Localization and responsiveness of a cowpea apyrase VsNTPase1 to phytopathogenic microorganisms

Apyrases (NTPases) are associated with both compatible and incompatible interactions between plants and microorganisms. Previously we reported that the ATPase activities of cell-wall-bound apyrases of several leguminous plants, such as pea, cowpea, soybean, and kidney bean, were enhanced by a glycoprotein elicitor and were inhibited in a species-specific manner by mucin-type glycopeptide suppressors secreted from a pea pathogenic fungus, Mycosphaerella pinodes. In this study, we isolated two apyrase genes, VsNTPase1 and VsNTPase2, from a cDNA library of Vigna sinensis Endl. cv. Sanjakusasage. Based on phylogenetic analysis, VsNTPase1 may belong to a group that responds to environmental stimuli. In a transient assay using DNA bombardment, a fusion protein of green fluorescent protein (GFP) and the N-terminal putative signal sequence of VsNTPase1 was distributed in the nucleus, cytoplasm (cytoskeletal structure), and cell wall. On the other hand, a fusion protein of GFP and the N-terminal putative VsNTPase2-signal sequence was localized in the cytoplasm, especially in small particles (perhaps mitochondria). A recombinant VsNTPase1 expressed in Spodoptera frugiperda 21 cells responded directly to signal molecules from several phytopathogenic microorganisms. Here, we discuss the role of apyrases in recognizing and responding to exogenous signals. The nucleotide sequences of VsNTPase1 and VsNTPase2 in this article have been submitted to DDBJ as accession numbers AB196769 and AB196770, respectively.     

Effect of Harpin from Four Pathovars of Pseudomonas syringae on Pea Defense Responses

To investigate the role of the proteinaceous elicitor, harpin, on host and nonhost plants, we isolated the harpin-coding gene, hrpZ, from Pseudomonas syringae pvs. pisi, glycinea, tabaci and tomato. Effects of the recombinant harpin proteins on pea plants were analyzed and compared with the effects of the corresponding bacterial treatment. After inoculation of pea with pea pathogen P. syringae pv. pisi, the bacterial population increased and the accumulation of PAL-mRNA and pisatin was inhibited. The nonpathogenic pathovars, glycinea, tabaci and tomato induced both defense responses in pea. However, none of the harpins induced the hypersensitive reaction or accumulation of PAL-mRNA and pisatin in pea. Harpins from P. syringae pvs. glycinea, tomato and pisi did induce these defense responses in tobacco, however, suggesting that externally applied harpins either are not recognized or are nonfunctional in pea plants. Received 27 June 2000/ Accepted in revised form 21 February 2001     

Use of three‐dimensional acceleration sensing to assess dairy cow gait and the effects of hoof trimming

The aim of this study was to assess the use of three‐dimensional acceleration sensing to describe the gait of dairy cows and the effect of hoof trimming. In Trial 1, a three‐dimensional acceleration sensor was attached to the carpal region of a front leg of six Holstein cows who were then walked for 9 m. Results showed that measures of acceleration could clearly show the presence of steps, which were divided into an acceleration phase, which involved the hoof descending to and ascending from the ground, and a non‐acceleration phase, which included the swing phase. Lissajous figures could succinctly depict the degree of acceleration for each cow and showed that one cow who was lame walked with greater vertical and lateral acceleration than the remaining cows. In Trial 2, 17 loose‐housed Holstein cows were walked for 20 m during the month before, once during the month after and once 2 months after hoof trimming with the sensor attached at the end of their thoracic vertebrae to measure acceleration of the whole body. To relate the acceleration of the body to movements of the legs, image analysis was used to the range of vertical movement of four major skeletal joints, using visual markers attached to them. Hoof trimming significantly (P < 0.005) decreased both the range of vertical movement of the joints of front and hind legs and the variance of lateral and forward acceleration at the end of the thoracic vertebrae, suggesting improved gait pattern smoothness. Acceleration sensing was able to characterize variation among gait patterns of the cows and could reproduce the pattern shown by image analysis. This study demonstrates that three‐dimensional acceleration sensing of either the carpal region of a front leg or the end of the thoracic vertebrae is useful to detect walking irregularities and evaluate the effectiveness of hoof trimming on walking ability of cattle.     

Effect of a novel rice fermented extract on gastric ulcers in horses

Gastric ulcers cause appetite loss, poor body condition, and colic in horses. This study investigated the protective effect of a rice fermented extract on the gastric mucosa in 17 healthy Thoroughbreds. For one month, horses in the rice fermented extract (nine horses) and control (eight horses) groups were orally administered a rice fermented extract (100%; 0.2 ml/kg, SID) and tap water (0.2 ml/kg), respectively. Gastric endoscopic images were obtained before and one month after rice fermented extract administration. The gastric ulcer score was lower after administration (median, 1; maximum, 2; minimum, 1) than before administration (median, 4; maximum, 4; minimum, 3) in the rice fermented extract group (P<0.05). In conclusion, the administration of a rice fermented extract for one month improves gastric mucosal lesions in Thoroughbreds with gastric ulcers.     

