Huet, H; Mahendra, S; Wang, J; Sivamani, E; Ong, CA; Chen, L; De Kochko, A*; Beachy, RN; Fauquet, C

Phytopathology, vol. 89, no. 11, pp. 1022-1027, Nov 1999

Rice tungro disease is caused by rice tungro bacilliform virus (RTBV), which is responsible for the symptoms, and rice tungro spherical virus (RTSV), which assists transmission of both viruses by leafhoppers. Transgenic japonica rice plants (Oryza sativa) were produced containing the RTSV replicase (Rep) gene in the sense or antisense orientation. Over 70% of the plants contained one to five copies of the Rep gene, with integration occurring at a single locus in most cases. Plants producing antisense sequences exhibited significant but moderate resistance to RTSV (60%); accumulation of antisense RNA was substantial, indicating that the protection was not of the homology-dependent type. Plants expressing the full-length Rep gene, as well as a truncated Rep gene, in the (+)-sense orientation were 100% resistant to RTSV even when challenged with a high level of inoculum. Accumulation of viral RNA was low, leading us to conclude that RTSV Rep-mediated resistance is not protein-mediated but is of the cosuppression type. Resistance was effective against geographically distinct RTSV isolates. In addition, RTSV-resistant transgenic rice plants were unable to assist transmission of RTBV. Such transgenic plants could be used in an epidemiological approach to combat the spread of the tungro disease.

Fray, RG; Throup, JP; Daykin, M; Wallace, A; Williams, P; Stewart, GSAB; Grierson, D

Nature Biotechnology [Nat. Biotechnol.], vol. 17, no. 10, pp. 1017-1020, Oct 1999

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30-84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenl) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carl. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.

Zhang, L; Xu, J; Birch, RG*

Nature Biotechnology [Nat. Biotechnol.], vol. 17, no. 10, pp. 1021-1024, Oct 1999

We generated transgenic sugarcane plants that express an albicidin detoxifying gene (albD), which was cloned from a bacterium that provides biocontrol against leaf scald disease. Plants with albicidin detoxification capacity equivalent to 1-10 ng of AlbD enzyme per mg of leaf protein did not develop chlorotic disease symptoms in inoculated leaves, whereas all untransformed control plants developed severe symptoms. Transgenic lines with high AlbD activity in young stems were also protected against systemic multiplication of the pathogen, which is the precursor to economic disease. We have shown that genetic modification to express a toxin-resistance gene can confer resistance to both disease symptoms and multiplication of a toxigenic pathogen in its host.

Chen, WP; Chen, PD; Liu, DJ; Kynast, R; Friebe, B; Velazhahan, R; Muthukrishnan, S; Gill, BS*

Theoretical and Applied Genetics [Theor. Appl. Genet.], vol. 99, no. 5, pp. 755-760, 22 Sep 1999

The possibility of controlling wheat scab (caused by Fusarium graminearum Schw.) was explored by engineering wheat plants for constitutive expression of pathogenesis-related (PR) protein genes. A rice thaumatin-like protein (TLP) gene (tlp) and a rice chitinase gene (chi11) were introduced into the spring wheat cultivar 'Bobwhite' by co-transformation of the plasmids pGL2ubi-tlp (ubiquitin/tlp//CaMV 35S/hpt) and pAHG11 (CaMV 35S/chi11//ubiquitin/bar). The transformation was by biolistic bombardment. Bialaphos was used as the selection reagent. The integration and expression of the tlp, bar, chi11 and hpt genes were analyzed by Southern, Northern and Western blot analyses. The four transgenes co-segregated in the T1 progeny of the transgenic plant and were localized at the telomeric region of the chromosome 6A long arm by sequential N-banding and fluorescent in situ hybridization (FISH) using pAHG11 or pGL2ubi-tlp as the probes. Only the transgenes tlp and bar, under the control of the ubiquitin promoter-intron, were expressed. No expression of the chi11 and hpt genes, controlled by the CaMV 35S promoter, was detected in T1 plants. After inoculation with conidia of F. graminearum, the symptoms of scab developed significantly slower in transgenic plants of the T1, T2 and T3 generations expressing the tlp gene than in non-transformed control plants. This is the first report of enhanced resistance to F. graminearum in transgenic wheat plants with constitutive expression of TLP.

