Isolation of a novel mutant gene for soil-surface rooting in rice (Oryza sativa L.)

Background

Root system architecture is an important trait affecting the uptake of nutrients and water by crops. Shallower root systems preferentially take up nutrients from the topsoil and help avoid unfavorable environments in deeper soil layers. We have found a soil-surface rooting mutant from an M₂ population that was regenerated from seed calli of a japonica rice cultivar, Nipponbare. In this study, we examined the genetic and physiological characteristics of this mutant.

Results

The primary roots of the mutant showed no gravitropic response from the seedling stage on, whereas the gravitropic response of the shoots was normal. Segregation analyses by using an F₂ population derived from a cross between the soil-surface rooting mutant and wild-type Nipponbare indicated that the trait was controlled by a single recessive gene, designated as sor1. Fine mapping by using an F₂ population derived from a cross between the mutant and an indica rice cultivar, Kasalath, revealed that sor1 was located within a 136-kb region between the simple sequence repeat markers RM16254 and 2935-6 on the terminal region of the short arm of chromosome 4, where 13 putative open reading frames (ORFs) were found. We sequenced these ORFs and detected a 33-bp deletion in one of them, Os04g0101800. Transgenic plants of the mutant transformed with the genomic fragment carrying the Os04g0101800 sequence from Nipponbare showed normal gravitropic responses and no soil-surface rooting.

Conclusion

These results suggest that sor1, a rice mutant causing soil-surface rooting and altered root gravitropic response, is allelic to Os04g0101800, and that a 33-bp deletion in the coding region of this gene causes the mutant phenotypes.     

Occurrence of Potato virus Y strain PVYNTN in foundation seed potatoes in Japan,and screening for symptoms in Japanese potato cultivars

In 2008 and 2009 seasons, a sudden increase in Potato virus Y (PVY) incidence was recorded in foundation seed potatoes in Hokkaido, northern Japan. This increase was obvious during the field inspection and the postharvest indexing. Molecular typing revealed that besides the previously reported strains of PVY^O and PVY^NA‐N, the most common strain identified was the recombinant PVY^NTN, with three characteristic recombinant junctions at the HC‐Pro, VPg and CP regions. No potato tuber necrotic ringspot disease (PTNRD) was observed in foundation seed potatoes in correlation with the presence of PVY^NTN. Moreover, an isolate with a typical PVY^NTN recombinant genome, namely Eu‐12Jp, did not induce PTNRD in 62 Japanese potato cultivars tested in both primarily and secondarily infected plants. Two cultivars carrying the extreme resistance gene Ry_chc were resistant to the infection with Eu‐12Jp, which presents potential sources of resistance to PVY^NTN. Eu‐12Jp induced systemic mottle in potato cultivars Desiree and King Edward carrying resistance genes Ny and Nc, respectively, but induced a hypersensitive reaction in potato cultivar Maris Bard, with the Nz hypothetical resistance gene typical of the PVY^Z strain group. Therefore, based on the genome structure and the reaction of the potato N resistance genes, Eu‐12Jp should be classified as PVY^Z‐NTN, as described for isolates from Idaho, USA recently. This is the first report of PVY^Z‐NTN in Japan and the sudden and increased occurrence of PVY^NTN/PVY^Z‐NTN represents a potential risk of PTNRD developing and increases the significance of PVY in Japan.     

Chlorfenapyr resistance in the spider mite Tetranychus urticae: stability, cross-resistance and monitoring of resistance

The two-spotted spider mite, Tetranychus urticae Koch, is a key pest of many agricultural crops. Studies of stability of resistance, cross-resistance relationships and monitoring of chlorfenapyr resistance were carried out with T. urticae to provide basic information necessary to define effective acaricide resistance management strategies for this pest. Chlorfenapyr resistance was shown to be stable in the absence of selection pressure under laboratory conditions. The activities of seven different acaricides against chlorfenapyr-resistant and -susceptible strains of T. urticae were evaluated. The results indicated possible positive cross-resistance between chlorfenapyr and the acaricides abamectin, propargite and etoxazole. No cross-resistance was detected for the acaricides milbemectin, fenpyroximate and diafenthiuron. A possible negatively correlated cross-resistance was observed between chlorfenapyr and spiromesifen. The evaluation of 21 T. urticae populations from several crops in the States of São Paulo, Mato Grosso, Goiás, and Bahia, in Brazil, indicated that the susceptibility of mites to chlorfenapyr was variable, with percentages of resistant mites ranging from 0.0 to 86.5%. The highest resistance frequencies were observed in ornamental plants in the State of São Paulo. Some populations from cotton and papaya also presented high frequencies of chlorfenapyr resistance. This is the first report on chlorfenapyr resistance in T. urticae on cotton and papaya in Brazil. Strategies for the management of acaricide resistance are discussed.     

Review of diagnostic plaque reduction neutralization tests for flavivirus infection

Flavivirus infections (including Japanese encephalitis, West Nile encephalitis and dengue fever/severe dengue) present a worldwide public health problem. Recent climate change may affect the geographical distribution of the arthropod vectors for these viruses and so the risk of flavivirus epidemics may increase. Many methods have been developed for the serological diagnosis of flavivirus infections, such as haemagglutination inhibition assay, enzyme-linked immunosorbent assay, and immunofluorescence in staining. However, the specificity of these assays varies.The plaque reduction neutralizing test (PRNT) using live viruses is currently the ‘gold standard’ for the differential serodiagnosis of flaviviruses. The specificity of results obtained with PRNT is better than that for other protocols and many laboratories apply the PRNT protocol to the differential serodiagnosis of flaviviruses. Here, recent refinements to the PRNT protocols with genetically modified recombinant viruses or reporter-harbouring virus-like particles are reviewed. Further, the problems associated with the differential serodiagnosis of flaviviruses using PRNT are discussed.     

