RESUMO
Agrobacterium tumefaciens supervirulent strain A281 induces a progressive necrotic response, rather than tumor formation, when inoculated on stems of several grape cultivars. The Ti plasmid, and specifically its T-DNA, is required for the process. In the present study, 40 T-DNA insertion mutants of A281 were generated via transposon mutagenesis and tested for their necrosis-inducing ability on grape stems in vitro. Ten mutants were attenuated in inducing necrogenesis. Restriction mapping and DNA sequencing revealed that at least two genes, tms1 and 6b, whose gene products are involved in the synthesis and activity modulation of auxin, are responsible for inducing necrogenesis. Double mutants of tms1 and 6b were totally non-necrogenic. The orientation of grapevine stem explants showed strong effects on the occurrence and progress of necrogenesis. Inoculation of Agrobacterium on physiological basal ends resulted in the greatest degree of necrogenesis. In addition, gene 5 of T-DNA, which modulates auxin responses in plants by the autoregulated synthesis of an auxin antagonist, was found to be separated from other TL-DNA genes by a novel insertion sequence, IS1312. Since a T-DNA borderlike sequence occurs in IS1312, gene 5 might not always be transferred into plants. Based on the accumulated data, we propose that the necrogenesis induced by Agrobacterium results from the sensitivity of grapevine cells to elevated levels of auxin or a precursor of auxin.
Assuntos
Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/patogenicidade , DNA Bacteriano/genética , Frutas/microbiologia , Genes Bacterianos , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , Dados de Sequência Molecular , Mutagênese Insercional , Necrose , Doenças das Plantas/microbiologia , Transformação GenéticaRESUMO
The presence of the Ti plasmid favorably influences the attachment of agrobacteria to grape callus cells, especially during the early stages of a 2-h incubation. Agrobacterium strains attached to a similar extent to both the crown gall-resistant cultivar (Catawba), Vitis labruscana, and the crown gall-susceptible cultivar (Chancellor), Vitis sp. Attachment of the virulent strain to grape callus cells is blocked by the avirulent strain HLB-2 in both the tissue culture cell suspension and the seedling root systems.