Real-time monitoring of translocation of selected type-III effectors from

Type-III (T3) effectors PthXo1 and AvrXa10 of Xanthomonas oryzae pv. oryzae are translocated into rice cells to induce virulence and avirulence on susceptible- and resistant-rice varieties Nipponbare and IRBB10, respectively. The translocation needs the bacterial T3 translocator Hpa1 and rice Oryza sativa plasma membrane protein OsPIP1;3. Here, we employed the beta-lactamase (BlaM) reporter system to observe PthXo1 and AvrXa10 translocation. The system was established to monitor effectors of animal-pathogenic bacteria by quantifying the BlaM hydrolysis product [P] and fluorescence resonance energy transfer (FRET) of the substrate. The feasibility of the BlaM reporter in rice protoplasts was evaluated by three criteria. The first criterion indicated differences between both [P] and FRET levels among wild types and OsPIP1;3-overexpressing and OsPIP1;3-silenced lines of both Nipponbare and IRBB10. The second criterion indicated differences between [P] and FRET levels in the presence and absence of Hpa1. The last criterion elucidated the coincidence of PthXo1 translocation with induced expression of the PthXo1 target gene in protoplasts of Nipponbare and the coincidence of AvrXa10 translocation with induced expression of the AvrXa10 target gene in protoplasts of IRBB10. These results provide an experimental avenue for real-time monitoring of bacterial T3 effector translocation into plant cells with a pathological consequence.