Functional Switch Of Rice Aquaporin OsPIP1;3 Between Physiological And Pathological Responses

The extensive studies boom on aquaporins(AQPs)have gained an important finding,which,however,is accompanied by shortcomings.The important finding is that the newly appreciated functions of AQPs not only surpasses the original definition of "water channel",with transporting substrate extending to other small molecule compounds such as CO2 and H2O2,but also relate to infection of eukaryotic hosts(including animals and plants)by pathogens and immune responses of the hosts.Regarding two shortcomings,one is the paucity in understandings of the structural basis for certain AQPs to mediate substrate transport.So far,conformations of on the ammonia transport channel AtTIP2;1 in Arabidopsis and water channels Aqyl in yeast and SoPIP2;1 in spinach have been dissected.Second,little is known about the substrate selectivity of most AQPs.Rice(Oryza sativa)is particularly prominent;it has 33 AQPs including 11 plasma membrane intrinsic proteins(PIPs),but none of them have been demonstrated with respect to transporting substrates.Recent studies in our laboratory found that rice OsPIP1;3 has both physiological and pathological functions,which encourages the author to explore the mechanisms involved.This dissertation will elucidate distinct roles of OsPIP1;3 in CO2 and H2O2 transport,and in translocation of the type Ⅲ effector PthXo1 from Xanthomonas oryzae pv.oryzae(Xoo).The author will describe a real-time,high-throughput technique used in monitoring of the bacterial effector translocation.Then,the author will further demonstrate the functional shift between CO2 transport and PthXol translocation in rice plants grown under regular conditions in contrast to infection by the pathogen.AQPs in the plant kingdom are categorized into five major protein families,and one of them,the PIP family is further divided into the PIP1 subfamily and the PIP2 subfamily,consisting of 11 members,which completely lack information on transporting substrates.By assays with Xenopus laevis oocytes following transformation with the OsPIP1;3 gene,H2O was excluded from substrate selectivity of OsPIP1;3.Then,the transport of H2O2 was evaluated on OsPIP1;3-overexpressing(OE)and OsPIP 1;3-nockout(KD)transgenic rice lines,which were tested in comparison with the wild-type(WT)plant of rice variety Nipponbare.These plants were treated with an H2O2 solution by leaf infiltration,followed by staining with dyes that can specifically probe H2O2,producing fluorescent compounds which are used in qualification of H2O2 concentrations in plants.In confocal microscopy of fluorescence emitted from H2O2-treated rice leaves,the externally applied H2O2 moved into rice cells gradually in 45 minutes as observed.The biggest and the smallest amounts of H2O2 transport was detected in OsPIP1;30E lines and OsPIP1;3KD lines,respectively,compared to the WT.Thus,OsPIP1;3 is an efficient facilitator of H2O2 transport across plasma membranes(PMs)of rice cells.In addition,by gas exchange measurements and chlorophyll fluorescence quenching assays performed on leaves of the WT,OE and KD plants,the author characterize OsPIP1;3 as a photosynthesis-essential CO2 transport channel.However,this role of OsPIP 1;3 is normally fulfilled in rice plants growing under regular conditions but is circumstantially suspended when the plants are infected by Xoo,due to the PM localization of the PIP.Because PIPs are located on plasma membranes(PMs),the PMs is in direct contact with the external environment,PIPs face constant risks of abduction by plant pathogens to induce virulence.Bacterial blight pathogen hijacks OsPIP1;3 to translocate T3 effectors into rice cells,as an essential step for virulence induction.Here the author elucidates the functional shift of OsPIP1;3 between the transport of CO2 and translocation of the bacterial T3 effector PthXo1,with the significance for photosynthesis performance and disease control.OsPIP1;3 overproduction enhances but its nullification inhibits mesophyll conduction to CO2 and net photosynthesis rate,suggesting that OsPIP1;3 is a physiologically relevant CO2 transport gate.This role is normally fulfilled in rice plants growing under regular conditions,but highly impaired in the plants under infection by Xoo.Rice infection compels OsPIP1;3 to switch its function from CO2 transport to translocation of PthXo1 into rice cells,allowing PthXo1 to induce virulence by activating its target gene in an OsPIP1;3-dependent manner.These results provide evidence for functional switch of a plant AQP between the physiological and pathological responses,offering a way to ensure photosynthesis and control disease by preventing bacteria from usurping the substrate transport gate.The T3 effectors PthXo1 and AvrXa10 of Xoo 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 Hpal and rice AQP OsPIP1;3.The author employed the β-lactamase(BlaM)reporter system to observe PthXol 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 BlaM reporter in rice protoplasts was evaluated by three criteria.One was differences in both[P]and FRET levels among wild types and OsPIP1;3-overexpressing and OsPIP1;3-silenced lines of both Nipponbare and IRBB10.The other one indicated differences of[P]and FRET levels in the presence and absence of Hpal.The last criterion elucidated the coincidence of PthXo1 translocation with induced expression of PthXo1 target gene in protoplasts of Nipponbare and the coincidence of AvrXa10 translocation with induced expression of 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.The innovation of this thesis is an experimental example to explain the physiological function and pathological function conversion of plant PIPs,and represents a way for AQPs to intervene in the relationship between plant growth and immune "tradeoff".The main disadvantage is that the molecular mechanism of OsPIP1;3 functional switching between CO2 transport and Xoo effector transport is relatively preliminary and needs further study.