Abstract
Agrobacterium-mediated plant galls are often misdiagnosed as nematode-mediated knots, even by experts, because the gall symptoms in both conditions are very similar. In the present study, we developed biosensor strains based on agrobacterial opine metabolism that easily and simply diagnoses Agrobacterium-induced root galls. Our biosensor consists of Agrobacterium mannitol (ABM) agar medium, X-gal, and a biosensor. The working principle of the biosensor is that exogenous nopaline produced by plant root galls binds to NocR, resulting in NocR/nopaline complexes that bind to the promoter of the nopaline oxidase gene (nox) operon and activate the transcription of noxB-lacZY, resulting in readily visualized blue pigmentation on ABM agar medium supplemented with X-gal (ABMX-gal). Similarly, exogenous octopine binds to OccR, resulting in OoxR/octopine complexes that bind to the promoter of the octopine oxidase gene (oox) operon and activate the transcription of ooxB-lacZY, resulting in blue pigmentation in the presence of X-gal. Our biosensor is successfully senses opines produced by Agrobacterium-infected plant galls, and can be applied to easily distinguish Agrobacterium crown gall disease from nematode disease.
Highlights
Agrobacterium-mediated plant galls are often misdiagnosed as nematode-mediated knots, even by experts, because the gall symptoms in both conditions are very similar
Rhizobium and Agrobacterium are very similar in many respects, and it is difficult to distinguish these genera using polymerase chain reaction (PCR)-based assays
Much effort has been made to avoid confusion between Rhizobium and Agrobacterium by designing the primer pairs used in PCR based on the gene located on the Ti plasmid of A. tumefaciens
Summary
Agrobacterium-mediated plant galls are often misdiagnosed as nematode-mediated knots, even by experts, because the gall symptoms in both conditions are very similar. Much effort has been made to avoid confusion between Rhizobium and Agrobacterium by designing the primer pairs used in PCR based on the gene located on the Ti (tumor-inducing) plasmid of A. tumefaciens Another fundamental problem of the PCR method is that bacterial pathogens generally multiply into large populations in diseased lesions, but crown gall bacterial pathogens persist at significantly lower populations away from lesions and PCR-mediated diagnosis in plant samples requires a threshold population density. A major limitation of routine PCR application to diagnose plant disease is that successful PCR can be prevented by the frequent occurrence of polyphenolic inhibitors and thermostable DNA polymerase inhibitors in plant tissues[12,15,16,17] To overcome these problems, a new approach to crown gall diagnosis is needed. Catabolic functions are activated in the presence of exogenous nopaline or octopine, and regulatory controls are mediated by the LysR-type transcriptional regulatory proteins NocR or OccR; the genes encoding these proteins are located in the opine transporter regions (noc and occ) of the C58 and 15955 strains, respectively[24,25]
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