Simple hand-held devices for the efficient infection of plants with viral-encoding constructs by particle bombardment.

An efficient method for infection of plants with a cloned potyvirus by particle bombardment has been described (Gal-On et al., 1995). A simplified method is described now whereby a vaccuum chamber and helium propulsive gas are not required to achieve a high efficiency of infection. The new device-the 'HandGun'--is hand-held, and easily constructed from readily available materials. With this technique it is possible to bombard soft plants and seedings that do not survive particle bombardment by other devices. bombardment of C. pepo plants with a full length clone of zucchini yellow mosaic potyvirus results in approximately 100% infection at 100 pg cDNA per plant using air or helium to propel the microprojectiles. The HandGun is 10(5)-fold more efficient than mechanical inoculation. Tungsten and gold were found to be the most efficient materials tested for use as microprojectiles. Crude extracts of plasmids from E. coli were found to be effective, as well as column-purified cDNA. A functional, simple version of the HandGun--'the Blowpipe'--was also constructed, which does not require an electrically controlled valve. Plants can be inoculated with plant viruses from sap with the HandGun.

Acifluorfen Enhancement of Cryptochrome-Modulated Sporulation following an Inductive Light Pulse.

Acifluorfen enhancement of blue-light mediated phototropism suggested that this diphenyl-ether herbicide augments the light reaction (TY Leong, WR Briggs 1983 Plant Physiol 70: 875-881). The separation of the possible direct interaction of acifluorfen with light reactions from interactions with dark pathways has been elucidated in this paper with Trichoderma harzianum. Acifluorfen at 30 micromolar, given for 5 hours in the growth medium, stimulated the conidiation of Trichoderma in response to blue light without apparently affecting growth. Enhanced conidiation could be elicited by dipping cultures into medium with acifluorfen both before as well as 0.5 hour after inductive blue light. This postphotoinduction stimulation indicates that acifluorfen does not directly augment the effect of light by interacting with cryptochrome(s) in Trichoderma. Instead, acifluorfen most probably interacted with the dark reactions following photoinduction.

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L. : The end-of-day response to phytochrome.

The control by phytochrome of hypocotyl elongation of light-grown Cucumis sativus L. after a white-light period was examined. The farred-absorbing form of phytochrome inhibits hypocotyl elongation. The response to phytochrome photostationary state (ϕ) is not linear; all values of ϕ from 0.004 to 0.13 promote growth maximally, in the range of values of ϕ from 0.13 to 0.22 there is a linear growth response, between values of ϕ of 0.22 and 0.35 there is again no differential effect, and for ϕ values above 0.35 there is a strong (near linear) effect of ϕ on elongation. A kinetic examination of events following the white-light period shows that the major recovery from the photoperiod requires 8.5 h of darkness. End-of-day far-red treatment produces a very different response pattern, with a minor growth stimulation within 28 min of treatment followed by a major effect after 80 to 90 min. Three hours after far-red treatment there is a transient decline in growth rate which persists for about 2 h. Over the whole time course there is a great stimulation of growth rate compared with the controls. A similar growth-rate pattern also occurs if the end-of-day ϕ is 0.48, although the magnitude of the growth stimulation is less. Two components are affected by end-of-day ϕ, namely the time at which growth recovers and the subsequent growth rate. In the long term, the latter accounts for most of the differences in elongation growth. The dark recovery when only the hypocotyl is irradiated requires 4 h, but end-of-day far-red treatment reduces this to about 1.5 h. The persistence of the far-red-absorbing form of phytochrome for many hours in darkness in these light-grown plants is also demonstrated.

Photocontrol of Hypocotyl Elongation in Light-Grown Cucumis sativus L. : Responses to Phytochrome Photostationary State and Fluence Rate.

