An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N-and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-P-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-P-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.

Four genes of rice, Oryza sativa L., conditioning resistance to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed between xa-5 and RFLP marker loci RZ390, RG556 or RG207 on chromosome 5. Xa-3 and Xa-4 were linked to RFLP locus XNpbl81 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker to Xa-lO, also located on chromosome 11, was the RAPD locus 0072o00 at a distance of 5.3 cM. From this study, the conventional map [19,28] and two RFLP linkage maps of chromosome 11 [14,26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes, Xa-4 + xa-5 and Xa-4 + Xa-lO. Lines carrying Xa-4 + xa-5 and Xa-4 + Xa-lO were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs &genes were resistant to more of the isolates than their single-gene parental lines. Lines carrying Xa-4 + xa-5 were more resistant to isolates of race 4 than were either of the parental lines ('quantitative complementation'). No such effects were seen for Xa-4 + Xa-lO. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N-and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-P-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-P-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.

No abstract

Bacterial blight (BB) caused by Xanthomonas oryzae pv oryzae (Xoo) is one of the most serious diseases of rice. The recessive gene xa-13 confers resistance to Philippine race 6 of Xoo. To tag xa-13 with molecular markers, RAPD analysis was conducted with the combined use of near-isogenic lines and bulked segregant analysis. From the survey of 260 arbitrary 10-nucleotide primers, one primer (OPAC05) was detected to amplify specifically a 0.9-kb band from the DNA of susceptible plants. The distance between the RAPD marker OPAC05-900 and xa-13 was estimated to be 5.3 cM. The RAPD marker was then mapped on chromosome 8 using a mapping population of doubled haploid lines derived from the cross of IR64/Azucena. The linkage between RFLP markers and the RAPD marker was analyzed using an F2 population of 135 plants derived from a cross between a near-isogenic line for xa-13, IR66699-5-5-4-2, and IR24. No recombinants were found between RZ28 and CDO116 and their distance from xa-13 was estimated to be 4.8 cM. RG136 was located at 3.7 cM on the other side of xa-13. The mapping of xa-13 with closely linked DNA markers provides the basis for marker-aided selection for rice improvement.