Kenji Oeda

Protein function. Chaperonin turned insect toxin.

Antlions are larvae of the Myrmeleontidae family that live on other insects by sucking out the body fluid from their prey, after first paralysing it with a toxin produced by salivary bacteria. Here we show that the paralysing toxin produced by bacterial endosymbionts in the saliva of Myrmeleon bore larvae is a homologue of GroEL, a protective heat-shock protein known as a molecular chaperone. The amino-acid residues critical for this proteins toxicity are located away from the regions essential to its protein-folding activity, indicating that the dual function of this GroEL homologue may benefit both the antlion and the endosymbiont.

Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G→A change at 291 in the first putative exon, resulting in a Val→Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G→A change at the equivalent position (5751) within exon 10.

Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci

In vitro assay indicated that the human lysozyme has lytic activity against phytopathogenic fungi and bacteria. A human lysozyme gene was placed under control of the constitutive CaMV 35S promoter and the resulting expression plasmid was introduced into two cultivars (cv.) of carrot, Kurodagosun (K5) and Nantes Scarlet (NS), by Agrobacterium tumefaciens-mediated method. Seven and fourteen transgenic plants of cv. K5 and cv. NS were regenerated, respectively, and the obtained transgenic carrots of T0 generation was tested for disease resistance against Erysiphe heraclei, a pathogenic fungi causing powdery mildew. Among the tested lines, the transgenic plant No. 12-1 and 8-1 of cv. NS showed a fairly strong resistance against E. heraclei. The strong disease resistance was also confirmed in T1 generation. Disease resistance against another pathogen of leaf blight, Alternaria dauci, were also tested using T1 transgenic lines. Significant enhanced resistance was observed in the No. 12-1 of cv. NS. Accumulation of synthesized human lysozyme protein was observed in this line, a finding consistent with observed disease resistance.

Characterization of genetic diversity of nuclear and mitochondrial genomes in Daucus varieties by RAPD and AFLP

Abstract The genetic diversity of nuclear genomes of five Daucus species and seven Daucus carota L. subspecies involving 26 accessions was characterized with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). AFLP produced more than four times as many discrete bands per reaction compared with RAPD analysis, while both AFLP and RAPD basically led to similar conclusions. The dendrograms constructed with both RAPD and AFLP revealed that all accessions of D. carota were grouped into a major cluster delimited from other Daucus species, in good agreement with the classification by morphological char-acteristics. All accessions of cultivated carrots [(D. carota ssp. sativus (Hoffm.) Arcang.] were clustered in the same group while the variation within D. carota was relatively extensive. Genetic diversity of mitochondrial genomes was also documented with RAPD for the same accessions. The mitochondrial dendrogram differed from that of the nuclear genome, suggesting that nuclear and mitochondrial genomes of some accessions had separate evolutionary histories.

Four Garlic Viruses Identified by Reverse Transcription-Polymerase Chain Reaction and Their Regional Distribution in Northern Japan

A specific and highly efficient indexing method for four major garlic viruses, GV1 carlavirus, GV2 potyvirus, onion yellow dwarf virus (OYDV), and mite-borne mosaic virus, was developed using the reverse transcription-polymerase chain reaction method (RT-PCR). When specific primers were synthesized to amplify the coat protein genes of these viruses, the amplified PCR bands had the same expected sizes. This indexing method can be performed using an extremely small amount of leaf tissue (50 mg) to obtain reproducible data. Diseased garlic plants collected from five fields in northern Japan were tested using this method. As GV2 was detected in all the fields, and high frequency of disease symptoms was obtained in the case of heavy infection, GV2 may well be a major garlic virus in this region. By contrast, GV1 was detected in only one of the five fields. OYDV and mite-borne mosaic viruses were also detected in various fields and at different frequencies. Judging from the virus indexing data and the disease symptoms, these two viruses appeared to cause severe symptoms in the case of a mixed infection with GV2. The frequency of virus re-infection to virus-free clones was low in isolated fields, and GV2 was the major virus that re-infected virus-free clones.

Mixed virus infections of garlic determined by a multivalent polyclonal antiserum and virus effects on disease symptoms.

A garlic virus-specific polyclonal antiserum was developed against a mixture of flexuous rodshaped virus particles isolated from mosaic-diseased garlic plants (15). This antiserum was used in Western blot analysis against tissues from mosaic-diseased garlic plants, at least seven viral coat protein (CP) bands (from 38 to 32 kDa) were identified. Using Western blot analysis with Potyvirus-specific antibodies and reverse transcription-polymerase chain reaction (RT-PCR) analysis, we concluded that three of the seven bands corresponded to CPs of Leek yellow stripe virus (LYSV) (38 kDa) and two different Onion yellow dwarf virus (OYDV) strains (35.5 or 34 kDa). The 35 kDa band corresponded to the CP of GV1-Carlavirus, and the other four bands, 36, 35 (not GV1), 33, and 32 kDa, were identified as the CPs of four mite-borne viruses, based on RT-PCR analysis. Based on the molecular weights of CP, mixed infections of Potyvirus, Carlavirus, and mite-borne viruses were characterized. LYSV causes apparent disease symptoms in garlic plants, however, little reduction in bulb weights. Conversely, garlic plants infected with three different mite-borne viruses expressed weak symptoms and yield losses. Mixed infections of OYDV, the mite-borne viruses, and LYSV caused severe disease symptoms and considerable reduction of bulb weights.

