T-DNA-encoded auxin formation in crown-gall cells

The T-region of Ti plasmids expresses two genes (No. 1 and 2) in crown-gall cells which are essential for auxin effects. It has been shown that gene 2 (=IaaH) codes for an amidohydrolase which converts indole-3-acetamide into indole-3-acetic acid and which is functional in bacteria and in crown-gall cells (Schröder et al. (1984), Eur. J. Biochem. 138, 387–391). In this report we describe a quantitative assay for the enzyme and its application to analyze the properties of the enzyme as expressed in plant cells and in Escherichia coli. The enzyme requires no cofactors, and the temperature optimum (30–37°C), pH optimum (8.5–9.5), and K_m (about 1 M) were very similar in both systems. Besides indole-3-acetamide, the enzyme also hydrolyzed indole-3-acetonitrile, esters of indole-3-acetic acid with glucose and myo-inositol, a-naphthaleneacetamide, and phenylacetamide, indicating that it may have a general function in converting substances of low auxin activity into those with high auxin activity. The results are discussed in relation to the possible function of T-DNA gene 1 which cooperates with gene 2 in evoking auxin effects in crown-gall cells.Abbreviations HPLC high-pressure liquid chromatography - T-DNA transferred DNA     

Hydrogen ion dependence of carrier-mediated auxin uptake by suspension-cultured crown gall cells

1. The dependence on external hydrogen ion concentration of the carrier-mediated components of the uptakes of indol-3-yl acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4D) by suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch. is examined. The initiall rates of uptake of IAA and 2,4 D from 0.30 mol/l solutions exhibit pH optima of approximately pH 5.0 and pH 4.0 respectively.-2. The inherent difficulties, especially at low pH, of estimating the carrier-mediated uptake component in the presence of the large diffusive uptake of undissociated acid are discussed.-3. At pH 5.0, the uptakes of the two auxins are characterised by similar Michaelis Constants (0.65 and 0.80 mol/l) and Maximum Velocities (0.87 and 1.0 pmol/min/mg cells) for IAA and 2,4D respectively.-4. A dependence of carrier-mediated auxin uptake on cations other than hydrogen ions could not be demonstrated. It is suggested that uptake of auxin anions may occur by co-transport with hydrogen ions.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-dichlorophenoxyacetic acid     

Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil

AIMS: To develop a specific, sensitive and rapid PCR-based method for detection of tumorigenic Agrobacterium in soil. METHODS AND RESULTS: Three newly designed primers complementary to tms2 gene amplified DNA of only the tumor-inducing agrobacteria of 113 strains tested, resulting in 617 bp and 458 bp products in the first and second rounds of semi-nested PCR respectively. As optimized method of pre-incubation of soil suspensions on selective medium, DNA isolation and two-round semi-nested PCR enabled detection of 1-2 bacterial cells in 1 g of soil. Using this method tumour-inducing Agrobacterium was detected in 67 of 69 samples of naturally infested soil originating from the field, where plants with crown gall symptoms occurred. The pathogen was detected only in two samples of 15 tested, collected from a nursery where crown gall symptoms were not observed. CONCLUSIONS: The semi-nested PCR-based method allowed for sensitive and rapid detection of tumorigenic agrobacteria in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is proposed for testing of soil in fields intended for nursery production of fruit trees, roses or other plants susceptible to crown gall, as well as a tool for ecological studies.     

Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer

The strongly conserved amino acid sequences of the P8 outer capsid proteins of Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) and the distribution of electrostatic potential on the proteins at the interfaces between structural proteins suggested the possibility that P8-trimers of RGDV might bind to the 3-fold symmetrical axes of RDV core particles, with vertical interaction between heterologous P3 and P8 proteins and lateral binding of homologous P8 proteins, thereby allowing formation of the double-layered capsids that are characteristic of viruses that belong to the family Reoviridae. We proved this hypothesis using chimeric virus-like particles composed of the P3 core capsid protein of RDV and the P8 outer capsid protein of RGDV, which were co-expressed in a baculovirus expression system. This is the first report on the molecular biological proof of the mechanism of the assembly of the double-layered capsids with disparate icosahedral lattices.     

A new Asphondylia species (Diptera: Cecidomyiidae) and a eulophid wasp (Hymenoptera) inducing similar galls on leaf buds of Schoepfia jasminodora (Schoepfiaceae), with reference to their ecological traits and a description of the new gall midge

Different gall inducers belonging to distinct insect orders are rarely known to induce similarly shaped galls on the same host plant organs. We report that Asphondylia tojoi Elsayed & Tokuda sp. nov. (Diptera: Cecidomyiidae) and Ceratoneura sp. (Hymenoptera: Eulophidae) induce galls on leaf buds of Schoepfia jasminodora Sieb. et Zucc. (Schoepfiaceae). We describe the gall midge species as new to science and report a phylogenetic analysis for known Japanese Asphondylia species. We also describe life histories of the two species, based on monthly surveys during 2015–2017: although both species are multivoltine, A. tojoi overwinters as first instars in galls, whereas Ceratoneura sp. possibly does so as adults outside the galls. In addition, the internal structure of galls differed between the two species. Galls containing A. tojoi consist of a single chamber with inner walls clearly covered with whitish fungal mycelia after the gall midges develop into second instars. Those containing the Ceratoneura sp. have multiple chambers with hard black inner walls. Although some eulophids are known to be inquilines of galls induced by Asphondylia species, we consider that the Ceratoneura sp. is probably a true gall inducer because of the different gall structure and absence of fungal mycelia in their galls. This is the first report detailing the annual life history of a Ceratoneura species. Asphondylia tojoi represents the first example of monophagous Asphondylia species with a multivoltine life history on a deciduous tree.     

