Recovery of nonpathogenic mutant bacteria from tumors caused by several Agrobacterium tumefaciens strains: a frequent event?

Abstract

We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor.

FIG. 1.

Genes carried by the different plasmids used in complementation analyses and positions of primers employed to check for the presence of the T-DNA regions in the mutants analyzed. For details, see Table 2.

FIG. 2.

RAPD pattern diversity obtained with colonies isolated from tumors of different hosts inoculated with strain IVIA325-4 and patterns obtained with mutants of strain IVIA1102. (A) RAPD analysis with primer OPE-2 of colonies obtained with strain IVIA325-4. Lane 1, strain isolated from Adafuel in tumor 1; lanes 2 to 4, isolates from the same host but from tumor 3; lane 5, isolate from Adafuel in tumor 4; lane 6, inoculated strain IVIA325-4; lanes 7 to 17, isolates from host cherry; lanes 7 to 11, isolates from tumor 1; lane 12, isolate from tumor 2; lanes 13 to 14, isolates from tumor 3; lanes 15 to 17, isolates from tumor 5. M, 123-bp molecular mass marker (Life Technologies); C(−), negative control. The arrows indicate the different patterns obtained with isolates from tumor 1 in cherry, the same shape indicates the same pattern, and the different arrows show the colonies that are different. (B) RAPD analysis of colonies obtained with strain IVIA1102 isolated from tomato with primers OPE-2 and OPE-7. Lanes 1 and 7, mutant T22; lanes 2 and 8, mutant T38; lanes 3 and 9, mutant T67; lanes 4 and 10, mutant T32; lanes 5 and 11, mutant T76; lanes 6 and 12, strain IVIA1102. M, 123-bp molecular mass marker (Life Technologies).

FIG. 3.

Mutant T1 contains an insertion of IS426 in a gene essential for the biosynthesis of 3-indoleacetic acid, iaaM. (A) Amplicons obtained with primer pair CYT21-CYT2. Nonpathogenic mutant bacteria T1 and P72 were isolated from tumors induced in tomato and pepper, respectively, by the wild-type strain C58. M, marker XVII (Life Technologies). (B) Position of IS426 in the T-DNA of mutant T1. The annealing sites of the different primers used for PCR and sequencing are indicated with arrows.

FIG. 4.

Plasmid profiles of the nonpathogenic mutants derived from tumors induced by strains C58 (A) and IVIA1102 (B). The positions of the pTi for C58 (210 kb) and IVIA1102 (120 kb) are indicated with arrows.

FIG. 5.

Presence of genes virD and tmr in nonpathogenic mutant bacteria. Mutants isolated from tumors induced in tomato by strain IVIA1102 were analyzed by multiplex PCR using primer pairs CYT1-CYT2 (CYT1-2) and VirDA-VirDE (VirDA-DE) (see the legend to Fig. 1 for details on amplified fragments). P, parental strain IVIA1102; M, 100-bp DNA size marker (Life Technologies); C(−), negative control. The sizes of the amplicons are detailed on the left.

FIG. 6.

Presence of the T-DNA region in nonpathogenic mutants derived from IVIA1102. Plasmid DNA was digested with HindIII and separated on an agarose gel before Southern hybridization; the probe used was the complete T-DNA region from strain C58, obtained from plasmid pTHE17. P, parental strain IVIA1102; M, marker λHindIII (Invitrogen).

FIG. 7.

Hybridization of uncut plasmids from strain IVIA1102 and mutants T22, T38, and T67 derived from it, using the single plasmid from IVIA1102 as a probe; plasmids were separated in an Eckhardt gel.