Nucleotide sequence of a gene for nitrite reductase from Arabidopsis thaliana

A nitrite reductase (NiR) gene was recovered from Arabidopsis thaliana genomic library by the homology with a cDNA of spinach NiR and sequenced. Based on the comparison with the spinach cDNA, the Arabidopsis NiR gene was concluded to contain 4 exons [exon 1 of 376 bp (beginning with ATG start codon), exon 2 of 355 bp, exon 3 of 289 bp and exon 4 of 741 bp (ending at TGA stop codon)] and 3 introns (intron 1 of 196 bp, intron 2 of 81 bp and intron 3 of 77 bp). This conclusion was confirmed by the analysis using the RT-PCR method. The deduced amino acid sequence of the coding region of the Arabidopsis NiR gene had high similarities with those of NiR genes of other plants including spinach.

Genetic variation of mitochondrial and nuclear genomes in carrots revealed by random amplified polymorphic DNA (RAPD)

Mitochondrial and nuclear genomic diversities of 8 carrot (Daucus carota ssp. sativus) varieties, including 6 pure lines and 2 cytoplasmic male sterile (cms) lines, were taxonomically identified using PCR with 19 RAPD primers. Dendrograms based on polymorphisms of both mitochondrial and nuclear genomes were constructed. According to the dendrogram of the mitochondrial genome revealed by RAPD, 4 differentiated clusters formed, in good accordance with the classification based on analyses with restriction enzyme digestion. Two cms lines were grouped into the same cluster, as genetically separated from the others. Thus, the cytoplasm donors of these male sterile lines were thought to be wild carrots. Conversely, RAPD analysis of the nuclear genome for these eight cultivars revealed no evident clusters although some cultivars were of a similar origin or place of cultivation. A correlation between nuclear and mitochondrial dendrograms was absent. RAPD has proved to be a useful tool for identifying mitochondrial and nuclear genomes. This technique will greatly aid in promoting efficient improvement of carrots.

DNA polymorphisms in Oryza sativa L. and Lactuca sativa L. amplified by arbitrary primed PCR

SummaryThirty-five rice (Oryza sativa L.) varieties, including 18 japonica, 5 javanica and 12 indica subspecies and 12 lettuce (Lactuca sativa L.) varieties were identified taxonomically, using PCR with originally designed 21 RAPD (Random Amplified Polymorphic DNA) primers and 8 sequence-specific primers, used for amplifying four specific DNA fragments. Use of these primers revealed polymorphisms among varieties in rice and lettuce and facilitates DNA fingerprinting. Dendrograms of both species based on polymorphisms were constructed and genetical relationships were established. In rice, half the number of amplified bands were polymorphic and almost all varieties differentiated. However, differentiation of minor genetic alterations among somaclonal variants or mutants and their mother varieties was not feasible. In L. sativa, 47% of the amplified fragments were polymorphic and all 12 varieties were differentiated. Some of the PCR fragments were variety or type specific, which could be used for indicators for type-selection. The dendrogram obtained showed differentiated clusters of crisphead, leaf and butterhead type, findings in good accord with the classification based on the genetic background.

Genetic variation of petaloid male-sterile cytoplasm of carrots revealed by sequence-tagged sites (STSs)

Abstract The mitochondrial DNA of various carrot lines was characterized by random amplified polymorphic DNA (RAPD) analysis, and six sequence-tagged sites (STSs) led to identification of the petaloid type of cytoplasmic male sterility (CMS). Using six STS primer combinations, we were able to classify five CMS lines into two groups and eight fertile carrots into six groups. Both the STS1 and the STS4 primer combinations differentiated CMS cytoplasms from the fertile cytoplasms, and the STS2 primer combination revealed two different types of CMS cytoplasms – of Wisconsin Wild and Cornell origins. Cybrid carrot lines with petaloid flowers which had been obtained by asymmetric cell fusion could also be separated from fertile cybrids by the STS1 primer combination. The STS1 fragment contained a homologous sequence with the orfB gene. DNA gel blot analysis indicated that homologous regions to the STS1 fragment existed in fertile types as well as the CMS types, although the restriction fragment size patterns differed. These observations demonstrate that rearrangements involving this region occurred in the mitochondrial genome. The STS4 fragment had a more complicated gene structure, including retrotransposon-like sequences and small segments of chloroplast genome.