Mechanism Of Auxin Transport In Salicylic Acid-induced Immune Response And Soybean Nodulation

The interactions between plants and microorganisms are complex and changeable.Such as pathogenic microorganisms activate plants to arouse defense strategies,while symbiotic microorganisms stimulate plants to activate nutrient circulation.The defensive and cooperative relationships developed between plants and microorganisms enable plants to cope with complex environmental changes leisurely.Auxin almost involved in all the process during plant growth and development,including response to harmful/beneficial microorganisms.When plants resist the invasion of pathogenic microorganisms,auxin controls plant growth and responds to salicylic acid-mediated plant immune signals,coordinating the balance between growth and disease resistance.When plants interact with soil microorganisms in symbiosis,auxin provides the necessary hormonal signals for the establishment of new organs of mycorrhizal symbiosis.The establishment and maintenance of auxin gradient mediated by polar auxin transport is the premise of plant and microorganism interaction,whether for disease defense or mutual symbiosis.However,a large number of unknown questions remain to be solved as to how auxin dependent plant growth responds to and effectively resist the invasion of harmful microorganisms and host the colonization of beneficial microorganisms.This study found that:1.During host–pathogen interactions,growth hormone auxin and defense hormone salicylic acid antagonize each other and mediate the growth and defense of plant roots.In this study,a series of advanced cell biology observation methods,such as Zeiss superdiffraction Airyscan confocal microscope variable Angle total internal reflection fluorescence microscope(VA-TIRFM),were used to observe membrane localized polarity efflux carrier PIN-formed 2(PIN2)in response to high concentration of salicylate at the protein single molecule level.The dynamics of PIN2 in membranel ipid raft and its translocation between membrane and cytoplasm regulate the maximization of root growth in disease resistance and defense.Remorin(REM)localized to membrane nanodomain and regulates the structure and assembly of membrane nanodomain.In this study,we focused on REM1 subfamily that is highly expressed in roots,in which REM1.2 showed similar expression pattern with auxin transporter PIN2,and participates in PIN2-mediated root gravity response.High salicylic acid and REM1.2 overexpression disturbed the distribution of PIN2 and auxin in the root apical meristem,resulting in noticeable reduction in root gravitropism.In rem1.2root,the effect of salicylic acid on PIN2 and auxin distribution and root geotropism was significantly reduced.On the one hand,salicylic acid regulates PIN2 protein aggregation and stabilization in membrane lipid raft microdomain through REM1.2-dependent assembly of ordered liquid phase and disordered liquid phase of lipid raft.On the other hand,salicylic acid inhibits clatirin-mediated endocytosis of PIN2 from cell membrane to cytoplasm.The aggregation and stabilization of PIN2 on the plasma membrane inhibited the circulation of PIN2 protein in the plasma membrane-intracellular and the dynamic diffusion of PIN2 molecules on the plasma membrane.Thus,under low salicylic acid conditions,the ordered liquid phase-disordered liquid phase and PIN2 protein were evenly distributed on the plasma membrane,and PIN2 protein effectively moved between the vesicle and plasma membrane through dynamic endocytosis and lateral diffusion.Under high salicylic acid condition,membrane lipid rafts formed aggregated membrane nanodomain,and ordered liquid phase arrangement increased,which reduced plasma membrane fluidity and restricted endocytosis and lateral diffusion of PIN2,resulting in high aggregation of PIN2 protein and inhibition of auxin transport and root gravitation.By regulating the dynamic range of PIN2 distribution on the surface of plant cells,salicylic acid regulates auxin flow and thus root growth,providing a new insight into the mechanism by which plants inhibit auxin transport in response to pathogen infection and thus balance defense-growth trade-off.2.During the process of establishing a symbiotic relationship between legumes and nitrogen-fixing rhizobia,auxin promotes the formation of symbiotic organ-nodules in cortex of legumes root.The formation of nodules is determined by the local auxin maximum in nodule primodium and depends on polar auxin transport to establish the auxin gradient.To overcome nitrogen deficiency,legume roots establish symbiotic interactions with nitrogen-fixing rhizobia and form a specialized organ(nodules).Similar to other organs,nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site,while auxin gradient is mainly established by polar auxin transport.However,how auxin regulates soybean determinate nodules development remains poorly understood.A unique cellular biological system was established to visualize auxin gradient and distribution during soybean nodule development by using CRISPR/Cas9 based gene knockout,auxin signal marker,high resolution confocal microscopy,soybean stable transformation,fluorescent protein labeling and shock sectioning.The results showed that nodule primordium initiation synchronized with auxin gradient establishment on nodule primordia tip(Stage I-II),then auxin melts away from the apical of nodule primordium(Stage III)and eventually concentrates on vascular bundle(IV).The Gm PIN1 subfamily in soybean mediates polar auxin transport and promotes the formation of nodule primordia.Gm PIN1 b mainly polar toward apical(Stage I-II),which mainly promoted the establishment of local auxin maximum in the apical of nodule primordium.The polarity index(signal ratio of apical relative to the lateral side)of Gm PIN1 b decreased(Stage III),showing a trend of lateral auxin transport,accompanied by the gradual weakening of auxin signal in the apical of primordium.In mature nodule(stage IV),Gm PIN1 b mainly restricted in nodule vascular bundle,while auxin also mainly concentrated in the vascular bundle.The upstream nodulation regulatory factor-flavonoids stimulate the diffusion of Gm PIN1 b from stele to cortical cells,and cytokinin promotes the redirection of auxin transport by regulating the polarity of Gm PIN1 b,and coordinate the initiation and development of nodule primordium.Gmpin1 abc triple mutants showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells.Moreover,overexpression of Gm PIN1 suppressed nodule primordium initiation.These findings reveal how Gm PIN-dependent auxin transport promotes the establishment of auxin maximum at the early stage and mediates the transfer of auxin maximum from the apical of nodule primordium to vascular bundle and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.