The effects of the calculated photostationary state of phytochrome (phi(c)) and the photon fluence rate on the elongation growth of the hypocotyl of light-grown seedlings of Cucumis sativus L. are examined. Two threshold responses to phi(c) are found at values of 0.06 and 0.43. At phi(c) = 0.06, there is no response at any fluence rate. In the phi(c) range 0.1 to 0.43, elongation growth does not respond to changes in phi(c). Above the second threshold (phi(c) = 0.43), there is a strong response to changes in phi(c). At all values of phi(c) at and above 0.1, there is a response to fluence rate. A linear relationship can be demonstrated between a factor comprised of the logarithm of phytochrome cycling rate (a fluence-rate-dependent process) and phi(c), and the growth response.

Photocontrol of Hypocotyl Elongation in De-Etiolated Cucumis sativus L. : Long Term, Fluence Rate-Dependent Responses to Blue Light.

Hypocotyl growth in Cucumis sativus L. cv Ridge Greenline is inhibited by increasing blue light (B) fluence rate in a near log linear fashion once a low fluence threshold is exceeded. Deviation from log linearity at the highest fluence rate used here is due to light perceived by the cotyledons and this effect is assigned to phytochrome. This response can be removed by Norflurazon treatment, without affecting the rest of the fluence response curve.There is also some activation of phytochrome by lower fluence rates of B, an effect which contributes to the overall inhibition of growth. Responses to photostationary state and cycling rate indicate, however, that B does not primarily act via phytochrome, but through a specific blue light photoreceptor.

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L. : A synergism between the blue-light photoreceptor and phytochrome.

An interaction is demonstrated between the effects of phytochrome and cryptochrome (the specific blue-light photoreceptor) in the inhibition of hypocotyl elongation of light-grown cucumber (Cucumis sativus L.) cv. Ridge Greenline seedlings. At certain fluence rates of blue light the total inhibition response is greater than the sum of the separate responses to each photoreceptor. The threshold for response to blue light is reduced at least 30-fold by additional red-light irradiation. The synergistic effect is demonstrated for two different fluence rates of red light. Synergism is mediated by phytochrome in both the cotyledons and the hypocotyl.

Improvement of Aconitum napellus micropropagation by liquid culture on floating membrane rafts.

An efficient method was developed using floating membrane rafts (Liferaft(™)) for the micropropagation of Aconitum napellus (Ranunculaceae), a cut flower crop with a low natural propagation rate. This was achieved by introducing shoot tips into culture on Murashige and Skoog's (1962) solid medium, or liquid medium-supported rafts, supplemented by different levels of benzyl adenine (BA). Optimum shoot proliferation on solid medium required 4mg/l BA, whereas for expiants supported on rafts optimal proliferation was achieved at 0.25mg/l BA. Maximum shoot proliferation was found using the floating rafts (propagation ratio of 4.2 per month), 45% higher than the maximum value on solid medium. A similar value could be obtained on solid medium after a period of 2 months. The optimal response to BA was similar for fresh weight gain and shoot length. Growth in a shallow layer of liquid in shake flasks gives a similar shoot multiplication rate to that on floating rafts; however, submerged leaves brown and die.

Recombination of engineered defective RNA species produces infective potyvirus in planta.

Recombination occurred between viral genomes when squash plants were cobombarded with mixtures of engineered disabled constructs of a zucchini yellow mosaic potyvirus. Single and double recombinants were detected in the progeny. Genes involved in the recombination process and the mechanisms of recombination were studied in potyviruses for the first time.

Degradation of the 32 kD Herbicide Binding Protein in Far Red Light.

White light (400-700 nanometers) supports the activity of photosystem I (PSI) and photosystem II while far red light (>/=700 nanometers) supports PSI almost exclusively. In intact fronds of Spirodela oligorrhiza, turnover of the 32 kilodaltons herbicide binding protein is stimulated under both these light conditions, although not in the dark or at wavelengths >730 nanometers. As is the case in white light, the far red light induced degradation of the protein is inhibited by DCMU. The means by which far red light operates is unclear. Hypotheses considered include: PSI activated proteolysis, PSI-induced formation of semiquinone anions, and PSI-generated free radicals.