High species richness and genetic diversity of the genus Caloglossa (Delesseriaceae,Rhodophyta) in New Ireland,Papua New Guinea

Distribution patterns and genetic diversity of Caloglossa species have been studied in various regions but are still poorly understood around the tropical western Pacific where many marine organisms show high species richness. We explored the diversity of Caloglossa species in New Ireland, Papua New Guinea using morphological and genetic analyses. Seven species of Caloglossa (C. adhaerens, C. leprieurii, C. ogasawaraensis, C. bengalensis, C. postiae, C. saigonensis and C. vieillardii) were collected from eight sites; the latter four species are new records in Papua New Guinea. All specimens collected were cultured to compare their morphology under the same culture condition. Reproductive structures of gametophytes and/or sporophytes were described for the first time in some species from these collections. In C. leprieurii, gall‐like structure appeared in most gametophyte strains but not in tetrasporophytes. Such galls were produced from wing cells, and spermatangia and/or procarps were formed on many galls. The LSU rDNA and rbcL analyses revealed high intraspecific diversity in many species although the number of samples was limited and the sampling sites were within 200 km. We revealed high species richness and intraspecific genetic variation in Papua New Guinea, suggesting a hot spot for the diversity of Caloglossa.     

Agrobacterium strain specificity and shooty tumour formation in eucalypt (Eucalyptus grandis ×E. urophylla)

Summary To develop a successful protocol forAgrobacterium-mediated transformation in plants it is essential to determine the most efficient bacterial strain/plant genotype interaction. In the present work, we evaluated the susceptibility ofEucalyptus grandis ×E. urophylla to fiveAgrobacterium rhizogenes and twelveA. tumefaciens wildtype strains. The results showed different degrees of virulence, according to the strain tested, indicating that transformation of this eucalypt hybrid by Agrobacterium-derived vectors is possible. All developed tumours showed an autonomous growth when transferred to a hormone-free medium. Some of these tumours formed shoots spontaneously, with a normal phenotype. Polymerase Chain Reaction (PCR) and Southern blot analyses were performed to confirm the absence of the oncogenic T-DNA in plants derived from these shooty tumours.     

Overexpression of a tryptophan decarboxylase cDNA inCatharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids inC. roseus by converting tryptophan into tryptamine. Hardly anytdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA ofAgrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which atdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced inC. roseus cells by infecting seedlings with an oncogenicA. tumefaciens strain. In the resulting crown gall tumour calluses harbouring thetdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing thetdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in thetdc cDNA overexpressing crown gall calluses are discussed.     

Coexistence and weak amensalism of congeneric gall-forming aphids on the Japanese elm

Closely related species of gall-forming aphids are often associated with a single host species. SixTetraneura species coexist on the Japanese elm,Ulmus davidiana, in Sapporo, northern Japan. This paper describes the probabilities of coexistence on macro- and microgeographic scales (i.e., on host trees and host leaves) and examines whether coexistence with conspecific or heterospecific galls on leaves or shoots has any effect on the fecundity of each aphid species using multiple regression. A Monte Carlo simulation showed that the frequency distribution of the numbers of species on individual host trees differed significantly from that expected from the null model. There were significantly positive or negative associations between species.Tetraneura radicicola andsorini always coexisted with other species on trees they infested. Multiple regression revealed that the coexistence of conspecific or heterospecific galls on individual leaves had no influence on the fecundity ofradicicola andsorini, but had a negative influence on that of sp. O. On average,radicicola andsorini produced a smaller number of offspring in galls than sp. O, and obviously consumed only a small part of resources available on the galled leaves. Evidence available suggests that although amensalism does arise between sp. O and other species, its influence in not so strong as to exclude sp. O competitively from theTetraneura community.     

EVOLUTION OF PARASITISM AMONG CLOSELY RELATED SPECIES: PHYLOGENETIC RELATIONSHIPS AND THE ORIGIN OF INQUILINISM IN GALL WASPS (HYMENOPTERA,CYNIPIDAE)

A new term, agastoparasitism, is proposed for parasitism among closely related species. Cynipid inquilines are typical agastoparasites. They cannot induce galls; instead their larvae live inside the galls formed by other cynipids. As in many other groups of agastoparasites, there are two competing hypotheses for the evolutionary origin of cynipid inquilines: either they arose from one of their cynipid hosts, and later radiated to exploit other gall-inducing cynipids (monophyletic origin), or they arose repeatedly, each inquiline from its host (polyphyletic origin). These hypotheses for the origin of cynipid inquilines were tested by a phylogenetic analysis of representative species of cynipid gall inducers and inquilines based on adult morphological characters. The analysis supported the monophyly of the inquilines and indicated an origin from gall inducers related to the genus Diastrophus, one of the current host groups. To examine whether the result of the analysis was influenced by convergent similarities among inquilines because of their similar mode of life, all putative apomorphies shared by some or all of the inquilines but not occurring in any of the gall inducers were removed. Despite this, the phylogenetic conclusions essentially remained the same, that is, the support for inquiline monophyly was not caused by convergent evolution. Based on these results, adaptive aspects of the evolutionary origin and maintenance of cynipid inquilinism are discussed, as well as general patterns in the evolution of agastoparasitism.