Production of bioactive bovine fibroblast growth factor 4 in E. coli based on the common nucleotide sequence of its structural gene in three breeds

Fibroblast growth factor 4 (FGF4) is considered a crucial gene in the proper development of bovine embryos. We recently determined the FGF4 gene sequence in eight cattle derived from three breeds and revealed a common nucleotide sequence of the structural gene encoding FGF4, which leads to the deletion and mutation of amino acid sequences in the mature FGF4 (Pro³²‐Leu²⁰⁶) compared with the sequence previously reported. In the present study, HisbFGF4, a 6× histidine‐tagged bovine FGF4 (Pro³²‐Leu²⁰⁶), was produced in Escherichia coli based on the validated nucleotide sequence and purified by heparin column chromatography. In primary bovine fibroblasts, HisbFGF4 showed significant mitogenic activity, whereas, intriguingly, the activity of a commercially available recombinant human FGF4 (Gly²⁵‐Leu²⁰⁶) produced in E. coli was weaker than that of HisbFGF4. In conclusion, the present study provides a simple method for the production of a bioactive bovine FGF4 derivative in E. coli utilizing its structural gene elucidated by us.     

Time-related morphological changes of porcine ovarian granulosa cells incubated with urea-EDTA solution.

Morphological profiles of porcine granulosa cells incubated with 10 mM Tris-HCl containing 1 M urea and 5 mM EDTA (urea-EDTA solution) were investigated. The percentages of granulosa cells incorporating the dye, trypan blue on incubation with urea-EDTA solution did not change during the initial 30 min. Thereafter, granulosa cells gradually took up the dye throughout the incubation. The amount of protein released from granulosa cells increased dramatically during initial 15 min of incubation, but decreased during the following 15-30 min of incubation. Thereafter, the amount of proteins released from granulosa cells increased gradually again. The releasing profile of 51Cr-bounded substances in granulosa cells increased markedly during the initial 15 min of incubation, and decreased during the next 15 min of incubation. Subsequently the amount of 51Cr released was enhanced. The min of incubation. Subsequently the amount of 51Cr released was enhanced. The plasma membranes of granulosa cells remained intact at 30 min of incubation, although chromatin clusters of granulosa cells disappeared. Thereafter, a number of cells showed signs of degeneration, including broken plasma membrane and cytolysis. The present study revealed that urea-EDTA solution is useful in extracting materials from porcine granulosa cells. The majority of the materials extracted from granulosa cells during the initial 30 min of incubation with urea-EDTA solution is considered to be from the cell surfaces and/or intercellular matrix.     

Phenotypic analysis of hepatic lymphocytes from healthy dogs

Phenotypes of lymphocytes from laparoscopically biopsied liver tissues of eleven healthy beagle dogs were analyzed. The proportion of CD3(+) lymphocytes (T cells), CD3 (-)CD21(+) lymphocytes (B cells) and CD3 (-)CD21(-) lymphocytes (non-T non-B lymphocytes), and the CD4(+)/CD8(+) ratio in the canine hepatic lymphocytes were 54.8 +/- 11.9%, 4.7 +/- 3.1%, 40.7 +/- 13.2%, and 0.33 +/- 0.12, respectively, while those in peripheral blood lymphocytes were 85.4 +/- 6.5%, 9.3 +/- 6.1%, 5.3 +/- 1.8%, and 1.64 +/- 0.36, respectively. These results indicated that the constitution of hepatic lymphocytes quite differed from that of peripheral blood lymphocytes in dogs, and suggested that the regional immunity in canine liver might be specific.     

Bovine apolipoprotein E in plasma: increase of ApoE concentration induced by fasting and distribution in lipoprotein fractions

Apolipoprotein E (apoE) is a protein constituent of lipoproteins, and acts as a receptor-binding ligand. Although the existence of bovine apoE in lipoprotein fractions has already been reported, quantitative studies on the changes of apoE in plasma and lipoprotein fractions are lacking. In the present study, an increase of a 38 kDa protein in the very low-density lipoprotein (VLDL) fraction obtained from fasted calves was detected. This 38 kDa protein was identified as bovine apoE by determination of the N-terminal amino acid sequence. Bovine apoE was purified and an enzyme-linked immunosorbent assay (ELISA) was developed. Using this system, the effect of fasting on the concentration of apoE in plasma and the distribution of apoE in lipoprotein fractions were investigated. After 3 days of fasting, the concentration of plasma apoE increased significantly (p<0.05) by 280 %, and was returned to the basal level by 3 days of refeeding. The lipoprotein fractions obtained from before and after fasting was separated by ultracentrifugation. ApoE was significantly increased in VLDL, low-density lipoprotein (LDL) and non-lipoprotein fractions by fasting (p<0.05). On the other hand, in high-density lipoprotein (HDL) fractions obtained from both before and after fasting, the level of apoE was very low compared to the other fractions. These results suggested that bovine apoE contents in triglyceride-rich lipoproteins are modulated by nutritional treatment and closely associated with triglyceride-rich lipoprotein metabolism.