Identification of a primary in vivo degradation product of the rapidly-turning-over 32 kd protein of photosystem II.

The 32 kd photosystem II protein of plant chloroplasts is rapidly turned over in the light. The initial events in the degradation of the 32 kd protein were studied. A 23.5 kd breakdown product was identified in Spirodela oligorrhiza membranes using immunological analysis. The 23.5 kd polypeptide was shown to be derived from the amino-terminal portion of the 32 kd protein using partial proteolytic fingerprinting. An in vivo precursor--product relationship between the 32 kd protein and the 23.5 kd polypeptide was kinetically demonstrated by radiolabeling and pulse-chase experiments. The cleavage site yielding the 23.5 kd polypeptide was localized to a functionally active region (between helices IV and V) of the 32 kd protein. We propose that an alpha-helix-destabilizing 'degradation' sequence, bordered by arginine residues 225 and 238, is involved in the formation of the 23.5 kd polypeptide.

The control of food mobilisation in seeds of Cucumis sativus L. : V. The effect of light on lipid degradation.

The degradation of neutral lipid and the development of lipase activity in cucumber cotyledons is stimulated by white light. Malate synthase and isocitrate lyase activities show no stimulation. Lipase activity and neutral lipid breakdown are also enhanced by red light, far-red light proving ineffective. Far-red light reverses the effect of red light indicating the involvement of phytochrome in the control of lipase activity. Although light stimulates neutral lipid degradation it appears that much of the additional lipid lost is used in the synthesis of polar lipid constituents. Furthermore, the influence of light on lipid degradation appears to be species dependent.

Identification of a novel plant virus promoter using a potyvirus infectious clone.

A putative promoter from the strawberry vein banding caulimovirus (SVBV) genome was identified by its ability to drive infection with full-length cDNA of the zucchini yellow mosaic RNA potyvirus (ZYMV). A high rate of infection was obtained with the cDNA under control of the SVBV promoter using particle bombardment technology. The SVBV promoter shows 60% homology to the cauliflower mosaic virus 35S promoter in the domain spanning the conserved motifs of CCACT (at -83) and the TATA box (at -31), to the transcription start. The 3'-end one-third of the putative promoter (328 bp) was sufficient to invoke full infectivity with the ZYMV clone, and drove transient reporter gene expression in Solanaceae and Cucurbitaceae transformed with a binary plant transformation vector. Stable expression of a reporter gene (GUS) under control of the truncated SVBV promoter was shown in transformed tobacco shoots in roots, leaves and stems.

Separate photosensitizers mediate degradation of the 32-kDa photosystem II reaction center protein in the visible and UV spectral regions.

A component of the photosystem II reaction center, the 32-kDa protein, is rapidly turned over in the light. The mechanism of its light-dependent metabolism is largely unknown. We quantified the rate of 32-kDa protein degradation over a broad spectral range (UV, visible, and far red). The quantum yield for degradation was highest in the UVB (280-320 nm) region. Spectral evidence demonstrates two distinctly different photosensitizers for 32-kDa protein degradation. The data implicate the bulk photosynthetic pigments (primarily chlorophyll) in the visible and far red regions, and plastoquinone (in one or more of its redox states) in the UV region. A significant portion of 32-kDa protein degradation in sunlight is attributed to UVB irradiance.

Particle bombardment drastically increases the infectivity of cloned DNA of zucchini yellow mosaic potyvirus.

