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     

Comparison of visual acuity and visual axis of three flatfish species with different ecotypes

ABSTRACT:   The visual acuity, visual axis and visual accommodation of pointhead flounder, slime flounder, and red halibut were determined to obtain basic knowledge for developing appropriate fishing gear and fishing methods for sustainable fisheries. Each of these species has a different ecotype in terms of habitat, depth and prey species. Thus, it was hypothesized that they may differ in terms of visual acuity, visual axis and visual accommodation. Few studies have compared these characters in flatfishes from different ecotypes. We used histological methods to determine visual acuity (i.e. cone cell density) and visual axis (i.e. cone cell distribution) in each of these species. The maximum visual acuity was 0.127 in pointhead flounder (total length, TL 344 mm), 0.092 in slime flounder (TL 372 mm) and 0.109 in red halibut (TL 336 mm). Based on the cone cell distribution in the retina, the visual axis was upward and forward in pointhead flounder, forward and downward in slime flounder, and downward in red halibut. Finally, the mean angle of lens movement was −2° in pointhead flounder, −13° in slime flounder and −32° in red halibut. This measurement of lens movement indicated that the average near-point distance was 0.87 × TL in pointhead flounder, 0.65 × TL in slime flounder and 1.02 × TL in red halibut. At similar TL (336–355 mm), the visual acuity of these species differs depending on the direction in which they are looking.     

Molecular cloning of CD4 from ginbuna crucian carp <Emphasis Type="Italic">Carassius auratus langsdorfii</Emphasis>

ABSTRACT:   The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii , a naturally occurring gynogenetic fish, is a useful model for studying T-cell-mediated immunity. CD4, a T-cell receptor (TCR) coreceptor, is a membrane-bound glycoprotein found on helper T-cells, and assists in the binding of major histocompatibility complexes. In the present study, full-length cDNAs encoding the CD4 molecule from the S3n strain of ginbuna crucian carp were cloned and characterized. 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE yielded two distinct cDNA clones of CD4 homolog from the ginbuna, and these sequences share 95% identity at the amino acid level. These ginbuna CD4 molecules consisted of a signal peptide, immunoglobulin superfamily (IgSf) like domains, a transmembrane domain, and a cytoplasmic domain similar to other known CD4. A tyrosine protein kinase p56^lck binding motif is conserved in the cytoplasmic tail of ginbuna CD4. Phylogenetic tree analysis indicated that ginbuna CD4 sequences are closely related to CD4L-1 from other fish species. Expression of ginbuna CD4 mRNA was detected in the gill, thymus, head kidney, trunk kidney and peripheral blood leukocytes, indicating that its expression pattern is similar to that of ginbuna TCRβ mRNA. The results suggest that ginbuna CD4 sequences are useful as molecular probes for helper T-cells.     

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.     

Abiotic and biotic factors affecting recruitment variability of walleye pollock (Theragra chalcogramma) off the Pacific coast of Hokkaido,Japan

Abiotic and biotic factors affecting the recruitment variability of the Japanese Pacific stock (JPS) of walleye pollock (Theragra chalcogramma) were examined using a bivariate regression and multivariate combined model. Of the abiotic variables around Funka Bay (spawning ground), February sea surface temperature (SST) and wind direction index showed significant bivariate relationships with recruitment. February SST was positively related to recruitment, suggesting that warmer water temperature in February favors JPS recruitment. On the other hand, the relationship between February wind direction index and recruitment predicts high JPS recruitment under predominant northwest winds in February. For the biotic variables in the Doto area (nursery ground), significant and negative bivariate relationships with recruitment were observed for catch per unit effort of Kamchatka flounder (Atheresthes evermanni), Pacific cod (Gadus macrocephalus), and walleye pollock, implying an important impact of predation by these groundfishes on JPS recruitment. The overall model incorporating these abiotic and biotic factors successfully reproduced the variability in JPS recruitment. Temperature and wind conditions around the spawning ground along with predator condition in the nursery ground appear to play a dominant role in the recruitment dynamics of JPS. Based on these results and prior knowledge, we propose a new hypothesis to explain the processes controlling JPS recruitment.     

Propionate tolerance test for determination of insulin secretion in a hyperglycemic Japanese Black steer

A propionate tolerance test (PTT) was used to determine the pathophysiology of a Japanese Black steer with hyperglycemia. In the hyperglycemic steer, a low insulin secretion was confirmed by a glucose tolerance test (GTT), so that the hyperglycemic steer was diagnosed as insulin-dependent diabetes mellitus. Although the plasma insulin concentration in the control cattle increased in response to propionate stimulation, a low insulin response to PTT was observed in the diabetic steer. The fact that both PTT and GTT determined that the diabetic steer had low insulin secretion suggests that the PTT might be an effective diagnostic tool for diabetes mellitus in cattle.