SummaryThe rice gene Xa21 confers resistance against Xanthomonas oryzae pv. oryzae (Xoo). Xa21 encodes a receptorlike kinase (XA21). We demonstrate that XA21 autophosphorylates residues Ser686, Thr688 and Ser689 in vitro. Substitution of these residues with alanines did not affect the autophosphorylation function of this kinase, but specifically destabilized the resistance protein in vitro and in vivo. Plants carrying these same substitutions in XA21 were compromised in their resistance to the normally avirulent Xoo Philippine race 6. Additionally, we show that wild-type XA21 and the kinase-dead mutant with the invariable Lys736 residue mutated to glutamic acid were also proteolytically degraded in protein extracts. Finally, we show a correlation between the in vitro degradation and in vivo instability of the proteins. We propose that autophosphorylation of Ser686, Thr688 and Ser689 functions to stabilize XA21 against the developmentally controlled proteolytic activity.

Transgenic rice plants were generated using particle bombardment to simultaneously introduce the rice Xa21 gene eective against bacterial blight and the Galanthus nivalis agglutinin (snowdrop lectin; gna) gene eective against sap-sucking insect pests, speci®cally the brown plant hopper. Using three plasmids, we cotransformed 5-to 10-d-old, mature seed-derived rice (Oryza sativa L.) callus of two elite Chinese rice cultivars, Eyi 105 and Ewan 5. The plasmids carried a total of four genes. The gna and Xa21 genes were carried on separate plasmids. The selectable marker hygromycin phosphotransferase (hpt) and the reporter gene b-glucuronidase (gusA) were linked on the same, co-integrate vector. We recovered over 160 independently derived transgenic rice plants. Over 70% of the transgenic plants carried all four genes, as con®rmed by polymerase chain reaction and/or Southern blot analysis. Furthermore, 70% of transgenic plants carrying all four genes also coexpressed all four genes, as con®rmed by growth on selection media (hpt), GUS histochemical assays (gusA), western blotting (gna) and reverse transcriptase-polymerase chain reaction (Xa21) analysis. The co-expression eciency reported for the four transgenes in our study is the highest ever found in any transgenic plant population generated through co-transformation. The linked genes (hpt and gusA) co-integrated with a frequency of near 100%, and we observed a co-integration frequency greater than 70% for the genes carried on separate plasmids. We observed no preferential integration of any particular gene(s). Genetic analysis con®rmed Mendelian segregation of the transgenes in subsequent generations. We report, for the ®rst time, generation and analysis of transgenic rice lines carrying genes eective against more than one taxa of pathogen or pest, substantiating that particle bombardment represents an eective way to introduce unlinked complex multiple traits into plants.Abbreviations: gna (GNA) = gene (protein) for Galanthus nivalis agglutinin; gusA (GUS) = gene (protein) for b-glucuronidase; hpt (HPT) = gene (protein) for hygromycin phosphotransferase; Xa21 (XA21) = gene (protein) for resistance to bacterial blight, RT-PCR = reverse transcriptase-polymerase chain reaction

Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.

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