An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80–90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.     

In vitro selection of Nicotiana sylvestris variants with limited resistance to TMV

Haploid tobacco (Nicotiana sylvestris) plants were inoculated with a yellow strain of tobacco mosaic virus (TMV-Flavum) and then exposed to 500 rads of acute gamma radiation. Leaf strips cultured on callus-inducing medium yielded two types of colonies: 1) yellow, virus-infected and 2) green, apparently healthy. Of the 3210 calli scored, approximately 5% were virus-free, and after regeneration, 0.2% were resistant at the plant stage. Later, adult plants, both TMV-resistant and TMV-susceptible, produced self-fertile, diploid flowers. Both seedling progeny and rooted cuttings from resistant stock plants showed resistance to TMV infection. This resistance was characterized by restricted virus multiplication and movement within the infected plant resulting in a 3–8 week delay in symptom expression.Journal Paper No. 10138 of the Michigan Agricultural Experiment Station.This work was supported in part by U.S.D.A. grant no. 79-59-2261-1-1-351-1.We thank Drs. M. Daub, R. Griesbach, and J. Hunsperger for helpful suggestions.The excellent technical assistance of Ms. Brenda Floyd and Sara Stadt is acknowledged.     

Plant development from isolated microspores of Zea mays L.

This paper reports the development of plants from mechanically isolated microspores of corn (Zea mays). Large populations of corn microspores were isolated using technology previously developed for rapeseed. Embryos and callus were developed from microspores in the late uninucleate stage. Scutellar-type embryos developed after two weeks and these could be transferred and germinated on a hormone free medium. However, the large majority of plants recovered from embryos developed only upon transfer to a corn embryogenic callus medium. These embryos produced shoots through organogenesis, and subsequently could be induced to form roots. Plants were developed from these colonies and grown in the greenhouse. The frequency of mature plants developed from the embryos was approximately 5 %. Non embryogenic callus which developed from some microspores have thus far either failed to develop or have developed only roots. Seed set has been obtained on some of the regenerated plants.     

Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10² viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.     

Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.     

Expression of tobacco mosaic virus RNA in transgenic plants

Summary Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in planta.     

Formation of anti-plant viral protein by Mirabilis jalapa L. cells in suspension culture

The extract of Mirabilis jalapa cultured cells and its precipitate fraction with 90% saturated ammonium sulfate showed an anti-plant viral activity comparable to that of the roots and leaves of the original plant. In the immunodiffusion experiment, the extract of cultured cells positively reacted with MAP (Mirabilis Anti-plant viral Protein) anti-serum. The changes in MAP formation during cell growth and the MAP content of roots and leaves were examined using enzyme-linked immunosorbent assay (ELISA). MAP formation proceeded almost in parallel with cell growth. The MAP content of cultured cells reached the highest level (0.6 mg/g dry weight) on the 9th day after inoculation, which was less than one-third of the content of the roots but three times larger than that of the leaves.Abbreviations MAP Mirabilis anti-plant viral protein - TMV tobacco mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - ELISA enzyme-linked immunosorbent assay Studies on the production of anti-plant viral substances of higher plant cells in suspension culture. Part 1     

Pathogen-inducible CaUGT1 is involved in resistance response against TMV infection by controlling salicylic acid accumulation

Capsicum annuum L. Bugang exhibits a hypersensitive response against Tobacco mosaic virus (TMV) P₀ infection. The C. annuumUDP-glucosyltransferase 1 (CaUGT1) gene was upregulated during resistance response to TMV and by salicylic acid, ethephon, methyl viologen, and sodium nitroprusside treatment. When the gene was downregulated by virus-induced gene silencing, a delayed HR was observed. In addition, free and total SA concentrations in the CaUGT1-downregulated hot pepper were decreased by 52% and 48% compared to that of the control plants, respectively. This suggested that the CaUGT1 gene was involved in resistance response against TMV infection by controlling the accumulation of SA.     

