Rice Genetics IV - IRRI books - International Rice Research Institute

Genetic dissection of the Xa21-mediated resistance pathwayTo identify genes essential for the function of resistance against multiple diseases, amutational approach was used. Both a chemical mutagen (diepoxybutane or DEB)and radiation (fast neutrons, FN) were used in the experiment. DEB has been reportedto cause deletion mutations at high frequency in Drosophila (Reardon et al 1987) andhas been used to generate mutants in several plant species (Salmeron et al 1994).DEB primarily causes deletions of less than 250 base pairs although deletions of up to8 kb have also been observed (Reardon et al 1987). Fast neutrons induce both basepair changes and deletions and translocation. Salmeron et al (1994, 1996) isolatedseveral Pto mutants using DEB and FN as mutagens and cloned the Prf gene, whichis a common component of the transduction of signals for Pto-mediated resistance toPseudomonas syringae pv. tomato as well as for sensitivity to the insecticide fenthion.A population of about 4,500 M2 families from IRBB21 (Xa21 donor) with DEB (3,500families) and FN (1,000 families) was used to screen for changes in disease resistanceresponse to both bacterial blight and rice blast. We recovered 31 mutants that havechanged from resistant to fully susceptible (10) or partially susceptible (21) to nineraces of the bacterial blight pathogen in the Philippines (Fig. 2).Polymerase chain reaction (PCR) analyses were used to detect deletions and rearrangementsin the Xa21 coding region. Two pairs of specific primers in the LRRregion and two pairs of specific primers in the kinase region of the Xa21 gene weredeveloped. We found that some mutants such as 3453, 3469, 3954, and N20-247 lostboth the LRR and kinase DNA fragments. Some lines such as N18-116-1 and N18-238-1 have rearrangements in the kinase region (Fig. 3).For Southern analysis, DNA of selected mutants was digested with the restrictionenzyme HindIII. Amplified fragments from the Xa21 LRR and kinase regions wereused as probes. We found that six FN-induced mutants had hybridization patternssimilar to those of three missing LRR-hybridizing bands (data not shown). ThreeDEB-induced lines had deletions in the Xa21 gene when the LRR fragment was usedas a probe. Line 3453 had lost three bands and the size of the remaining bands wasdifferent from that of the Xa21 donor line IRBB21. Lines 3469 and 3954 lost sevenbands and had the same hybridization pattern as the recurrent parent IR24. When thekinase fragment was used in Southern hybridization, all 10 mutants with deletions inIRBB21IR24MutantMutantFig. 2. Bacterial blight reaction of identified mutants. Two-month-old plants were used for inoculationwith Philippine isolate POX99A. Disease reaction was recorded 2 wk after inoculation.340 Guo-Liang Wang et al