The Function And Underlying Mechanism Of PSK Peptide Receptor PSKR1-CPK28 Interaction In Tomato Growth And Defense

Tomato(Solanum lycopersicum)is an important economic vegetable crop in China.It is favored by consumers because of its great flavor and nutrition.It is widely cultivated all over the country and has achieved supply all year round.However,the rough and extensive management in cultivation brought low yields and high disease incidences frequently.Thus,it is facing major industrial demands for yields and defenses improvement.Therefore,it is important to explore the mechanism of the coordinated regulation of growth and biotic stress resistance.The peptide hormone PSK(phytosulfokine)as plant endogenous signalling molecules regulates both plant growth and defense,its intercellular signalling remains largely unknown.In this study,we explore the mechanism of PSK-regulated tomato growth and resistance by analyzing target protein interaction modification and N metabolism.The role and mechanism of N fertilizer and N metabolic genes in bacterial leaf spot resistance were also explored.The main findings are as follows:1.The peptide hormone PSK regulates growth and resistance,but the mechanism is unclear.We take advantage of model plants tomato to explore the role of PSK and its receptor PSKR1 in regulating growth and defense against Pst DC3000.We found that PSK-PSKR1 signalling exhibits inverse effects in tomato by promoting seedling growth but repressing defense against Pst DC3000.Tomato pskr1 mutants and OE-PSKR1 overexpression lines were obtained by CRISPR/Cas9-mediated gene editing and transgenic overexpression approaches,respectively.By observing the root growth and dry mass accumulation,it was found that PSKR1 promoted the growth of tomato seedlings.Exogenous application of PSK promoted root length,whereas the roots of pskr1 mutant lines were less responsive to PSK.The different lines were challenged with Pst DC3000 inoculation,we found that pskr1 plants showed increased resistance,while the resistance of OE-PSKR1 plants decreased.2.PSKR1 senses PSK and transmit the signal,but the mechanism of PSK-PSKR1 regulating bacterial leaf spot resistance is still unclear.We found that PSKR1 and CPK28 function together and that CPK28 may work downstream of PSKR1 in the same pathway that regulates plant growth and defense by affecting N metabolism.There is a close relationship between PSK signal and Ca2+ signal.We found that PSKR1 interacted with Ca2+-dependent protein kinases CPK28 using a yeast two-hybrid screen library.By performing Bi FC,pull down and Co-IP assays,we confirmed that PSKR1-CPK28 complex is mainly localized in the plasma membrane,and the binding was increased following the PSK treatment.PSK-induced growth promotion is not only comprised in the pskr1 mutant phenotype,but also in the cpk28 and the pskr1 cpk28 double mutant backgrounds.Notably,in addition to being phenotypically similar,OE-PSKR1-induced growth enhancement and susceptibility to Pst DC3000 were decreased in the OE-PSKR1 cpk28 lines.Further study showed that single and double mutants of pskr1 and cpk28 affects the in-planta nitrogen status.3.Based on the hypothesis that PSKR1-CPK28 signalling pathway may play a role in the regulation of nitrogen assimilation that regulates seedling growth and defense responses,we further found that CPK28 phosphorylate GS2 at S334 and S360,each is associated with defence regulation and growth response,respectively.We have previously found that CPK28 potentially interacts with N assimilation key enzyme GS2 using a Y2 H screen library.This protein-protein interaction was verified using Pull down and Co-IP approaches.The intensity of the interactions between CPK28 and GS2 was increased by the presence of PSK and PSKR1.GS2 promotes growth but repress defenses against Pst DC3000.LC-MS/MS analysis revealed that the serine-334(S334)and serine-360(S360)sites on the GS2 protein are phosphorylated by CPK28.We generated stable genetic complementary GS2,non-phosphorylatable S334 A and S360 A lines,as well as phospho-mimic variants S334 D and S360 D in the gs2 mutant background,found that S360 phosphorylation site associated with growth response and S334 associated with defense responses.Based on the transcriptome analysis,we propose that GS2-S334 phosphorylation-mediated defense is linked to oxidation-reduction processes,systemic acquired resistance and amino acid metabolism,while the GS2-S360 phosphorylation enhances growth by regulation of the balance between nitrogen and carbon metabolism.4.Nitrogen(N)influences a myriad of physiological processes while its effects on plant defenses and the underlying mechanisms are largely unknown.In this study,we found that tomato immunity against Pst DC3000 was generally higher under low N as well as NO3-as the sole N source compaired with mixed NO3-/NH4+(70/30,mol/mol)under the same N concentration.Here,the interaction between tomato and pathogens was examined under four N regimes(sole NO3-or mixed NO3-/NH4+ of total 1 and 7 m M N,denoting low and high N regimes,respectively)followed by inoculation with Pst DC3000.After Pst DC3000 infection,the expression of N assimilation key genes,such as NR,Ni R and Fd-GOGAT,decreased significantly.The disease susceptibility was reduced by silencing N metabolism genes such as Ni R and Fd-GOGAT,while increased in Ni R1-overexpressed plants.Low N as well as the silencing of N metabolism genes increased the SA(salicylic acid)levels and transcripts of its maker genes,and low N-enhanced defense was blocked in Nah G transgenic tomato plants that do not accumulate SA,while exogenous SA application attenuated the susceptibility of OE-Ni R1.These results demonstrated that the N modulated defense was dependent on the SA defense pathway.The study provides insights into the mechanisms of how nitrogen fertilization and metabolism affect plant immunity in tomato,which might be useful for designing effective agronomic strategies for the management of N supply.In summary,this study found a molecular signalling pathway that PSK receptor PSKR1 interacted with CPK28 which phosphorylate GS2,speculating that CPK28 may mediate PSK signal to regulate N metabolism and then regulate plant growth and defense against Pst DC3000.This study can not only create novel tomato germplasms through gene editing based on the molecular sites discovered from original innovation,but will also provide theoretical supports for application of peptide hormones.