GrainGenes

KANSAS STATE UNIVERSITY

Departments of Agronomy and Biochemistry

Throckmorton and Burt Halls, Manhattan, KS 66506-5501, USA.

The expression of a rice chitinase gene in transformed wheat plants.

Xu Gu¹, S. Muthukrishnan², and G.H. Liang¹.

¹Department of Agronomy and ²Department of Biochemistry.

Most higher plants can synthesize a group of pathogenesis-related (PR) proteins when they are infected by pathogens or under abiotic stresses. Chitinase, one of the PR proteins, hydrolyzes the FONT SIZE=2 FACE="Symbol"a-1,4 linkages of the N-acetyl-D-glucosamine polymer, chitin. Chitin is a major component of the cell walls of many fungi. Chitinase is suggested to have a protective role against fungal pathogens. In recent years, transgenic plants of tobacco, barley, and rice have shown that elevated levels of chitinases, brought about by introduction of a transgene or by microinjection, increased their resistance to some fungal pathogens, such as powdery mildew and sheath blight pathogens. In this study, transgenic wheat plants in which a rice chitinase gene was incorporated are shown to express the rice chitinase constitutively.

Plant material and tissue culture. Wheat plants (Pavon 76) were grown in a greenhouse, and immature embryos were removed aseptically 12 to 14 days after pollination and placed on a MS medium containing 2 mg/l 2,4-D; 100 mg/l inositol; 100 mg/l casein hydrolysate; and 3 % sucrose.

Plasmid DNA. The pAHC20-G11 plasmid containing the rice chitinase gene, G11, was constructed at KSU. The vector pAHC20 was kindly provided by Dr. P.H. Quail (University of California, Berkeley, USA). This vector contains the selectable marker gene Bar, which encodes the enzyme phosphinothricin acetyltransferase that inactivates phosphinothricin, the active ingredient of the herbicide bialaphos, by acetylation. The marker gene was driven by the maize ubiquitin promoter, and the G11 rice chitinase gene was under the control of the CaMV 35S promoter.

Microprojectile bombardment and plant regeneration. Prior to bombardment, plasmid DNAs were precipitated and adsorbed to M17 tungsten particles following the procedure recommended by the manufacturer (Bio-Rad) and Weeks et al (1993). Thirty immature embryos were placed in the center of a `100 x 15 mm' petri dish containing MS medium. After 5-7 days of culture, the embryo-derived calli were bombarded with DNA-coated tungsten particles. The distance from the stopping screen to the target was 10-13 cm, and the rupture disc strength was 900 and 1,100 psi.

After bombardment, embryo-derived calli were transferred to MS medium with 1-2 mg/l bialaphos for selection of resistant lines. For regeneration, resistant calli with green spots or green shoots selected from the bialaphos-containing medium were transferred to regeneration medium containing 1 mg/l bialaphos and cultured under 16 h/8h light-dark period at 24-26_C until the plantlets were 3-5 cm tall. Those shoots without a well-developed root system were transferred to a rooting medium containing half-strength MS elements with 1 mg/l bialaphos. Plantlets transplanted in soil were grown in a growth chamber at 18-21_C with 16h/8h light-dark photoperiod.

Western blot analysis. Protein extraction, gel electrophoresis, and western blotting were as described by Lin et al. (1995). Typically, 50-150 µg of total protein extracted from callus tissue or plant leaves was used in the assay.

PAT assay. Activity of phosphinothricin acetyltransferase (PAT) was assayed by a thin-layer chromatographic method as described previously (Spencer et al. 1990; Weeks et al. 1993).

Results.

Bombardment and selection of plantlets. In different transformation experiments, a total of 1,556 embryos was bombarded and selected on medium containing bialaphos. Some calli turned to yellow-brown, and other calli proliferated and differentiated. The frequency of shoot induction varied from 30-50 % in different experiments. Shoots without a well-rooted system were transferred to a rooting medium containing 1 or 2 mg/l bialaphos without hormone. About 10 % of the shoots were able to form roots on the rooting medium. The herbicide-sensitive plantlets could initiate roots, but the roots were thin, short, and grew slowly. Herbicide-resistant plantlets could thrive in the rooting medium for more than 1 month, whereas the nonresistant plantlets died after 3 weeks. The frequency of regeneration varied from 2-5 % depending on the transformants and selection pressure.

Expression of chitinase in different tissue. Two weeks after bombardment, fresh protein was extracted from bombarded calli to detect expression of chitinase. From a total of 50 calli analyzed, four had the expected 35 kDa chitinase, corresponding to the rice chitinase gene G11. A second chitinase band of about 30 kDa also was seen in transformed calli.