枯草芽孢杆菌L-脯氨酸合成途径中glnA、proB、proA基因功能探究

【目的】从基因水平探究枯草芽孢杆菌渗透压调节因子L-脯氨酸合成途径中glnA、proB、proA基因的功能,通过分子改造实现对代谢途径的人工扰动。【方法】从枯草芽孢杆菌WB600出发,通过向胞内引入一系列基因敲除或过表达,分别构建了proB和proA基因过表达的重组菌WB601和WB602、glnA基因缺失的重组菌WB603以及在此基础之上过表达proB基因的重组菌WB604。借助菌株胞外和胞内游离脯氨酸积累的表型分析影响途径的关键节点。【结果】在非胁迫条件下,重组菌WB601和WB602胞外脯氨酸含量分别是原始菌的2.21倍和2.82倍,单位细胞胞外脯氨酸得率分别是原始菌的4.09倍和9.80倍,胞内游离脯氨酸含量分别是原始菌的1.91倍和3.34倍;重组菌WB603胞外脯氨酸含量上升至1221.43 mg/L,是原始菌的6.28倍,单位细胞胞外和胞内游离脯氨酸得率分别为原始菌的9.13倍和3.66倍;而重组菌WB604胞外脯氨酸含量最高达1391.65 mg/L,相比菌株WB603,其胞外脯氨酸含量及单位细胞得率分别提高了13.94%和14.10%,且胞内游离脯氨酸含量提高了32.60%。在5%Na Cl胁迫条件下,重组菌WB601和WB602的胞外脯氨酸含量分别是原始菌的1.94倍和1.54倍,单位细胞胞外脯氨酸得率分别是原始菌的2.15倍和2.19倍;重组菌WB603胞外脯氨酸含量及其单位细胞得率分别是原始菌的4.16倍和7.29倍;相同条件下,相比于重组菌WB603,重组菌WB604的胞外脯氨酸含量及其单位细胞得率分别提高了32.61%和5.54%。此外,实验组菌株的胞内游离脯氨酸含量均高于非胁迫时,并达到相对平衡状态。【结论】proB和proA基因的过表达均能显著提升细胞合成脯氨酸的能力,并且能增强细胞的耐盐性;glnA基因的缺失能增强脯氨酸合成途径,提高脯氨酸的积累;两种效应的正向叠加可进一步提升细胞脯氨酸合成能力。     

RNA-dependent RNA Polymerase Activities in Tomato Plants Susceptible or Resistant to Tobacco Mosaic Virus

RNA-dependent RNA polymerase activities were measured in healthyand tobacco mosaic virus (TMV)-infected tomato plants, to investigatethe possibility that altered activity might be involved in theoperation of the Tm-I gene for resistance to TMV. Healthy, susceptibleand resistant plants had similar levels of enzyme activity.Infection with TMV strain 0, which is inhibited by Tm-I, causeda 2-fold increase in activity in susceptible plants but no increasein Tm-I plants. Infection with a number of strain 1 isolates,which overcome Tm-I resistance, led to a 2 to 4-fold increasein enzyme activity in resistant plants. RNA-dependent RNA polymerase, Tm-I resistance gene, tobacco mosaic virus, tomato, Lycopersicon esculentum     

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.     

The Effect of Arachidonic Acid and Viral Infection on the Phytohemagglutinin Activity during the Development of Tobacco Acquired Resistance

The effects of arachidonic acid (AA) on the development of viral infection and the activity of phytohemagglutinins in Nicotiana tabacum L. plants were studied. Cv. Samsun NN was used, which displayed a genotypically determined hypersensitive response to tobacco mosaic virus (TMV) infection. When tobacco leaf disks were treated with 10^–9 to –10^–7 M AA, viral reproduction was suppressed by 90–100%. The AA concentration of 10^–8 M was optimal for the improvement of plant virus resistance. Tobacco leaves maintained virus resistance for at least two weeks. Both AA treatment and TMV inoculation were accompanied by an enhanced lectin activity, which may indicate the involvement of lectins in the development of plant defense responses. Lectin accumulation was observed in the intact plants developing systemic resistance and in the detached leaves characterized by local resistance.     

TGBp3 triggers the unfolded protein response and SKP1‐dependent programmed cell death

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8‐kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV‐p3), and we noted the up‐regulation of SKP1 and several endoplasmic reticulum (ER)‐resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV‐p3, but not TMV or PVX. Such lesions were the result of TGBp3‐elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR‐related gene expression occurred within 8 h of TMV‐p3 inoculation and declined before the onset of PCD. TGBp3‐mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro‐survival mechanism. Anti‐apoptotic genes Bcl‐xl, CED‐9 and Op‐IAP were expressed in transgenic plants and suppressed N gene‐mediated resistance to TMV, but failed to alleviate TGBp3‐induced PCD. However, TGBp3‐mediated cell death was reduced in SKP1‐silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.     

Factors affecting the use of chloramphenicol acetyltransferase as a marker for Brassica genetic transformation

The CAT gene which codes for the enzyme chloramphenicol acetyltransferase was found to be ineffective as a reporter gene in cells and tissues of Brassica species. High levels of endogenous CAT activity were found to be widespread among this genus and did not appear to be distributed in a tissue- or cell-specific manner. Moreover, the presence of an inhibitor of CAT activity was discovered in Brassica napus and Brassica juncea. This inhibitor appeared to act selectively on bacterial CAT in transgenic plants. These findings provided an explanation for difficulties experienced in the detection of transgenic CAT activity in B. napus.     

Chemical suppression of the symptoms of two virus diseases

Carbendazim applied at the rate of 2 g per plant to the roots of tobacco (Nicotiana tabacum cv. White Burley) plants before infection with tobacco mosaic virus (TMV) caused very considerable reduction in the severity of disease symptoms in systemically infected leaves but did not affect their virus content. Leaves of untreated, infected plants had a greatly reduced chlorophyll content 100 days after infection whereas the chlorophyll content of leaves of infected plants treated with carbendazim was similar to that of normal uninfected leaves. Carbendazim had no effect on the infectivity of TMV in vitro or on the local lesion reaction of N. glutinosa plants when inoculated with TMV. Carbendazim was applied to lettuce cv. Cobham Green at a total rate of o-i g per plant before and after they were infected with beet western yellows virus and the plants were then grown on in the field. At harvest time (50 days after infection) almost all the treated virus-infected plants were of a normal green appearance, whereas the untreated controls were almost all very severely yellowed and unmarketable.     

