Multiple virus resistance in transgenic plants conferred by the human dsRNA-dependent protein kinase

We have developed a new strategy for engineering resistance to multipleviruses in plants. The strategy exploits the human double stranded (ds)RNA-dependent protein kinase (PKR). PKR is one of theinterferon-induced enzymes. It confers viral resistance in mammals byinhibitingviral replication through the inactivation of the translational initiationfactor, eIF-2, upon activation by dsRNA. The humanPKR gene was fused to the promoter of theArabidopsis blue copper binding protein gene(BCB) that is induced rapidly in response to wounding. Thechimeric gene cassette was introduced into tobacco plants. Expression of thePKR gene in transgenic tobacco plants was demonstrated byRNA gel blot analysis and autophosphorylation assay of anM _r 68,000 protein. The transgenic plantsexpressing the PKR gene showed significantly reduced viralsymptoms or no viral symptoms at all, when challenged by different plant RNAviruses, such as Cucumber mosaic virus, Tobaccoetch virus, or Potato virus Y. Thus, expressionof a single component in the human interferon pathway, thePKR gene, can effectively confer resistance to multipleviruses in transgenic plants.     

The value of a tree species (Caryocar brasiliense) for a stingless bee Melipona quadrifasciata quadrifasciata

The expansion of crop lands and increased logging for charcoal production in the Brazilian savannahs (cerrados) has reduced richness and abundance of Meliponini bees. This may be a consequence of limitation in the availability of potential nesting substrate. The role of a cerrado-tree (Caryocar brasiliense) in providing nesting substrate for Melipona quadrifasciata quadrifasciata was evaluated. Tree (p= 0.006) and branch (p= 0.001) diameters, number of suitable branches (n= 513), height of the trees and availability of trees suitable for bee nesting were all important to the conservation of M. quadrifasciata. However, the high availability of nesting substrate did not seem to limit nest density nor cause the clumped pattern of nest distributions found for the study site. Nests (n= 48) were found mainly in individuals of C. brasiliense (n= 46) suggesting an active tree selection. In addition, nests located on the highest branches (mean = 4.6 m, sd = 1 m, n= 46) had lower probability of being extirpated by human honey collectors.     

Yeast communities associated with stingless bees

The yeast communities associated with the stingless bees Tetragonisca angustula, Melipona quadrifasciata and Frieseomelitta varia were studied. The bees T. angustula and F. varia showed a strong association with the yeast Starmerella meliponinorum. M. quadrifasciata more frequently carried a species related to Candida apicola, but also vectored low numbers of S. meliponinorum. Some of the yeasts isolated from adult bees were typical of species known to occur in flowers. Other yeast species found in adult bees were more typical of those found in the phylloplane. S. meliponinorum and the species in the C. apicola complex, also part of the Starmerella clade, may have a mutualistic relationship with the bees studied. Many yeasts in that group are often found in bees or substrates visited by bees, suggesting that a mutually beneficial interaction exists between them.     

Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera l)

Different modes of cell death have been revealed in the regressing hypopharyngeal glands of worker honey bees. The hypopharyngeal gland, which is well developed in young nursing bees to produce protein for larval food, was seen to regress naturally in foraging adult worker bees. A range of techniques including histology, cytochemistry, in situ TUNEL, Annexin V and Comet assays indicated that cells within the gland demonstrate progressive symptoms of apoptosis, necrosis and a vacuolar form of programmed cell death. The latter mode of cell death did not display chromatin margination, but was accompanied by an enhanced level of autophagic and hydrolytic activity in which a cytosolic source of acid phosphatase became manifest in the extra-cisternal spaces. Normal and annexin-positive cells were found to occur in the younger nursing bees, whilst necrosis and an aberrant vacuolar type of apoptosis predominated in the older foraging bees. The relevance of these results to the classification of programmed cell death is discussed.     

Effect of deleterious mutation-accumulation on the fitness of RNA bacteriophage MS2

Abstract.— RNA viruses show the highest mutation rate in nautre. It has been extensively demonstrated that, in the absence of purifying selection, RNA viruses accumulate deleterious mutations at a high rate. However, the parameters describing this accumulation are, in general, poorly understood. The present study reports evidences for fitness declines by the accumulation of deleterious mutations in the bacteriophage MS2. We estimated the rate of fitness decline to be as high as 16% per bottleneck transfer. In addition, our results agree with an additive model of fitness effects.     

Intron Conservation in a UV-Specific DNA Repair Gene Encoded by Chlorella Viruses

Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers. The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V. The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions. Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base). In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical. The introns in the pdg gene have 5′-AG/GTATGT and 3′-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene. However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical. These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences. Received: 13 May 1999 / Accepted: 20 August 1999     

Pollinator preferences and the persistence of crop genes in wild radish populations (Raphanus raphanistrum, Brassicaceae)

Crop-weed hybridization can potentially influence the evolutionary ecology of wild populations. Many crops are known to hybridize with wild relatives, but few studies have looked at the long-term persistence of crop genes in the wild. This study investigated one factor in the hybridization process in radish: differential pollinator visitation to wild radish (Raphanus raphanistrum) vs. crop-wild F1 hybrids (R. sativus x R. raphanistrum). Wild genotypes had yellow flowers, a recessive single-locus trait, whereas hybrids always had white or pale pink flowers. In experimental arrays in northern Michigan, total pollinator visitation was significantly biased toward wild plants when the frequencies of wild and hybrid plants were equal. Syrphid flies, the most frequent visitors, preferred wild plants while bumble bees showed no preference. This pattern was also observed when hybrid plants were overrepresented in the array (12 hybrid:2 wild). In contrast, when hybrid plants were rare (2 hybrid:12 wild), neither morph was preferred by any pollinator group. Later in the summer, pollinators were also observed in a large experimental garden with nearly equal frequencies of wild and hybrid plants. Cabbage butterflies (Pieris rapae) strongly overvisited wild plants, while bumble bees showed a slight preference for hybrids. Taken together, these studies suggest that F1 hybrids may not be at a disadvantage with regard to pollinator visits when they occur at low frequencies or when bumble bees are frequent flower visitors. Thus, variation in the proportion of white-flowered morphs among wild radish populations could be influenced by different histories of crop-to-wild hybridization, as well as by variation in the composition of local pollinator taxa.     

