Functional characterization of the Arabidopsis disease resistance gene RPS4
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The Arabidopsis disease resistance gene RPS4 activates defense responses to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 in a gene-for-gene specific manner. Like other plant TIRNBS-LRR resistance genes, RPS4 produces multiple transcripts via alternative splicing. Alternative RPS4 transcripts are predominantly generated by intron retention. First, the biological significance of these alternative transcripts in disease resistance was analyzed. It was shown that alternative RPS4 transcripts are required for complete function and that RPS4-mediated resistance requires the combined presence of multiple transcripts encoding both full-length and truncated open reading frames. Interestingly, the dominant alternative transcript is the only alternative transcript whose abundance relative to the regular transcript undergoes dramatic and dynamic changes during the resistance response. Furthermore, RPS4 expression is induced by AvrRps4 and an unrelated effector, HopPsyA, in an EDS1-dependent manner. These data suggest that rapid gene induction and changes in transcript ratios might be under coordinated regulation that are important to fine-tune RPS4-mediated resistance. Our previous data showed that removal of one intron abolished RPS4 function. However, no significant changes of transcript ratios in intron-deficient transgenic rps4-1 plants were observed compared to rps4-1 expressing a wild type genomic transgene, suggesting that the artificial removal of one intron has no effect on the splicing frequency of other introns. In consistent with our previous data, analyses on secondary RNA structures suggest that alternative RPS4 transcripts function at protein level. Of the three expected truncated RPS4 proteins, only one was detected and stable in vivo, indicating that RPS4 protein stability or activity is regulated. In summary, RPS4 function is regulated at multiple levels including gene expression, alternative splicing and protein stability or activity.
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