INTERACTION BETWEEN THE MAIZE PATHOGEN COCHLIOBOLUS HETEROSTROPHUS RACE T AND TOBACCO EXPRESSING THE T-URF13 (BMT TOXIN RECEPTOR) GENE FROM TEXAS MALE-STERILE MAIZE
A EL-MALEH1, C KOEHLER2, J HUANG2, AM MYERS2 and E HACK1
1Department of Biological and Nutritional Sciences, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK; 2Department of Biochemistry, Iowa State University, Ames, IA 50011, USA
Background and objectives
Results and conclusions
For pathogenicity tests, a small amount of C. heterostrophus race T mycelium was placed on leaf surfaces of cultured tobacco shoots. On some transgenic shoots, a halo appeared around the inoculation point and slowly enlarged. Eventually, fungal mycelium grew from the lower surface of the leaf and secondary infections occurred on the stems. On non-transgenic shoots, no host-pathogen interaction was evident. Susceptible shoots were cloned for further investigations. The presence in them of the T-urf13 gene was confirmed by PCR analysis.
When the fungus was allowed to grow on medium supporting cultured shoots, susceptible shoots were systemically bleached, indicating systemic spread of toxin, while control shoots were unaffected. To test the effect of toxin directly, shoot culture protoplasts were treated with partially purified BmT toxin and then with the fluorescent dye DiOC6, which accumulates only in coupled mitochondria. Toxin concentrations that eliminated mitochondrial fluorescence of protoplasts from susceptible shoots had no effect on control protoplasts.
Shoots from a susceptible line were rooted in soil and inoculated with race T culture filtrate. The treated areas became bleached after about 16 h and necrotic at 24 h. There was no evidence of systemic spread of the toxin. This localized response to toxin suggests that tobacco plants can resist its effects. There was no visible effect of treating control plants with the culture filtrate, or toxin-sensitive plants with culture filtrate of a toxin-non-producer isolate. Attempts to infect potted plants with the fungus were unsuccessful.
Our results show that expression in tobacco of a gene coding for the maize mitochondrial T-urf13 gene fused to a mitochondrial targeting peptide can confer sensitivity to BmT toxin and, moreover, a degree of susceptibility to the fungus C. heterostrophus race T, which is normally a pathogen of maize. Susceptibility was only observed, however, in shoot cultures. This could be because these cultures have a generally low ability to resist pathogens, or because they are more sensitive to the toxin than plants transferred to soil.