Mechanism Of Receptor Like Kinase FERONIA Response To RALF23 Signal In Regulating Low-Pi Stress In Plants

Phosphorus is an essential macronutrient for plant growth and development and it is one of the major factors that limits crop production worldwide.The concentration of inorganic phosphate(Pi),the main form of phosphorus that can be absorbed by plants,is typically less than 10 μM in soils.To better cope with Pi-deficient environments,plants have evolved adaptive Pi starvation responses.Common strategies for improving Pi uptake include altering root morphology,such as the rapid elongation of lateral roots(LRs)and activating Pi starvation related transporters and transcriptional regulators.PHR1 is a master transcription factor involved in the Pi starvation in Arabidopsis thaliana.Under Pi starvation conditions,PHR1 binds to the PHR1-binding site(P1BS)element in the promoters of Pi starvation-induced(PSI)genes,activating their expression and promoting Pi uptake.PHR1 also regulates jasmonic acid(JA)and salicylic acid(SA)pathway genes at the transcriptional level to modulate plant immunity in A.thaliana.PHR1 also negatively regulates immune reactions triggered by the bacterial epitope flg22,a pathogen-associated molecular pattern(PAMP)that induces PAMPtriggered immunity(PTI).Although PHR1 plays an important role in immunity suppression during the Pi starvation in A.thaliana,the molecular pathway underlying the PHR-mediated inhibition of plant immunity and the understanding of how this process benefits plants in relieving Pi starvation remain unclear.In plants,the elicitation of PTI relies on the recognition of PAMPs by cell surface-localized receptor kinases.For instance,the receptor flagellin sensing 2(FLS2)perceives flg22 interacted with coreceptor BAK1 and activates the immune responses,including the activation of MAPK,ROS bursts and the induction of defense-responsive genes expression.As a receptor kinase on plasma membrane,FERONIA(FER)is one of the well-studied signaling networks in plants so far.FER modulates many biological processes in plants(e.g.,growth,reproduction and stress responses).FER is a receptor of RALFs and perception of RALF23 by FER inhibits plant immunity by disrupting the complex between FLS2 and BAK1.Besides that,RALF23-FER also interferes with JA signaling to modulate plant immunity.This revealed that the RALF23-FER axis regulates the colonization of rhizosphere microorganisms by mediating PTI immunity.Based on this,this study uses a series of molecular,biochemical and genetic experiments approaches and aims to(1)test whether RALF23-FER is involved in low-Pi stress mediated suppression of plant immunity;(2)explore whether PHR1 targets and activates RALF expression during the Pi starvation;(3)determine whether RALF23 promotes the colonization of rhizosphere bacteria that help plants resist LP stress;(4)determine if the root microbiota of fer-4 mutant alleviates the Pi starvation;(5)investigate whether FER is involved in Pi uptake.The results of this study are as follows:(1)The Pi starvation suppresses the PTI responses,which includes MAPK inhibition and ROS burst suppression in Col-0 plants.While the PTI suppression was nearly completely abolished in fer-4 mutant root under LP condition.This suggests that FER is involved in Pi starvation mediated immunity suppression in roots.RALF23 significantly inhibits ROS production in Col-0 roots,while RALF23 treatment has no impact on fer-4 mutants,suggesting that the RALF23-FER complex is involved in immunity suppression under LP condition.(2)PHR1 activates RALF expression during the Pi starvation.We analyzed the promoter regions of all 37 RALFs in A.thaliana and found that RALF1,RALF4,RALF22,RALF23,RALF33 and RALF34 contain P1 BS,which was cleaved by S1 P and inhibit flg22-triggered immunity.(3)PHR1-mediated RALF activation suppresses FLS2-BAK1 complex formation to inhibit immunity during the Pi starvation.We immunoprecipitated the FLS2-BAK1 complex together with anti-FLS2 in Col-0 and phr1 mutants.The results showed that LP treatment reduced the flg22-induced FLS2-BAK1 complex association in the Col-0 but not in the phr1 mutant.Complementation of PHR1-Myc in phr1 protoplasts or RALF23/34 treatment partially recovered Pi starvation mediated suppression of immune complex formation,suggesting that PHR1 is required for Pi starvation mediated immune complex inhibition.(4)LP and RALF23 treatment promotes the colonization of rhizosphere bacteria that help plants resist LP stress.The microbial composition of Col-0,fer-4 and phr1 mutants after normal phosphorus(HP),LP and RALF23 treatments were compared by 16 S r RNA-based sequencing,and we found that low-Pi stress and RALF23 treatment could recruit a specialized root microbiota,such as Pseudomonas,Bacillus,Geodematophilus and Methylobacillus,in the rhizosphere of Col-0,while there was no significant difference in the rhizosphere of fer-4mutants,indicating that RALF23-FER plays an important role in the regulation of rhizosphere microbial colonization under LP conditions.By inoculating Col-0,fer-4 and phr1 mutant seedlings grown under HP and LP conditions with Pto DC3000 and B.subtilis for 3 weeks,the shoot fresh weight and lateral root number of the aerial parts after different treatments were detected.Inoculation with Pto DC3000 and B.subtilis significantly increased the shoot fresh weight and lateral root number of Col-0 and phr1 mutants,indicating that RALF23-FER is involved in regulating rhizosphere microbial colonization to alleviate low-Pi stress.(5)fer-4 mutants enriche beneficial microbes under low-Pi stress.We found fer-4 mutants enrich beneficial microorganisms according to the 16 S r RNA-based sequencing dates,and we collected the microbiome population from the rhizosphere of 3-week-old Col-0 and fer-4 plants by shaking the soil in sterile water and applied them to Col-0,fer-4 and phr1 mutants grown in HP and LP conditions,respectively.Indeed,treatment with the fer-4 microbiome significantly enhanced plant growth in Col-0 and phr1 mutants compared to the Col-0 microbiome treatment,suggesting that the mutation of FER assembles a rhizosphere microbiome that alleviates Pi starvation alleviation.(6)The RALF23-FER-GRP8 pathway directly regulate Pi absorb.GRP8 interacts with FER and was phosphorylated by FER.RALF23 treatment suppressed FER-GRP8 complex formation,increased the ability of GRP8 binding to WRKY75 mRNA,enhanced the stability of WRKY75 mRNA,and promoted the synthesis of WRKY75 protein to uptake more Pi.That indicates that FER is directly involved in the regulation of Pi absorption.This study establishes a signaling pathway that balances immunity and nutrient absorption in plants under low-Pi stress and provides a model for the coordination of host-microbe homeostasis through modulating plant innate immunity after perturbation.Besides,it also provides a theoretical basis for cultivating new varieties for high Pi uptake.