Fossil Clusiaceae from the late Cretaceous (Turonian) of New Jersey and implications regarding the history of bee pollination

The Turonian flora from Sayreville New Jersey includes one of the world's most diverse assemblages of Cretaceous angiosperm flowers. This flora is made even more interesting by its association with a large insect fauna that is preserved by charcoalification as well as in amber. Floral diversity includes numerous representatives of Magnoliidae, Hamamelididae, Rosidae, Dilleniidae, and Asteridae (Ericales sensu lato). Included are hypogynous, five-merous flowers with uniseriate hairs on the pedicels and stamens in bundles most frequently borne opposite the petals. There is considerable variation in filament length, and some filaments are branched. On some anthers, strands of residue, suggesting the former presence of a liquid of unknown nature, partially occlude the apparent zone of dehiscence. In other cases, open anthers are fully occluded by an amorphous substance. pollen is rarely found associated with anthers, but is common on stigmatic surfaces. pollen is prolate and tricolporate with reticulate micromorphology. The superior syncarpous ovary is five-carpellate with axile/intruded parietal placentation and numerous anatropous ovules/carpel. Ovary partitions have closely spaced, parallel ascending channels (secretory canals?), and there are apparent secretory canals/cavities in receptacles, sepals, and petals. Individual stigmas are cuneiform with a central groove and eccentrically peltate. Styles are short and fused. In aggregate, the stigmas form a secondarily peltate stigma. Seeds have a reticulate sculpture pattern, a pronounced raphe, and funicular arils with sculpture similar to the seeds. phylogenetic analyses of several data matrices of extant taxa place this fossil in a monophyletic group with the modern genera Garcinia and Clusia within the Clusiaceae. As such, these fossils represent the earliest fossil evidence of the family Clusiaceae. Some modern Clusiaceae are notable, in particular, for their close relationship with meliponine and other highly derived bee pollinators; the fossil flowers share several characters that suggest a similar mode of pollination. This possibility is consistent with other floral and insect data from the same locality.     

Infectibility and sensitivity of UK raspberry, blackberry and hybrid berry cultivars to Rubus viruses

Six blackberry or hybrid berry cultivars and 19 raspberry cultivars were assessed for their infectibility with, and sensitivity to, graft inoculation with 10 distinct viruses found infecting Rubus in the UK. Cultivars were grafted with each of, two isolates of the pollen borne raspberry bushy dwarf virus (RBDV), five aphid borne viruses: black raspberry necrosis, raspberry leaf mottle (RLMV), raspberry leaf spot (RLSV), rubus yellow net and raspberry vein chlorosis (RVCV); and isolates of the nematode transmitted nepoviruses, arabis mosaic, raspberry ringspot, strawberry latent ringspot and tomato black ring. All tested cultivars were infectible with a resistance breaking isolate of RBDV but only about half of that number with the Scottish type isolate of the virus. The raspberry cvs Autumn Bliss, and occasionally Glen Garry and Glen Prosen, developed leaf yellowing symptoms following infection with RBDV, but none of the other infected cultivars showed obvious leaf symptoms when kept in a heated glasshouse during the growing season. All tested cultivars were infectible with each of the four viruses transmitted in nature by the aphid, Amphorophora idaei. Most were infected symptomlessly, but seven cultivars developed severe leaf spotting symptoms due to infection with RLMV or RLSV. All but one of the raspberry cultivars were infectible with RVCV, which is transmitted in nature by the aphid Aphis idaei, and almost all infected plants developed leaf symptoms; only one of the hybrid berry or blackberry cultivars tested was infected with RVCV. In tests with the four nepoviruses, all tested cultivars, except Tummelberry, were infectible with at least one or more of these viruses. However, cultivars responded differently to challenge inoculation with different isolates of individual nepoviruses. Several cultivars developed chlorotic leaf mottling following infection with some nepovirus isolates. The implications of these results for virus control are discussed in the light of the changing pattern of virus and virus vector incidence in the UK.     

Do Solitary Bees Osmia cornuta Latr. and Osmia lignaria Cresson Use Proximal Visual Cues to Localize Their Nest?

Female Osmia repeatedly return to their nest to provision it with food and building material. The present study investigates the bees' nest localization performance by modifying visual cues in the near-vicinity of the nest. Each of several arrays of nesting holes was surrounded by four geometrical shapes. Removing some or all of the shapes reduced the proportion of direct returns but never prevented a bee from finding the appropriate array and, within that array, its own nest. The more the pattern was modified, the higher was the error score and the longer the delay, but no bee failed finally to find its nest. Shifting the pattern as well as the whole array of holes shifted homing orientation accordingly. There was no effect of the positions of the removed shapes, of contrast inversion, or of modifying the holes array. Our data are discussed in the light of the snapshot theory.