Nishizawa, Y; Nishio, Z; Nakazono, K; Soma, M; Nakajima, E; Ugaki, M; Hibi, T

Theoretical and Applied Genetics [Theor. Appl. Genet.], vol. 99, no. 3/4, pp. 383-390, 24 Aug 1999

Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R0), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines.

Sela, I; Vardi, E; Stram, Y

US Patent: 5939603, , 17 Aug 1999

The invention relates to chimeric plasmids comprising at least one non-sttural potyvirus gene or fragment thereof, which gene is capable of being expressed in a plant transformed with the chimeric plasmid, the thus transformed plant being resistant to an infection by a plant potyvirus. The invention also relates to vectors comprising a chimeric plasmid according to the invention, particularly microorganisms transformed with the chimeric plasmids of the invention. Potyvirus-resistant plants having cells containing in their genomes the chimeric DNA sequences according to the invention, as well as seeds and cells of a potyvirus-resistant plant containing in their genome the said chimeric DNA sequences are also described and claimed, as well as methods of protecting plants against potyvirus infection wherein genomes of cells of the plant are provided with a chimeric DNA sequence according to the invention, whereby the calls become resistant to an infection by a potyvirus.

Xu, D; Collins, GB; Hunt, AG; Nielsen, MT

Crop Science [Crop Sci.], vol. 39, no. 4, pp. 1195-1202, Aug 1999

Eighty transgenic tobacco (Nicotiana tabacum L.) lines expressing either the tobacco vein mottling virus (TVMV) coat protein (CP) gene or the alfalfa mosaic virus (AMV) CP gene were evaluated for their agronomic performance with or without virus challenge. All TVMV CP lines were characterized previously for their virus resistance following challenge with three different potyviruses. In the absence of virus challenge, the cured leaf yield of more than 80% of the TVMV CP lines was not significantly different from their non-transgenic counterparts; half of the poor performing lines were in one of the five genetic backgrounds. Similarly, most of the negative variation for yield among the AMV CP transgenic lines was within a single genetic background. Variation for other agronomic traits, including plant height, leaf size, and stalk diameter closely followed the results for yield. Following virus challenge, cured leaf yield of the most resistant transgenic lines was similar to the non-challenged treatment. Among the AMV CP transgenic lines, yield loss following challenge with AMV was reduced from 22 to 30%. These results suggest that the primary selection criterion in a population of transgenic tobacco lines containing a viral CP gene should be for virus resistance, but it must be recognized that variation for agronomic performance may exist among the resistant selections, particularly in some genetic backgrounds.

Lorito, M; Scala, F

Journal of Plant Pathology [J. Plant Pathol.], vol. 81, no. 2, pp. 73-88, Jul 1999

The development of plant transformation and gene cloning techniques has opened new avenues for augmenting disease and insect resistance in crops. The concept of 'Genetically Acquired Resistance' now encompasses a variety of strategies based on the transgenic expression in plant of genes from many different origins. Some of the most potentially useful transgenes capable of enhancing resistance to attack by microbes, viruses and insects have been obtained from bacteria and fungi, which are regarded as rich sources of desirable traits for plant genetic improvement. The main research directions have included: (i) the enhancement of the plant anti-microbial and insecticidal arsenal by the constitutive or inducible synthesis of various pathogen inhibiting compounds; (ii) the alteration of the plant response to pathogens by the appropriate induction of the local or systemic defence mechanisms, or by the interference with plant-pathogen signalling and (iii) the inactivation of pathogen toxins or the improvement of plant resistance to them. Although transgenic plants that are insect-resistant or bear other useful traits obtained from microbes, such as herbicide-resistance or enhanced food quality, are being commercially marketed, genetically-engineered crops exhibiting resistance to fungal or bacterial diseases have yet to reach the marketplace. The main focus of this paper addresses the application of bacterial or fungal genes to improve plant resistance to insect, fungi, bacteria and viruses, and the usefulness of these microbial genomes in the development of new transgenic technologies. In addition, perspectives dealing with environmental concerns and important biosafety questions resulting from these technologies are briefly discussed.

Monti, MM; Valanzuolo, S; Cassani, G; Colombo, M

Journal of Plant Pathology [J. Plant Pathol.], vol. 81, no. 2, pp. 113-122, Jul 1999

Transgenic 'San Marzano' tomato lines transformed with a chimeric gene expressing a benign satellite RNA of Cucumber Mosaic Virus (CMV) were tested under conditions of natural infection, in southern Italy. Agronomic performance, virus susceptibility and spread of transgenic satellite RNA were monitored during the test. ELISAs and yield measurements showed that virus incidence was slightly lower in transgenic plants compared to controls. Satellite RNA was detected in melon, zucchini and pepper plants grown in the surroundings of the release site. Spread of the benign satellite RNA from transgenic tomatoes to surrounding host plants did not increase disease incidence or disease phenotype.

