Function Analysis For Small Gtpase Activating Protein During Nodulation In Lotus Japonicus

The release of flavonoid by legume plant, which can induce the rhizobium to product nod factors, is the first step to initiate nodulation. Nod factor receptor land5form a heterodimer at the membrane and directly bind nod factor in Lotus japonicus. NFR5was transphosphorylated by NFR1kinase domain, which seems essential to make it function. A small GTPase ROP6interacted with NFR5and specifically expressed in nodule primordias and young nodules.3RacGAPs was indentified in previous research by our lab, which were LjRacGAPl, LjRacGAP2and LjRacGAP3. All of these3RacGAPs interacted with ROP6constitutively active mutant. Base on above backgrounds, in this report, protein interaction, expression analysis and subcellular location were used to analyze biological functions of these3RacGAPs, and results were showed as below.1. RacGAP1, RacGAP2and RacGAP3interacted with ROP6-CA in yeast two-hybrid system.2. Real-time PCR showed that only RacGAP2expression up-regulated in nodules. Promoters of these three genes were used to drive the expression of GUS in hairy roots respectively. RacGAP2specifically expressed in nodule primordias and young nodules, however, not any RacGAPl or RacGAP3expression was detected in nodule.3. RacGAPl and RacGAP3LORE1inserted mutant didn’t show any defects about nodulation. Therefore RacGAP1and RacGAP3were not considered involving in symbiosis.4. BiFC was used to assess the interaction between RacGAP2and ROP6in tobacco leaves. RacGAP2interacted with ROP6and its constitutively active mutant at dots in plasma membrane and cytoplasm, which seems to be vesicles.5. RacGAP2was located to dots in plasma membrane and cytoplasm when expressed its GFP-fused protein in tobacco leaves. But situations were different when co-expressed with ROP6or NFR5. RacGAP2was located to big vesicles except of the small dots when NFR5existed. All RacGAP2assembled at the medium size dots where ROP6located to when co-expressed with ROP6. These results indicated that the interaction among RacGAP2, ROP6and NFR5related to vesicles trafficking.6. Overexpression of RacGAP2by Hair root transformation didn’t lead to any phenotype change including infection thread, nodule number and nodule form. Down-regulation of RacGAP2gene by RNA interference significantly increased the nodule number. It suggested that RacGAP2may act as a negetive regulator in the process of symbiosis in L. japonicus.