Research Description

Map-based Cloning of a Salicylic Acid Tolerant Mutant

Like most organisms, plants must avoid disease in order to thrive. Plants are essential to food supplies around the world and the agricultural market is vital constituent of our present day economy. Thus, plant disease has wide ranging negative effects on the economy and society as a whole. Plant disease itself is rare because plants have evolved effective mechanisms for combating pathogens. Salicylic acid and the protein NPR1 have been shown to be essential for pathogen resistance (1). By understanding how these components act we can better understand and strengthen plant defense responses.

In the model plant Arabidopsis, the mutant npr1 lacks a functional NPR1, which causes npr1 to be more susceptible to disease than wild type plants. npr1 also dies when grown on media supplemented with salicylic acid, whereas wild type survives. By mutating seeds of npr1 and screening for SA-tolerant plants, the mutant 6-9 was isolated. 6-9 is heritable and recessive (unpublished observations), and restores SA tolerance and pathogen resistance to npr1. The mutant also exhibits yellowing of young leaves. My research will further genetically analyze and clone the 6-9 mutation. This may illuminate how SA and NPR1 activate plant defenses.

Salicylic acid tolerance and leaf discoloration may be caused by the same mutation or two different mutations. I am now performing genetic analysis to determine if the yellowing phenotype can be used to select mutant plants for map-based cloning.

If I find that the yellowing and SA tolerance phenotypes are inherited together, and thus probably caused by the same mutation, I will proceed with map-based cloning of the 6-9 mutation.

First I will carry out bulked segregant analysis to determine the rough location of the mutation. Mutant plants that are Columbia ecotype will be crossed to the ecotype Landsberg and plants exhibiting yellowing in the F2 generation will be used for mapping. DNA will be extracted from ~100 individual mutant plants in the F2 mapping population and pooled. Control DNA from F1, Landsberg, and Columbia plants will also be used. Using PCR and gel electrophoresis, 22 loci will be tested for simple sequence length polymorphisms (SSLPs) that occur between the Columbia and Landsberg ecotypes. Since the 6-9 mutation was generated in the Columbia ecotype, the preferential amplification of Columbia DNA from the pooled template at a given loci suggests the 6-9 mutation is nearby. In this manner the approximate location of the mutation can be determined. By developing new markers in the vicinity of the mutation the position can be resolved further.

Once the mutations have been roughly mapped through bulked segregant analysis, I will carry out three-point mapping by testing DNA from individual plants using more markers, further narrowing the possible interval where 6-9 could be (2). Future work will include complementation of the mutant and detailed characterization of the mutant gene product, though this is beyond the scope of my proposed research.

1) Cao H, Bowling SA, Gordon S, Dong X. Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 1994, 6:1583-1592.

2) Lukowitz W, Gillmor C, and Scheible W. Positional Cloning in Arabidopsis. Why It Feels Good to Have a Genome Initiative Working for You. Plant Physiology 2000, 123:795–805.