Physiological And Transcriptional Regulation Mechanism Of Silicon-mediated Resistance Of Tomato To Ralstonia Solanacearum

Tomato(Solanum lycopersicum)is an economically important vegetable crop worldwide.Bacterial wilt caused by Ralstonia solanacearum is a destructive disease that limits tomato production throughout the world.Several studies showed that Si could enhance the resistance of tomato to bacterial wilt,but the exact molecular mechanism remains unknown.Meanwhile,previous studies mainly focused on the biochemical and physiological responses of tomato stems to R.solanacearum,few studies have focused on the underground parts.The bacterial wilt-susceptible tomato genotype HYT was used in this study.We investigated the biochemical and physiological responses of Si-treated and non-Si-treated tomato roots to R.solanacearum infection;the dynamic changes of three major stress hormones [salicylic acid(SA),jasmonic acid(JA),and ethylene(ET)] from different treatments at various time points after inoculation were measured by using gas chromatograph(GC)and ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)technology,in order to better understand their roles;we employed RNA-Seq to compare and characterize transcriptional responses of Si-treated and non-Si-treated tomato roots to R.solanacearum infection.The mechanisms of Si-mediated tomato resistance to R.solanacearum at physiological,biochemical,and transcriptional level were outlined.The results are presented as follows:1.Si can increase tomato resistance against R.solanacearum.Si treatments delayed the development of bacterial wilt disease symptom.Compared with the controls,Si pretreatment significantly reduced the disease index of tomato plants by 4.8-64.5% at 2-7 days post-inoculation(dpi).Si application could enhance tomato plant resistance to R.solanacearum.The amount of bacterial population in roots,leaves and stems of the Si-treated tomato plants was slightly lower than that in untreated control group,but no significant difference was observed for bacterial population between treatments.The Si concentration in all three organs of the Si-treated tomato plants was significantly higher than that in untreated control group.Si was mainly accumulated in tomato roots.2.Si treatment could increase the activities of defense-related enzymes and the contents of TSPs and LTGA derivatives and also affect sucrose metabolism.In the +Si group,the activities of peroxidase(POD),polyphenol oxidase(PPO),phenylalanine ammonia-lyase(PAL),lipoxygenase(LOX)and the contents of total soluble phenolics(TSPs)and lignin-thioglycolic acid(LTGA)derivatives derivatives in the roots were significantly increased.Si application can increase the resistance of tomato plants to R.solanacearum by affecting the activities of defense-related enzymes.Basal resistance response such as the reinforcement of root cell walls by the LTGA derivatives might partly contribute to Si-mediated tomato resistance against R.solanacearum.Sucrose content in the roots increased significantly at 1 and 2 dpi in +Si-treated plants in comparison with the control,and no differences were observed at 3 and 7 dpi between treatments.Sucrose content in tomato leaves and xylem sap were significantly higher in the +Si plants at 2,3,7 dpi than in the control.Under our experimental condition,application of Si does not lead to reduction of sucrose content.The activities of SS,NI,and AI increased with time,whereas SPS activity continuously decreased.These preliminary data suggest that high sucrose contents and changes in the activities of sucrose metabolism-related enzymes may contribute to alleviating the water-deficit stress caused by infection,thereby indirectly and partially increasing the tolerance of tomato to bacterial wilt.3.The burst of ET production was delayed,and SA and JA contents were altered in Si treatment.At 1 dpi,the untreated plants emitted 2.7-fold more ET compared with the +Si plants in response to pathogen infection;ET production was 2.3-fold higher in the +Si plants at 7 dpi than in the untreated plants;Si treatments delayed the burst of ET production in tomato roots;the delay of ET release in +Si tomato plants is associated with delayed necrotrophic phase progression and symptom appearance.SA occurred at significantly higher basal levels in the controls at 1 dpi.For the +Si plants,SA content was only significantly higher(7.3-fold)at 7 dpi in comparison with those in the controls.The JA content in the +Si plants increased gradually from 1 dpi to 3 dpi,and this content was significantly higher in the +Si plants at 1,2,3 dpi compared with the controls.4.Si treatment altered the expression of many genes.RNA sequencing(RNA-Seq)technology was employed to analyze the root transcriptome of Si-treated(+Si)and non-Si-treated(-Si)tomato plants at 1,3,and 7 dpi of R.solanacearum.Approximately 187.21 million of reads were generated for the six samples(+Si1,+Si3,+Si7,-Si1,-Si3,and-Si7).PCA and Pearson correlation analysis suggested that the response to infection at the transcriptional level in the controls at 7 dpi was similar to that in the +Si3 plants.Hence,the response to R.solanacearum infection of the +Si plants was quicker than of the untreated plants.Differentially expressed genes(DEGs)were identified by pairwise comparisons of transcriptome datasets(+Si1 vs.