An infectious full-length cDNA clone of the RNA genome of the potyvirus zucchini yellow mosaic virus (ZYMV) was constructed under the control of the cauliflower mosaic virus 35S promoter. All squash, cucumber, melon and watermelon plants inoculated with the cloned cDNA of ZYMV by particle bombardment become infected. Bombardment technology is 10(6)-fold more effective than mechanical inoculation. Due to the great increase in efficiency, ineffective constructs now became infective (i.e. cDNA under the control of the 35S promoter without the NOS terminator; with an addition of 127 nucleotides at the 5' end of the viral cDNA; uncapped transcripts), and the infectivity of capped-transcripts was maximized. Inoculation by particle bombardment produced visual symptoms rapidly (3-4 days), allowing the detection of viral coat protein and virions after 2 and 3 days in systemically infected leaves and inoculated cotyledons respectively.

Breakage of resistance to Cucumber mosaic virus by co-infection with Zucchini yellow mosaic virus: enhancement of CMV accumulation independent of symptom expression.

Resistance to the cucumovirus Cucumber mosaic virus (CMV) in cucumber cv. Delila was manifested as a very low level of accumulation of viral RNA and capsid protein, and an absence of CMV-induced symptoms. In addition, resistance was observed at the single cell level, with a reduction in accumulation of CMV RNAs, compared to accumulation in cells of the susceptible cucumber cv. Bet Alpha. Resistance to CMV in cv. Delila was broken by co-infection with the potyvirus Zucchini yellow mosaic virus (ZYMV). Resistance breakage in cv. Delila plants was manifested by an increase in the accumulation of (+) and (-) CMV RNA as well as CMV capsid protein, with no increase in the level of accumulation of ZYMV. Resistance breakage in the resistant cultivar by ZYMV also occurred at the single cell level. Thus, synergistic interactions known to occur between a potyvirus and a cucumovirus led to resistance breakage during a double infection. However, resistance breakage was not accompanied by an increase in disease symptoms beyond those induced by ZYMV itself. On co-inoculation with an asymptomatic variant of ZYMV-AG an enhancement of CMV infection occurred without disease manifestation. Consequently, intensification of viral RNA and capsid protein accumulation can occur without a corresponding increase in disease development, suggesting that different host genes regulate viral accumulation and disease development in the CMV-resistant cucumber plants.

Shoot production in squash (Cucurbita pepo) by in vitro organogenesis.

Seedling-derived cotyledon explants of squash ( Cucurbita pepo L.) of commercial cultivars True French, Ma'yan and Goldy were regenerated in vitro on Murashige and Skoog medium augmented with 1 mg/l benzyladenine. After 4 weeks in culture small shoots and buds regenerated only on the most proximal cotyledon edge. Culture on an elongation medium with a reduced cytokinin concentration (0.1 mg/l) with or without 1 mg/l gibberellic acid (GA(3)) facilitated the recovery of shoots. Fresh shoots could be recovered at each subculture of the regenerating mass. Peak productivity was during the third cycle of subculture, and shoot production ceased after the fifth subculture. Culture on elongation medium supplemented with GA(3) was 55% more effective with respect to overall shoot production than that on medium without GA(3), with 22 shoots recovered in total per explant from the former. Regeneration occurred under both light and dark conditions. All of the shoots tested were diploid. The shoots were rooted and transferred to the greenhouse where they grew and flowered normally.

A cDNA clone from a defective RNA of citrus tristeza virus is infective in the presence of the helper virus.

A naturally occurring defective RNA of 2379 nt (D2.3) from the VT strain of citrus tristeza closterovirus (CTV) was cloned and sequenced. The D2.3 RNA is a fusion of two regions of 1521 and 858 nt from the 5' and 3' ends of the CTV genome, respectively. A cDNA clone of D2.3 RNA was tagged by the insertion of a 0.47 kb chimeric DNA fragment and the recombinant cDNA was inserted downstream of the cauliflower mosaic virus 35S promoter. The resulting construct was bombarded into CTV-infected tissue, which was then grafted onto virus-free plants. The presence of recombinant RNA in systemically infected leaves was demonstrated by RT-PCR. Sequencing the RT-PCR products synthesized from double-stranded RNA confirmed the presence of the chimeric segment used for tagging. This is the first report of an infectious cDNA molecule derived from CTV D-RNA.