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Minisymposium 11: Plant-symbiont interactions

Abs # 24001: Cloning and identification of genes involved in plant growth promotion by a Burkholderia species (PsJN)

Presenter: Wang, Keri , wangkeri@agr.gc.ca
AuthorsWang, Keri  (A)   Conn, Kenneth  (A)   Lazarovits, George  (A)  
Affiliations: (A): Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON N5V 4T3

Rhizobacteria have been shown to dramatically affect plant health, development, and environmental adaptation in a variety of ways. The genus Burkholderia is a major group among the rhizobacteria that are known to increase crop yield by biological control of soil borne diseases. A Burkholderia sp (PsJN) isolated more than 10 years ago has been shown to provide stable and direct promotion of plant root growth under gnotobiotic conditions. Depending on the potato cultivar used, increases in the root weight may be by 6 to 10 times larger than uninoculated controls. The molecular mechanism is not known, but the utility of such genes may provide numerous benefits to agricultural production, particularly for northern climates. Field tests with PsJN showed that plant growth varied with plant species, cultivars and soil conditions. In this study, we examined the genes involved in growth promotion of potato by creating non-growth promoting mutants of PsJN using Tn5 transposon mutagenesis. One of 2,400 Tn5 mutants, H41, showed the same ability to colonize the potato plants as the strain PsJN, but was devoid of growth-promotion activity. The interrupted gene was isolated from PsJN lambda library and identified by subcloning into a broad host-range plasmid vector pRK415. Complementation tests showed a full restoration of growth-promotion activities with a 7.2 Kb DNA segment. DNA sequence analysis revealed four full open reading frames (ORFs). Three of these were putative genes conferring in vivo biosynthesis of one product potentially involved in plant growth promotion. The product was not a phytohormone such as indole-3-acetic acid etc. The ORF1 and ORF2 were linked together and shared the same promoter while ORF3 was located in downstream of ORF2. Reverse-transcription PCR confirmed that ORF1 and ORF2 were transcribed as an operon in wild-type PsJN. These genes were cloned into an expression vector pGEX-5X-3 for protein expression and purification. The purified proteins encoded by these genes showed functional enzymatic activities. The product of this enzymatic pathway was demonstrated to restore growth promotion of the mutant H41 when supplemented into the tissue culture medium. We concluded that the identified genes ORF1-3 were involved in plant growth promotion activities but we surmise that there are more genes controlling growth promotion. Several other mutants are now being examined for the basis of their loss of growth promotion.

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