Polyphosphate kinase is essential for swarming motility, tolerance to environmental stresses, and virulence in Pseudomonas syringae pv. tabaci 6605

Polyphosphate kinase (PPK), encoded by the ppk gene, is a principal enzyme responsible for synthesis of inorganic polyphosphate (poly P) from ATP in many Gram-negative bacteria. In order to elucidate the functions of poly P in Pseudomonas syringae pv. tabaci 6605, an in-frame deletion mutant of the ppk gene (ppk) was constructed. The ppk mutant did not accumulate poly P, whereas the wild-type strain accumulated a large quantity. The mutant had reduced swarming motility, even though it retains swimming motility like the parental strain. The mutant exhibited increased sensitivity to prolonged incubation and environmental stresses, such as heat shock and oxidative stress and reduced exopolysaccharide (EPS) production compared to the wild-type. Northern blot analysis revealed that expression of the rpoS gene, encoding the stationary phase sigma factor RpoS, was reduced in ppk in the logarithmic phase, indicating that rpoS is regulated by the ppk gene. The poly P deficient mutant had significantly reduced ability to cause disease in its host tobacco plant and in planta growth of the mutant was also significantly reduced in host tobacco leaves as compared to the wild-type strain. Thus, our results suggest that poly P plays an important role in the virulence of P. syringae pv. tabaci 6605.     

Glycosylation of flagellin from Pseudomonas syringae pv. tabaci 6605 contributes to evasion of host tobacco plant surveillance system

Pseudomonas syringae pv. tabaci (Pta) possesses a genetic region composed of two open reading frames (ORFs), fgt1 and fgt2, that are involved in glycosylation of flagellin. The deletion mutant Δfgt1 produced non-glycosylated flagellin, and exhibited reduced ability to cause disease in the host tobacco plant. Flagellin is known to induce plant defense responses, and the recognition of flagellin by Arabidopsis thaliana is mediated by a conserved N-terminal region, flg22, in flagellin and a leucine-rich repeat domain in the FLS2 receptor. Because flg22 localizes inside the flagellum, polymerized flagellum needs to be dissociated to be recognized. Therefore, the effect of glycosylation on flagella stability was investigated. The polymerized flagella from glycosylated flagellins were more resistant to heat treatment than those from non-glycosylated flagellins, suggesting that the glycosylation of flagellin contributes to the structural stability of flagella and prevents exposure of the flg22 region. Polymerized flagella from Pta Δfgt1 flagellin and depolymerized and glycosylated flagellin from Pta wild type induced cell death and callose deposition, and inhibited seedling growth in tobacco more effectively, whereas polymerized flagella from Pta wild-type flagellin caused a low level of these responses. These results suggest Pta might have evolved the flagellin glycosylation system to evade detection and defense response of a host by increasing flagella stability and suppressing their dissociation.     

Recurrence analysis of intraoperative acridine orange-photodynamic therapy for dogs with intranasal tumors

Intraoperative acridine orange-photodynamic therapy (AO-PDT) and cribriform plate irradiation are used to treat canine intranasal tumors. The purpose of this study was to evaluate the effects of AO-PDT on intranasal tumors and the recurrence rate of tumors after this treatment. Treatments with AO-PDT were performed on 38 dogs through a narrow window of the dorsal nasal cavity. The median progression-free interval was 12 mo and recurrence was detected in 21 dogs. Based on computed tomography, recurrence in 16 dogs was biased to the following areas: lateral (n = 10), medial (n = 2), ventral (n = 0), rostral (n = 0), and caudal (n = 8). Side effects were mild and included subcutaneous emphysema and rhinitis. The median survival time was 24 mo. Although AO-PDT with cribriform irradiation is an effective treatment for intranasal tumors, AO-PDT techniques should be improved to treat the nasal cavity more uniformly and thoroughly.     

Taxonomic identity and the effect of temperature and irradiance on the photosynthesis of an indoor tank-cultured red alga Agardhiella subulata from Japan

We determined through morphological and rbcL phylogenetic analyses that a previously unidentified, but introduced species of macroalga, which has been easily cultivated in indoor tanks in Japan, is Agardhiella subulata (Solieriaceae). Additionally, the photosynthetic biology of this alga was examined by inducing photosynthetic activity under a variety of water temperatures and photosynthetic active radiation (PAR) to clarify the optimal conditions needed for its efficient cultivation. Photosynthetic activity was evaluated by using both dissolved oxygen (DO) and pulse amplitude modulated-chlorophyll fluorometric (PAM) techniques, and focused on elucidating temperature and PAR levels that would potentially maximize productivity. The DO method revealed that the net photosynthetic rates at 24 °C quickly increased as PAR increased, and approached a P _max of 27.8 mg O₂ g _ww ^?1  min^?1 (95 % Bayesian credible interval, BCI 23.8–32.1). The maximum gross photosynthetic rate occurred at 26.7 °C (BCI 24.4–28.3 °C). However, PAM experiments indicated that for the maximum quantum yield, the optimal temperature was 23.7 °C (BCI 22.7–24.6) and the maximum relative electron rates occurred when the water temperature was 31.0 °C (BCI 30.6–31.5). This study suggests that the broad tolerance of maximal photosynthetic activity to temperature (22.7–31.5 °C) is one of the main reasons why this alga can be successfully cultivated year-round.