Those plantlets that survived in soil were analyzed by western blots. Protein was extracted from the leaves of transformed plants. A protein extract of a transgenic rice plant containing the G11 chitinase was used as positive control, and an extract from a nontransformed plant was used as a negative control. Aliquots containing an equal amount of protein were separated in a 12 % SDS-PAGE followed by western blot analysis. Out of seven transformants tested, the chitinase antibody detected the presence of one immuno-reactive polypeptide with an apparent molecular weight of 35 kDa in plants #1, #2, and #7 and in the positive control, but not in other plantlets and nontransformed control. The intensities of the 35 kDa band were different in extracts of individual T0 plants with #1, #2, and #7 plants showing a higher level and #3 a lower amount of protein. In addition to the 35 kDa band, a 28 kDa chitinase band also was found in transformed plants as in the case of transgenic rice plants (Lin et al. 1995).

Bar gene expression. The leaves of regenerated plants were homogenized to obtain a crude extract, and PAT activity was analyzed using methods previously described (Spencer et al. 1990, Weeks et al. 1993). The level of PAT activity, as measured by production of the acetylated form of phosphinothricin, varied among plants. PAT activity was detected in three of the transgenic plants, and the level of PAT activity was significantly lower than the Ubi-Bar transformed calli of `Bobwhite' (kindly provided by Dr. J. Troy Weeks). Cotransformation and coexpression were found between the Bar gene and the rice chitinase gene. Those plants that expressed rice chitinase also showed PAT activity.

References.

Becker D, Brettschneider R, and Lorz H. 1994. Plant J 5:299-307.

Christensen AH, Sharrock RA, and Quail PH. 1992. Plant Mol Biol 18:675-689.

Christou P, Tameria LF, and Kofron. 1991. Bio/Technology 9:957-962.

Datta SK, Peterhans A, Datta K, and Potrykus I. 1990. Bio/Technology 8:736-740.

Lin W, Anuratha CS, Datta K, Potrykus I, Muthukrishnan S, and Datta SK. 1995. Bio/Technology 13:686-691.

Nehra NS, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauer L, Baga M, and Kartha KK. 1994. Plant J 5:285-297.

Peng J, Kononowicz H, and Hodges TK. 1992. Theor Appl Genet 83:855-863.

Spencer TM, Gordon-Kamm WJ, Daines RJ, Start WG, and Lemaux PG. 1990. Theor Appl Genet 179:625-631.

Vasil V, Brown SM, Re D, Fromm ME, and Vasil IK. 1991. Bio/Technology 9:743-747.

Vasil V, Castillo A, Fromm ME, and Vasil IK. 1992. Bio/Technology 10:667-674.

Wan Y and Lemaux PG. 1994. Plant Physiol 104:37-48.

Weeks JT, Adernson OD, and Blechl AE. 1993. Plant Physiol 102:1077-1084.

Evapotranspiration Laboratory

Department of Agronomy, Kansas State University, Waters Hall, Manhattan, KS 66502, USA.

M.B. Kirkham.

News.

Jingxian Zhang has received his Ph.D. He is working as a postdoctoral fellow in the Plant Molecular Genetics Laboratory headed by Dr. Henry T. Nguyen, Department of Plant and Soil Sciences, Texas Tech University, Lubbock, TX 79401, USA. Dr. Zhang is screening cereals for osmotic adjustment to identify genotypes that will grow well under dry conditions.

Publications.

Zhang JS, Cui S, Li JM, Wei JK, and Kirkham MB. 1995. Protoplasmic factors, antioxidant responses, and chilling resistance in maize. Plant Physiol Biochem 33:567-575.

Zhang JX and Kirkham MB. 1995. Sap flow in a dicotyledon (sunflower) and a monocotyledon (sorghum) by the heat-balance method. Agron J 87:1106-1114.

Zhang JX and Kirkham MB. 1995. Water relations of water-stressed, split-root C4 (Sorghum bicolor; Poaceae) and C3 (Helianthus annuus; Asteraceae) plants. Am J Bot 82:1220-1229.

Zhang JX and Kirkham MB. 1995. Enzymatic responses of the ascorbate-glutathione cycle to drought in sorghum and sunflower. Plant Sci 113:139-147.

Kirkham MB and Kirkham D. 1995. Chloride and water content in the root zone of barley grown under four salt-water irrigation regimes. In: Vadose Zone Hydrology: Cutting across Disciplines (Silva D ed). Proc Inter Conf, Kearney Foundation of Soil Science and Hydrologic Science, University of California, Davis. Pp. 75-76.

