HomeInternational Rice Research Notesvol. 25 no. 2 (2000)

Improvement of conjugation methods for Xanthomonas oryzae pv. oryzae strain DY89031 to identify avrXa21 clones

P. K. Sharma | F. G. Da Silva | Y. Shen | P. C. Ronald

Discipline: Agriculture

 

Abstract:

Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, causes bacterial blight, a destructive disease of rice. Xoo strain PXO99A (race 6), an avirulent strain on plants carrying the Xa21 gene, is hypothesized to carry the avrXa21 gene. Attempts to identify avrXa21 by transferring genomic library clones (plasmid or cosmid) of Xoo strain PXO99A to Xoo strain DY89031 (virulent on plants carrying the Xa21 gene) were not successful because strain DY89031 was not a good recipient strain. The conjugation methods developed for Xoo strain PXO99A failed to transfer cosmid pHM1 and plasmid pUFRO27 clones to Xoo strain DY89031, thus delaying identification of avrXa21. In this report, conjugation methods described by Choi and Leach (1994) and Simon et al (1989) were modified to give a high frequency of conjugation in Xoo strain DY89031.

Xoo strain DY89031 was streaked on peptone sucrose agar (PSA) plates that were incubated at 30 °C for 3Ð4 d. Cells were scraped from plates and resuspended in 100 mL of nutrient broth (NB) to give O.D.600 = 0.25Ð0.3. Cells were incubatedfor 5Ð6 h on a rotary shaker (150 rpm) at 30 °C to O.D.600 = 0.5Ð0.6. Donors Escherichia coli S17-1 (pUFRO27) for biparental mating and E. coli DH10B (pUFRO27) for triparental mating containing different-size inserts were grown in NB at 37 °C for 4 h to give O.D.600 = 0.5. Helper E. coli DH10B (pRK2013) was grown in NB at 37 °C for 4 h to O.D.600 = 0.5. For biparental matings, 1.5 mL of Xoo strain DY89031 was mixed with 0.1 mL of donor, while for triparental matings, 1.35 mL of Xoo strain DY89031 was mixed with 0.15 mL of helper and 0.1 mL of donor in an eppendorf tube. The mating mixture was centrifuged for 2 min at 8,000 rpm. The pellet was resuspended in 40 mL of sterilized distilled water, spotted on well-dried PSA plates, and allowed to dry in the laminar flow chamber. After incubation at 30 °C for 48 h, the mating mixture spots were resuspended in 0.2 mL sterilized distilled water and were spread on PSA plates containing kanamycin (25 mg mLÐ1) and cephalexin (20 mg mLÐ1). Transconjugants appearing after 72 h were purified on the same medium and stored in 10% glycerol in microtiter plates for inoculation experiments.