Non-specific Lipid Transfer Protein NtLTP4 As A Positive Regulator Involved In Abiotic And Biotic Stress In Nicotiana Tabacum

As they have a sessile lifestyle,plants face a multitude of environmental stresses.Enviromental stress is broadly consist of biotic and abiotic stress,and biotic stress contains microbial and insect infections and abiotic stress largely includes salinity,drought and high or low temperature.Environmental stress is ever-present threat to plant agriculture worldwide and has an important effect on plant growth and development,which is a major obstacle to achieving optimal yield and quality.During their long term evolution,plants have evolved high plasticity and elaborate mechanisms to sense environmental stress and protect themselves from environmental stress,including activation of Mitogen-Activated Protein Kinase Cascade and several ion channels,up-regulation of pathogenesis-related proteins and generation of many protective proteins.Therefore,unraveling stress-associated genetic resources and dissecting stress tolerance molecular mechanisms,and further using genetic engineering or other biological techniques,to improve genetic traits and introduce new greater stress tolerance species for cultivation is urgently needed.Non-specific lipid transfer proteins(nsLTPs)are abundant,small cysteine-rich and lipid-binding proteins that are vital stress-associated proteins,which are involved in various physiological processes in plants.In the previous study,commom tobacco(Nicotiana tabacum,NC89)was used to study the function unknown gene NtLTP4,overexpression of NtLTP4 could enhance the salt and drought stress tolerance.In this study,we will elaborate the molecular mechanism of NtLTP4 involved in abiotic and biotic stress,and the results were as following:(1)NtLTP4 widely responded to various environmental stressesThe transcript of NtLTP4 was dramatically induced by abiotic stresses,biotic stresses and stress-related hormones.Expression pattern analysis exhibited that NtLTP4 could be involved in multiple signaling pathway,and play vital roles in plant growth and development,especially in response to environmental stresses.(2)NtLTP4 was a small secreted proteinThe transient expression of the GFP-fused Nt LTP4 proteins were analysed in Agrobacterium-infiltrated Nicotiana benthamiana leaves.Confocal fluorescence imaging revealed that Nt LTP4-GFP fusion was primarily observed at the cellular boundary.Subsequently,onion epidermal cells carrying the 35S::NtLTP4-GFP plasmid emitted fluorescence only in the peripheral cell layers upon plasmolysis.Nt LTP4 localized into the extracellular matrix might be linked to signal transduction pathway and exchange lipids between membranes.(3)NtLTP4 positively regulated plants abiotic stressTo study the function of the function unknown gene NtLTP4,we produced NtLTP4-overexpressing and RNAi transgenic tobacco plants.NtLTP4-overexpressing lines all showed apparently enhanced salt and drought stress resistance compared with WT,regardless of seed germination,root elongation or water loss rate was assayed.We observed a strikingly higher Na~+contents in WT than OE lines.Under salinity condition,the expression levels of HKT1 and NHX1 were higher than those in WT lines.Therefore,the salt-tolerance phenotype of OE lines might be due to the increased levels of HKT1 and NHX1 to maintain Na~+homeostasis.Moreover,lower transpiration rate and higher net photosynthesis rate were detected in OE lines compared with those in WT plants after drought stress,which suggested that NtLTP4 might enhance tolerance to drought stress in N.tabacum through reducing water loss.(4)NtLTP4 acted as positive regulator in biotic stressAfter biotic stress treatment,transgenic plants increased resistance to R.Solanacearum compared with wild type plants.In addition,after R.Solanacearum infection,pathogen-related proteins dramatically induced in transgenic plants,and the ROS content and stomatal aperture were relatively lower in OE lines than in WT,which suggested that NtLTP4enhanced R.Solanacearum resistance by suppressing ROS accumulation and inducing stomatal closure.(5)NtLTP4 acted downstream of WIPK casacade pathway and interacted with WIPKThe results of yeast-two-hybrid(Y2H),firefly luciferase complementation imaging(LCI),co-Immunoprecipitation(co-IP)and bimolecular fluorescence complementation(BIFC)proved that Nt LTP4 could interact with WIPK(wound-induced protein kinase).Virus induced gene silencing system(VIGS)and overepression system were used to analyze the regulation mechaniasm of WIPK on NtLTP4.WIPK was downregulated in NtLTP4 RNAi(ntltp4)through VIGS to obtain ntltp4/wipk,and ntltp4/wipk was more sensitive due to the functional redundancy,suggesting that both WIPK and NtLTP4 played vital role in plant innate immunity.Moreover,WIPK was downregulated in NtLTP4-OE lines through VIGS to obtain NtLTP4/wipk,NtLTP4 overexpression partially improves R.solanacearum tolerance in wipk and the phenotype of NtLTP4/wipk was similar with NtLTP4-OE lines.These observations indicated that NtLTP4 acted downstream of WIPK in R.solanacearum resistance and plant innate immunity.(6)WIPK positively regulates protein levels of Nt LTP4 in tabaccoNt LTP4 protein levels were lower in wipk mutant and evidently higher in WIPK overexpression lines.Thus,a functional WIPK seems to be important for acquiring the full Nt LTP4 overexpression-related improved biotic stress tolerance and WIPK could maintain the protein stability of Nt LTP4.And cell-free assay got the same result.However,the transcript levels of NtLTP4 in WIPK-OE lines and wipk were almost the same.Therefore,WIPK positively regulated Nt LTP4 in protein level.(7)NPK1-NtMEK2-WIPK-Nt LTP4 was involved in plant pathogen resistanceTo further obtain intact signaling pathway,yeast-two-hybrid assays showed that the upstream components of WIPK were NPK1 and NtMEK2,and therefore Nt LTP4 was involved in pathogen resistance throuth NPK1-Nt MEK2-WIPK cascade pathway.In conclusion,our study indicates that NtLTP4 enhances environmental stress tolerance in N.tabacum.The tolerance to salt stress may be coordinated with the induced expression levels of HKT1 and NHX1 to detoxify the Na~+,and NtLTP4 also regulates of the transpiration rate and stomatal conductance to respond to drought stress.Further studies show that NtLTP4is involved in regulating stomatal aperture and pathogen immunity downstream of NPK1-NtMEK2-WIPK cascade.Our results illuminate the molecular mechanisms of NtLTP4positively regulates stomatal immunity and R.solanacearum resistance through MAPK signaling cascade pathway,NPK1-NtMEK2-WIPK cascade,which has important theoretical and applied research value in plants.