Cloning the Cf-9 Gene of Tomato by Transposon Tagging

Transposon tagging was used by Jones et al. in 1994 (Science 266:789) to clone the Cf-9 gene from tomato. Cf-9 provides resistance against the races of C. fulvum that express the Avr9 avirulence gene. The interaction between the host and pathogen follows the gene-for-gene theory of host resistance. In this system the tomato Cf-9 gene interacts with the avirulence gene Avr9 of the C. fulvum to provide resistance.

This interaction has been extensively analyzed. For example, the final product of the Avr9 avirulence gene is a 28-amino acid peptide that induces necrotic lesions when it is injected into the tomato plant. Previously, a unique response was noted when an engineered version of the pathogen Avr9 gene was introduced into a tomato line via plant transformation techniques. If the line contained the recessive susceptibility allele at the Cf-9 gene the plants remained healthy. But if the Avr9 gene was transformed into a line with the dominant Cf-9 allele, the plants rapidly became necrotic and died. (These results were reported in 1994 by Kosack et al. in the Proceedings of the National Academy of Science, USA 91:10445.)

Jones et al. previously developed a tomato line that contained a maize Ds element 4 cM from Cf-9. To activate the element, this line was crossed to a line that contained a maize Ac element. This new line was crossed to a transgenic line that contained the Avr9 gene. The following diagram is based on Figure 1 of the Jones et al. Science article and shows the crossing pattern.

Progeny Class 1

Progeny Class	Phenotype
Class 1	These plants all die because the Avr9 is necrotic in combination with the Cf-9 allele
Class 2	These plants are stable and show no necrosis because Ds has inactivated the Cf-9 allele.
Class 3	This class contains plants that show a variegated-necrotic phenotype. The plants are variegated because the Ds element has excised (with the aid of the Ac element) in sectors of the plant. Necrosis will develop in those sectors of the plant.

A total of 160,000 progeny seeds were grown, and a total of 63 independent mutations (that is plants that survived with no necrosis or expressed variegated necrosis) were detected. These mutations feel into three classes. 21 class 3 progeny were detected that expressed variegated necrosis (and thus contained an active Ac element). 33 class 2 stable progeny that expressed no necrosis were found. Finally, 9 stable progeny were detected that did not contain a Ds were found. The variegated class is important because it is clear that these contain a Ds element in the Cf-9 gene. It was further shown that 16 of the stable mutants contained a Ds element in Cf-9 becuase it could be moved when crossed to a tomato line containing an Ac element. Therefore a total of 37 independent Ds insertions events into the Cf-9 were found with this experiment. A mapping experiment showed that 28 of the 37 mapped within a 3 kb region of the tomato genome.

One mutant line was used to actually isolate the Cf-9 gene. (The details of that will not be presented here.) Analysis of this gene revealed that it had similarities to two other cloned plant disease resistance genes. Specifically, these are the RPS2 gene of Arabidopsis that confers resistance to the bacterial pathogen Pseudomonas syringae [Bent et al. (1994) Science 265:1856] and the N gene of tobacco that provides resistance to the tobacco mosaic virus [Whitham et al (1994) Cell 78:1101]. That genes with similar structure have been found to confer resistance against fungal, bacterial and viral pathogens from three different speices suggests that common mechanisms of disease defense may be at work in plants.

Copyright © 1998. Phillip McClean