Bi, Y; Cammue, BP; Goodwin, PH; KrishnaRaj, S; Saxena, PK*

Plant Cell Reports [Plant Cell Rep.], vol. 18, no. 10, pp. 835-840, 16 Jun 1999

Scented geranium (Pelargonium sp. `Fren-sham') was transformed with a gene encoding an antimicrobial protein (Ace-AMP1) from onion through an Agrobacterium-mediated transformation system. The binary vector pFAJ3033 contained the coding region of the Ace-AMP1 preproprotein-encoding cDNA. Transformants were verified by polymerase chain reaction and Southern blot analysis. Transgenic plants expressing high levels of Ace-AMP1 were identified by immunoblots and those plants were shown to have increased resistance to Botrytis cinerea leaf infection.

Xue, B; Ling, K-S; Reid, CL; Krastanova, S; Sekiya, M; Momol, EA; Suele, S; Mozsar, J; Gonsalves, D; Burr, TJ*

In Vitro Cellular & Developmental Biology - Plant [In Vitro Cell. Dev. Biol. Plant], vol. 35, no. 3, pp. 226-231, Jun 1999

To facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating root-stocks: Couderc 3309, Vitis riparia 'Gloire de Montpellier', Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture and grape regeneration were accomplished with four media. Embryogenic calluses from anthers were induced in the initiation medium [MS basic medium containing 20 g sucrose per L, 1.1 mg 2.4-dichlorophenoxyacetic acid (2,4-D) per L, 0.2 mg N super(6)-benzyladenine (BA) per L, and 0.8% Noble agar]. The percentage of anthers that developed into embryogenic calli ranged from 2 to 16.3% depending on the rootstock. Calluses with early globular stage embryos were cocultivated with Agrobacterium tumefaciens strain C58Z707 containing the gene constructs of interest. The genes were sense-oriented translatable and antisense coat protein genes from GFLV and GLRaV-3, a truncated HSP90-related gene of GLRaV-3 (43K), and a virE2 del B gene from A. tumefaciens strain C58. Twenty independent transformation experiments were performed on five rootstocks. After 3-4 mo. under kanamycin selection, secondary embryos were recovered on differentiation medium (1/2 MS salts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Embryos that were transformed were regenerated on a medium containing MS salts with 20 g sucrose per L, 4.6 g glycerol per L, 1 g casein hydrolysate per L, and 0.8% Noble agar. Elongated embryos were then transferred to a rooting medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, and 0.65% Bacto agar. A total of 928 independent putative transgenic plants were propagated in the greenhouse. All plants were tested for neomycin phosphotransferase II expression by enzyme-linked immunosorbent assay (ELISA). The presence of transgenes was assessed by polymerase chain reaction and Southern analysis. ELISA revealed various levels of expression of GFLV coat protein in transgenic plants of Couderc 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to the virus and crown gall diseases.

Wang, Y; Nowak, G; Culley, D; Hadwiger, LA; Fristensky, B*

Molecular Plant-Microbe Interactions [Mol. Plant-Microbe Interactions], vol. 12, no. 5, pp. 410-418, May 1999

To identify genes effective against the blackleg fungus Leptosphaeria maculans (Phoma lingam), we have transformed canola (Brassica napus) with four pea (Pisum sativum) genes under constitutive control by the cauliflower mosaic virus 35S promoter: PR10.1, chitinase, DRR206, and defensin. Transgenic lines containing single-copy T-DNA insertions for each gene were screened for both seedling (cotyledonary) and adult plant resistance. Lines for which pea DRR206 mRNA was expressed showed decreased disease scores, compared with non-expressing transgenic lines. Transgenic plants expressing pea defensin showed a slight enhancement of resistance, while for PR10 and chitinase transgenics there was little or no enhancement of resistance. Resistance to L. maculans cosegregated with DRR206 transgenes. Extracts from DRR206 and defensin transgenic plants inhibited fungal germination in vitro. DRR206 transgenic plants also demonstrated decreased hyphal growth at inoculation sites. While the precise function of DRR206 remains to be determined, these results suggest that it does play an important role in defense against fungi.