-Si1,+Si3 vs.-Si3,and +Si7 vs.-Si7).A total of 1265 DEGs(398 upregulated and 867 downregulated)were identified in the +Si1 vs.-Si1 comparison,1143(483 upregulated and 660 downregulated)in the +Si3 vs.–Si3 comparison,and 4015(2218 upregulated and 1797 downregulated)in the +Si7 vs.-Si7 comparison.The pairwise comparisons demonstrated that the genes related to ROS,cell wall reorganization and phenylpropanoid biosynthesis are significantly affected during multiple stages of infection in Si-treated tomato roots.5.Si treatment can activate pattern(PAMP)-triggered immunity(PTI)-related genes expression,Si-mediated resistance against R.solanacearum in tomato involves multiple hormonal pathways.On the basis of functional annotation and trend analysis of DEGs,DEGs associated with Si-mediated tomato resistance to R.solanacearum were identified.In the present study,several PTI-related genes(e.g.FLS2,EFR,CMLs,ACA2,ACA12,WRKYs)were upregulated in +Si tomato plants;these findings indicated that the activation of PTI-related genes contributes to Si-mediated tomato resistance to R.solanacearum.Some SA-and systematic acquired resistance(SAR)-related genes(e.g.PR1,PR2,PR5,PALs)were upregulated in the +Si plants.These results confirmed that the SA-dependent SAR pathway is involved in Si-mediated tomato resistance to R.solanacearum.Salicylic acid-binding protein 2(SABP2)is essential for the establishment of SAR.Notably,in +Si plants,the DEG encoding SABP2 was continuously downregulated.Hence,long-distance SAR signal transduction is partly repressed in the +Si plants,but resistance was still retained.We proposed that Si-mediated resistance against R.solanacearum in tomato is also involved in an SA-and SAR-independent mechanism.Most of the ET synthesis-related genes(e.g.ACS,ACO)were differentially upregulated,ET signaling-related genes(e.g.ERF1,EIN3)showed different expression patterns;these results suggest that ET biosynthesis and signal transduction in +Si plants are complicated.LOX is a JA-mediated defense marker gene,the DEG encoding LOX was upregulated in the +Si plants.We provided additional evidence to support the concept that the ET-and JA-related pathways are involved to some extent in Si-mediated resistance against R.solanacearum in tomato.Auxin-homeostasis-related DEGs(e.g.PIN10,LAX5,Sl PIN7)were assigned into different expression patterns.Auxin-related pathway may also contribute to Si-mediated resistance against R.solanacearum.Cytokinin(CK)-,brassinosteroid(BR)-and gibberellin(GA)-related genes were also differentially expressed in +Si tomato.Besides,abscisic acid(ABA)signaling and senescence-associated genes(SAGs)may be suppressed to some extent in Si treatment.Our results suggest that Si-mediated resistance against R.solanacearum in tomato involves multiple hormonal pathways.6.Si treatments also alter the expression of genes associated with tolerance and adaptation to water-deficit,salinity stress and oxidative stress.In the present study,DEGs(e.g.TAS14,RD22,CDPK29,CORA-like)associated with tolerance and adaptation to water-deficit and salinity stress were upregulated in Si treatment.We proposed that the alleviated adverse effect of water-deficit stress and subsequent salinity stress caused by infection is one of the mechanisms for Si-mediated tomato resistance against R.solanacearum.Meanwhile,POD,PPO,and GST genes were differentially upregulated in +Si tomato.Thus,mitigation of oxidative stress and stabilization of membranes by various activated antioxidant enzymatic systems might partly contribute to bacterial wilt resistance.In addition,one DEG with homology to respiratory burst oxidase homolog(RBOH)was induced in Si treatment,ROS may be an active signal in Si-mediated tomato resistance.Taken together,Si treatment affects the regulation of many DEGs involved in phytohormone synthesis,hormone homeostasis,signal transduction,pathogen resistance,stress adaptation and tolerance,oxidation resistance,and senescence regulation.Si-mediated resistance involves mechanisms other than SA-and JA/ET-mediated stress responses.Si treatment may enhance tomato resistance to R.solanacearum infection in three ways: activating PTI-related responses;altering disease resistance and tolerance of tomato plants by influencing multiple hormone(e.g.,SA,JA,ET,and auxin)signaling pathways;and alleviating adverse effects(e.g.,senescence,water-deficit and oxidative stress)caused by infection.The proposed hypothetical model may help in understanding the mechanism of Si-enhanced tomato resistance against R.solanacearum.These results provide a comprehensive transcriptome analysis of Si-mediated tomato resistance,which will also provide an important basis to further characterize the role of Si.