Relationships between tobacco mosaic virus biosynthesis and the nitrogen metabolism of the host

1. Comparisons of the nitrogen content of TMV-infected and uninfected tobacco leaf discs at various times after inoculation show that virus synthesis is associated with a net increase in protein content. This excess protein is due to: (a) TMV, (b) an excess in insoluble protein which develops soon after inoculation and ends about 100 hours before cessation of TMV synthesis, and (c) an excess in soluble non-virus protein, which is variable in size and which only occurs during the time of virus synthesis. A deficiency in non-protein nitrogen occurs during the time when virus appears. 2. Isotope experiments with N¹⁵-labelled nutrient show that: (a) The bulk of TMV nitrogen is derived from the free ammonia of the host tissue. (b) Amino acid residues of TMV protein are not derived from the corresponding free amino acids in the host. (c) The appearance of TMV is preceded by the synthesis of an insoluble precursor of the virus which is then converted into TMV or some soluble intermediate protein. This effect is associated with a cell particulate which represents a small fraction of the total insoluble protein. (d) Infected tissue synthesizes de novo small amounts of soluble non-virus protein, which may represent intermediates in TMV synthesis. (e) Infected tissue fails to synthesize a rapidly turned-over soluble protein which is synthesized in comparable uninfected tissue. (f) TMV synthesis is preceded by a temporary enhancement of the metabolic stability of an insoluble protein component. 3. The results lead to the conclusion that TMV formation is due to diversion of some part of the host's protein-synthesizing apparatus from its normal course.     

The effect of virus infection on morphology and protein components of pollen grains

Morphology of pollen grains collected from healthy and virus infected plants ofChenopodium quinoa L.,Chenopodium album L. andNicotiana tabacum L. cv. Samsun was investigated using scanning electron microscopy (SEM). Pollen grains from tobacco plans infected with tobacco mosaic virus (TMV) were smaller, with rounded shape and conspicuous deformation of aperture unlike oval and smooth pollen grains from healthy plants. No morphological alterations were observed inC. quinoa andC. album plants infected with TMV and cucumber mosaic virus (CMV). Polyacrylamide gel electrophoresis of pollen proteins revealed substantial quantitative and qualitative differences in protein components of pollen grains collected from healthy and virus infected plants     

Changes in Glucose-6-Phosphate Dehydrogenase, Ribonucleases, Esterases and Contents of Viruses in Potato Virus Y Infected Tobacco Superinfected with Tobacco Mosaic Virus

Effects of the superinfection with tobacco mosaic virus (TMV) on susceptible tobacco plants infected with potato virus Y (PVY) were determined. Dynamic changes in the TMV and/or PVY contents, the ribonucleases (RNases), the phosphomonoesterase (PME), the phosphodiesterase (PDE) and the glucose-6-phosphate dehydrogenase (G6P DH) activities were studied. The PVY infection caused a substantial reduction in the multiplication of TMV. The content of TMV in the PVY inoculated leaves amounts to 6 and 9 % in the PVY systemically infected leaves when compared with single TMV. Surprisingly, the challenging virus (TMV) enhanced the content of inducing virus (PVY) in the locally inoculated leaves up to 130 – 141 %. In contrast, the reduction of PVY content down to 35 – 40 % by TMV was seen in the PVY systemically infected leaves. The activities of the RNase, the PME, the PDE and the G6P DH were increased (when compared with the healthy plants) during the acute phase of single virus multiplication (PVY or TMV). The increase in the activities of the enzymes in the leaves with mixed infection was at least as high as the sum of the increases of single infections. Moreover, a higher increase than the sum was seen for G6P DH and PDE (by about 20 – 35 %). This revised version was published online in July 2006 with corrections to the Cover Date.     

Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in shape Nicotiana tabacum

We evaluated the concept for protection of plants against virus infection based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mosaic virus (TMV)-specific scFv antibody, which binds to intact virions, was integrated into the plant expression vector pSS and used for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Xanthi-nc. Regenerated transgenic tobacco plants were analysed by northern blot, western blot and ELISA to assess expression and functionality of recombinant antibody (rAb) fragments. A significant increase of scFv levels in T₁ progeny was obtained for plants secreting apoplastic scFv antibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T₁ progeny producing scFvs in different plant cell compartments showed different levels of resistance upon inoculation with TMV. The most dramatic reduction of necrotic local lesion numbers upon virus infection was observed in T₁ plants expressing scFv fragments in the cytosol. Infectivity could be reduced by more than 90%, despite the observation that protein expression levels for functional scFv antibodies were very low. Furthermore, upon inactivation of the N-resistance gene at elevated temperature, a significant portion of the T₁ progenies inhibited systemic virus spread, indicating that expression of TMV-specific cytosolic scFvs confers virus resistance in these transgenic plants. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulation of virus particles is affected by cytosolic scFv expression.