia marker: hypoxyprobe-1 adducts). Our results showed that tissue hypoxia expressed as positive staining for hypoxyprobe-1 adducts preceded neuronal degeneration. Platelets and granulocytes were detected close to the hypoxyprobe-1 adducts positive area. These results suggested that the hypoxic environment could persist even after reperfusion of MCA, because of vascular obstruction with accumulation of platelets and granulocytes.     

<Emphasis Type="Italic">White clover mosaic virus</Emphasis>-induced gene silencing in pea

Double-stranded RNAs formed in secondary structures and replicative intermediates of viral genomes are thought to strongly elicit RNA silencing. This phenomenon is known as virus-induced gene silencing (VIGS). VIGS is a powerful tool for modifying gene expression in host plants. We constructed a virus vector based on White clover mosaic virus (WClMV) and demonstrated VIGS of phytoene desaturase (PDS) in pea. Photobleaching of tissues, caused by VIGS of PDS, was observed in restricted areas of upper leaves and stems. We confirmed that the PDS mRNA and subgenomic RNAs of WClMV were reduced in the photobleached tissues.     

Characterisation and histopathological observations of a selected Brazilian precocious line of Eimeria acervulina

Precocious lines of Eimeria acervulina "Cu" and "I" strains were obtained after 25 passages of oocysts in chickens that showed a shortening of the prepatent period for first oocyst output from 96 h to 81 and 82 h, respectively. Both precocious lines were evaluated for pathogenicity using as criteria weight gain, lesion score and total oocyst production. Infection of the "Cu" precocious line in chickens showed a high weight gain, low lesion score and low oocyst production, when compared to parent strain infected chickens. However, the results did not show a significant difference in relation to the criteria used above for the E. acervulina "I" precocious line when compared to its parent strain. This suggests a low degree of attenuation for the "I" strain but good attenuation for the precocious "Cu" line. The histopathological observations of chickens infected with the E. acervulina "Cu" parent strain and precocious line, comparing life cycle and intestinal lesions, showed: (1) parasite stages only in the border cells of infected chicken intestinal villi, for the precocious line; (2) parasite stages in the border cells of the intestinal villi and submucosa cells near the Lieberkühn glands of the intestine; and (3) high degree of inflammatory cells around the parasites in chickens infected with the parent strain. The "Cu" strain was also characterized for sensitivity against eight anticoccidial drugs. Sensitivity was observed for four anticoccidial drugs and partial resistance for four other drugs, although the strain had never had previous contact with anticoccidial drugs, suggesting the presence of a natural resistance factor. This Brazilian E. acervulina "Cu" precocious line showed attenuation for pathogenicity in chickens, suggesting that it could be a suitable strain for use as a live vaccine in Brazil.     

CD38 gene disruption inhibits the contraction induced by alpha-adrenoceptor stimulation in mouse aorta

CD38 is an ectoenzyme with ADP-ribosyl cyclase and hydrolase activities, which synthesizes cyclic ADP-ribose from NAD and hydrolyzes cyclic ADP-ribose to ADP-ribose. It has been shown that cyclic ADP-ribose is a potent Ca(2+) mobilizing messenger in many cells. To know the physiological role of cyclic ADP-ribose in vascular smooth muscle, we examined the effects of various agonists in the aorta isolated from CD38 knockout (CD38(-/-)) mouse. Western blot analysis showed that CD38 protein was detected in the aorta isolated from wild-type (CD38(+/+)) mouse, but not from CD38(-/-) mouse. In the aortae isolated from both CD38(+/+) and CD38(-/-) mice, KCl, phenylephrine and norepinephrine induced concentration-dependent contraction. KCl produced similar concentration-dependent responses in the aortae from both CD38(+/+) and CD38(-/-) mice. Maximum force of contraction induced by KCl (65 mM) was same in the size. Phenylephrine- and norepinephrine-induced contractions were, however, significantly smaller in the aortae from CD38(-/-) mice than in those from CD38(+/+) mice. 5-Hydroxytryptamine, endothelin-1, caffeine and thapsigargin-induced contractions were not significantly different in these two aortae. These results suggest that CD38 gene disruption inhibits alpha-adrenoceptor-induced vascular contractions and cyclic ADP-ribose-mediated signal transduction system is committed in these responses.     

Positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector

The stability of the inserted genes in the viral expression vector varied depending on the sequence introduced and the position of insertion. Infectious cDNA to Clover yellow vein virus (pClYVV) was modified to insert a foreign gene at two independent sites: one, along with a polylinker, between the NIb and CP genes (pClYVV/CP/W) and the other between P1 and HC-Pro (pClYVV-Pst/CP). The green fluorescent protein (GFP) gene was inserted into either pClYVV/CP/W or pClYVV-Pst/CP. GFP gene was stably maintained and expressed in both vectors following serial passages in plants. Progeny viruses from both constructs accumulated in similar amounts and at rates of 70%–80% of that of the wild-type virus. On the other hand, progeny viruses carrying the human interferon- (hIFN) gene cloned in pClYVV-Pst/CP were genetically unstable owing to frequent deletions of the cloned gene during passage through plants. In contrast, the hIFN sequence cloned in pClYVV/CP/W was stably maintained in viruses after several passages in broad bean plants, and the progeny virus accumulated at the rate of about 50%–100% of that of the wild-type virus. The nucleotide sequence analyses indicated that the genetic instability of the inserted sequence results from homologous recombination of viral vector and inserted DNA sequences; it is not due to the inserted sequence alone.     

Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.     

Ratio of rice reflectance for estimating leaf blast severity with a multispectral radiometer

 Rice reflectance was measured to determine the spectral regions most sensitive to leaf blast infection with a multispectral radiometer. As disease severity increased, reflectance also increased in the 400–500 nm (blue), 570–700 nm (red), and 900–2000 nm regions but decreased in the 500–570 nm and 700–900 nm regions. The increased reflectance in the blue and red regions may be attributed to decreased chlorophyll and carotenoid contents in response to the blast infection. The maximum and minimum reflectance differences occurred at 680 nm and 760 nm for the nondiseased and diseased rice, respectively. The spectral location of maximum sensitivity was 675 nm regardless of disease severity. Rice reflectance ratios were evaluated as indicators of leaf blast severity. Two ratios, R550/R675 (reflectance at 550 nm divided by reflectance at 675 nm), and R570/R675 quantified the significant disease severity. These wavelengths were selected based on the sensitivity minima and maxima. The ratios of nondiseased rice plants varied depending on growth stage. The variation in ratios must be considered when they are used to estimate leaf blast severity. Received: April 2, 2002 / Accepted: August 12, 2002