Department of Plant Pathology

Throckmorton Hall, Manhattan, KS 66502-5502, USA.

Loss of herbicidal activity in biocontrol bacteria.

M.M. Pyle and J.E. Leach and P. Harris and P.W. Stahlman KS).

A previous report (Pyle et al. 1995) detailed the effects of temperature and soil moisture on the survival in the downy brome (Bromus tectorum L.) rhizosphere of two potential biocontrol bacteria, Pseudomonas putida FH160R and Stenotrophomonas maltophilia FH131R. In this update, we report attempts to compensate for the loss of herbicidal activity of FH160R, as well as to identify other potentially effective bacteria. Except where noted, water agar and soil assays were conducted as previously described (Pyle et al. 1995).

In initial greenhouse assays, FH160R reduced downy brome biomass by as much as 70 % (Harris and Stahlman, 1990); however, the herbicidal activity of FH160R has abated severely over time. We approached this problem in two ways. First, we attempted to restore phytopathological activity to FH160R. Dr. S. Lam at CIBA-Geigy mobilized the plasmid pCIB137 into FH160R by conjugation. pCIB137 consists of the 2kb XhoI fragment containing the global regulatory sequence gacA cloned into the XhoI site of pVK100; plasmids containing such sequences have been shown to restore traits such as antibiotic activity to other pseudomonads (Gaffney et al. 1994). The presence of the plasmid failed to restore the lost herbicidal activity to FH160R in water agar or soil assays.

We also examined additional bacterial strains for herbicidal activity. More than 400 unidentified strains collected from Agricultural Research Center-Hays were tested in water agar assays; approximately 100 of these strains were tested in soil. These strains inhibited downy brome in the water agar assays but had no herbicidal effect when pipetted onto soil in pots containing wheat and downy brome seed. We also tested strains having biocontrol activity in other systems. Pseudomonas fluorescens Pf5 (Howell and Stipanovic 1979) and four Tn5 mutants of Pf5 (Kraus and Loper 1992) demonstrate biocontrol activity against plant fungal pathogens. Strains producing the compounds coronatine (Bender et al. 1991), phenazine-1-carboxylate, and 2,4-diacetylphloroglucinol (Thomashow et al. 1988) were tested, as were nonproducing mutants of these strains. Although some of these strains were bioactive against brome in the water agar assay, none maintained this herbicidal effect in soil.

Acknowledgments. Many thanks to Steve Lam for his work with FH160R, and to Fanny Rodriguez, Bill Pfender, Carol Bender, Linda Thomashow, and Mark Mazzola for providing strains and/or purified biocontrol compounds. Thanks to Anne Fischer and Jianfa Bai for their technical assistance.

References.

Pyle MM, Mazzola M, Harris P, Stahlman PW, and Leach JE. 1995. The effect of temperature and soil moisture on the ecology of two bacteria in the rhizosphere of downy brome. Ann Wheat Newslet 41:238-240.

Harris PA and Stahlman PW. 1990. Selective control of winter annual grass weeds in winter wheat with soil bacteria. Agron Abstr p. 250.

Gaffney TD, Lam ST, Ligon J, Gates K, Frazelle A, Di Maio J, Hill S, Goodwin S, Torkewitz N, Allshouse AM, Kempf H-J, and Becker JO. 1994. Global regulation of expression of antifungal factors by a Pseudomonas fluorescens biological control strain. Mol Plant Microbe Inter 7:455-463.

Howell CR and Stipanovic RD. 1979. Control of Rhizoctonia solani on cotton seedlings with Pseudomonas fluorescens and with an antibiotic produced by the bacterium. Phytopath 69:480-482.

Kraus J and Loper JE. 1992. Lack of evidence for a role of antifungal metabolite production by Pseudomonas fluorescens Pf5 in biological control of Pythium damping-off of cucumber. Phytopath 82:264-271.

Bender CL, Young SA, and Mitchell RE. 1991. Conservation of plasmid DNA sequences in coronatine-producing pathovars of Pseudomonas syringae. Appl Env Micro 57:993-999.

Thomashow LS and Weller DM. 1988. Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. J Bact 170:3499-3508.

The Wheat Genetics Resource Center

Departments of Plant Pathology and Agronomy and the USDA-ARS, Throckmorton Hall, Manhattan, KS 66506-5502, USA.

T.S. Cox, W.W. Bockus, B.S. Gill, R.G. Sears, T.J. Martin, W.F. Heer, J.H. Long, T.L. Harvey, W.J. Raupp, and D.L. Wilson.