Attenuated Response to Agrobacterium Strains1

We have examined the response of the hormone-resistant mutants axrl and axr2 of Arabidopsis thaliana to inoculation by Agrobacterium tumefaciens and Agrobacterium rhizogenes. Our results indicate that recessive mutations in the axrl gene affect the frequency of tumor formation after inoculation with either Agrobacterium strain. In addition, tumors produced on axrl plants were smaller than those growing on wild-type plants. These results indicate that the product of the AXR1 gene is important for both crown gall and hairy root tumor formation. In contrast, the dominant axr2 mutation has a more severe effect on the development of crown gall tumors than on hairy root tumors. Crown gall tumors produced on axr2 plants had a different morphology than wild-type tumors and did not grow when they were removed from the explant. In contrast, a large number of hairy root tumors were produced on wild-type and axr2 plants, and both types of tumors grew when they were removed from the explant. Like the roots of axr2 plants, roots produced on axr2 explants lacked root hairs. Virulent strains of Agrobacterium tumefaciens and A. rhizogenes cause the formation of crown gall and hairy root tumors, respectively, on susceptible dicotyledonous plants (2, 5, 12). In both cases, tumorigenesis is due to the action of plant-specific oncogenes that are present on the Ti or Ri plasmid and transferred from the bacteria into the plant genome during infection (23, 29). T-DNA transfer involves a set of genes called the vir genes and appears to be identical in both A. tumefaciens and A. rhizogenes (29). However, the oncogenes differ between the two bacterial species (5). The formation of a crown gall tumor involves the action of three genes on the T-DNA called iaaM, iaaH, and ipt (1, 6). The products of the iaaM and iaaH genes produce IAA from tryptophan in a two-step process (6), whereas the ipt gene product synthesizes isopentenyl AMP, a precursor to a number of biologically active cytokinins (1). Hence, tumor formation is primarily the result of a change in the levels ofIAA and cytokinin in the affected plant tissue. Until recently, changes in hormone level were thought to have a limited role in the formation of a hairy root tumor (5). A number of groups have shown that tumor formation by A. rhizogenes 'This research was supported by a National Institute of Health Predoctoral Fellowship (GM07757) to C.L. as well as National Science Foundation grant DCB-900499 1 and National Institute of Health GM43644 to M.E. depends on the synergistic action of three genes called rolA, roiB, and rolC (18, 21, 22, 27). Some experiments indicated that the rol genes, particularly rolB, act by increasing auxin sensitivity in transformed tissue (19, 21). However, experiments by Spena and his colleagues (3, 4) indicate that rolB and rolC encode enzymes that hydrolyze IAA and cytokinin glucosides, respectively. Thus, the rol genes appear to promote hairy root tumor formation either by increasing the intracellular levels of IAA and cytokinin or by decreasing the levels of hormone glucosides. It is not clear how these changes also result in an increase in auxin sensitivity. Very little is known about the plant genes required for tumorigenesis. Increased levels ofIAA and cytokinin probably stimulate cellular proliferation by acting through hormone reception/signal transduction pathways. However, the components of these pathways have not been identified. In addition, the differences in morphology between crown gall and hairy root tumors suggest that different plant genes may be important for development of these two types of tumors. Phytohormone-resistant mutants have been identified in a number of plant species including Arabidopsis thaliana, tobacco, and tomato (7). An analysis of the response of these mutant plants to inoculation with Agrobacterium species may provide new information on the mechanisms oftumorigenesis as well as the function of the mutant genes. For example, an auxin-resistant mutation in tobacco has been shown to affect response to inoculation by Agrobacterium (25). In Arabidopsis, the axrl2 and axr2 mutations both confer resistance to auxin (8, 9, 28). The axrl mutations are recessive and confer a number of morphological abnormalities, including defects in stem elongation and apical dominance (8). In addition, experiments with stem explants indicate that mutant tissue requires a higher concentration of auxin to induce callus growth than does wild type (9). The axr2 mutation is dominant and confers resistance to cytokinin, ABA, and ethylene in addition to auxin (28). Mutant plants also display a number of growth changes, including defects in shoot and root gravitropism, reduced root length, and lack of root hairs. In this study, we examine the effects of the axrl and axr2 mutations on the process of crown gall and hairy root tumor formation. Our results show that these mutations affect the frequency of tumor formation as well as tumor morphology. In addition, 2 Genetic nomenclature: according to guidelines established at the Third International Arabidopsis Meeting, Michigan State University, April 1987, wild-type gene symbols are capitalized (i.e. AXR2) and mutants are represented with lower case symbols (i.e. axr2).