Studys On The Mechanisms Of Inducing Systemic Resistance To Pseudomonas Syringae And Meloidogyne Incognita By Bacillus Cereus AR156 In Plant

Bacillus cereus AR156(short for Bc AR156)is a plant growth-promoting rhizobacterium originally isolated from the rhizosphere of tree soil from garden,Nanjing,China,can protect tomato against bacterial wilt caused by Ralstonia solanacearum and the root-knot nematode Meloidogyne incognita.Bc AR156 is a sequenced and patented strains,and has been registered as a biopesticide against root-knot nematode in tomato.Previously,we found that Bc AR156 can induced systemic resistance to Pseudomonas syringae pv.tomato DC3000 by simultaneously activating SA,JANET two signaling pathway and show NPR1 dependent.At present,the biocontrol mechanisms of Bc AR156 is still unclear.For example,why Bc AR156 can simultaneously activate SA,JA/ET two signaling pathway,while other ISR induced factor only can active SA or JA/ET one signaling pathway?Besides,how the Bc AR156 was percept or recognized by plant and trigger ISR in plant?How the plant small RNA function on the regulation of Bc AR156 induced systemic resistance?In addition,the Bc AR156 show high biocontrol efficacy to root knot nematode,but the prevention and control of root knot nematode biocontrol mechanism is still not clear.It will be beneficial for deeply investigating the B.cereus mechanism and accelerating the commercial process of B.cereus biopesicides.This study was set up by using Bc AR156 as biological strain,the Arabidopsis and tomato as a plant model;Pst DC3000 and root knot nematode as pathogens to deeply understand the mechanisms of Bc AR156 to bacteria and root knot nematode disease.In our study,we parsesed the Bc AR156 induced systemic resistance mechanism from the transcriptional factor area.Moreover,we found the extracellular polysaccharides(EPS)of Bc AR156 could act as novel microbe-associated molecular patterns(MAMPs)and function in the early perception status of the ISR of Bc AR156,these parts results indicated that how the rhizobacteria were percepted by plant and triggered ISR to pathogen.Other whiles,we also found that plant small RNA,miRNA,miR472,miR825/825*,can act as a regulators,function on the process of Bc AR156 induced systemic resistance.The study of nematode biocontrol mechanisms shown that,Be AR156 can induce plant root structure change and accelerate the maturity of the root system to induced plant resistance to root knot nematode,on the other hand,Be AR156 also can suppress the effector coding gene expression to effect the invasion process of root knot nematode,so that can successfully control the root knot nematode disease.1.The study of transcription factors WRKY70 and WRKY11 served as regulations in B.cereus AR156 induced systemic resistance.By using Arabidopsis as a model system,we identified two transcription factors WRKY11 and WRKY70 as important regulators involved in ISR triggered by Be AR156.The results revealed that Be AR156 treatment significantly stimulated the transcription of WRKY70,but suppressed that of WRKY11 in Arabidopsis leaves.Furthermore,they were shown to be required for Be AR156 enhancing activation of cellular defense responses and transcription level of plant defense response gene.Overexpression of the two transcription factors in Arabidopsis also showed that they were essential for Be AR156 to elicit ISR.Be AR156-triggered ISR was completely abolished in the double mutant of the two transcription factors,but still partially retained in the single mutants,indicating that the regulation of the two transcription factors depend on two different pathways.The target genes of the two transcription factors and epistasis analysis suggested that WRXY11 regulated Be AR156-triggered ISR through activating JA signaling pathway,and WRKY70 regulated the ISR through activating SA signaling pathway.In addition,both WRKY11 and WRXY70 modulated Be AR156-triggered ISR in a NPR1-dependent manner.In conclusion,WRKY11 and WRKY70 played an important role in regulating signaling transduction pathways involved in Be AR156-triggered ISR.Also,this study is the first to illustrate the mechanism by which a single rhizobaterium elicits ISR by simultaneously activating SA-and JA/ET-signaling pathways.2.B.cereus AR156 extracellular polysaccharides served as a novel micro-associated molecular pattern to induced systemic immunity to Pst DC3000 in ArabidopsisBe AR156 could act as novel microbe-associated molecular patterns(MAMPs)and function in the early perception status of the ISR of Be AR156.The results revealed that Be AR156 EPS could induce systemic resistance to Pst DC3000 in Arabidopsis.The defense-related genes PR1,PR2,and PR5 and mitogen-activated kinases(MAPK)cascade marker gene MPK6 were concurrently expressed in the leaves of EPS-treated plants.Moreover,the cellular defense response markers such as hydrogen peroxide accumulation,callose deposition,and defense-associated enzyme were induced upon challenge inoculation in the leaves primed by EPS-treated plants and induced higher resistance to Pst DC3000 in Col-0 than that in the jar1 or etr1 mutants.The protection was absent in the NahG transgenic plants and nprl mutant,suggesting an activation of the salicylic acid(SA)-and the MAPK-dependent signaling pathways with NPR1-dependent by Bc AR156 EPS.In conclusion,Bc AR156 EPS play an important role in MAMP perception during the process of rhizobacteria-triggered ISR.3.The regulation of small RNA on rhizobacteria induced systemic resistance in plants Here,by comparing small RNA profiles of Pseudomonas syriingae pv.tomato(Pst)DC3000-infected Arabidopsis with and without Bc AR156 pretreatment,we identified a group of Arabidopsis microRNAs(miRNAs)that are differentially regulated by Bc AR156 pretreatment.Northern blot analysis confirmed that miR472,miR825 and miR825*,three miRNA products from two single miRNA gene,were significantly down-regulated in Pst DC3000-infected plants with Bc AR156 pretreatment,in contrast to the samples without Bc AR156 pretreatment.The target prediction information shown that miR825 targets two ubiquitin-protein ligases,while miR472 and miR825*targets toll-interleukin-like receptor(TIR)-nucleotide binding site(NBS)and leucine-rich repeat(LRR)type resistance(R)genes.The expression of these target genes was negatively correlated with the.expression of miR472,miR825 and miR825*.Moreover,transgenic plants knocking down the expression of the three miRNAs displayed enhanced resistance to Pst DC3000 infection,whereas transgenic plants overexpressing miR472,miR825 and miR825*were more susceptible.Taken together,our data indicates that Bc AR156 pretreatment trigger ISR to Pst DC3000 infection by suppressing miR472,miR825 and miR825*and activating the defense related genes they targeted.4.Study on the mechanisms of B.cereus AR156 biocontrol root knot nematode disease by regulating the plant root developmentIn our study,it was found that,when use the Be AR156 to treat the root of tomato,it can induce plant root structure change,accelerate the maturity of the root system,so as to reduce the probability of the nematode into the plant and reduce the number of insect population in the root of plant,thus that will assist plant to achieve the effect of preventing and controlling root knot nematode.In order to explore the mechanism of Bc AR156 on prevention and control of root knot nematode,we study from the root structure development perspective,and employed transcriptome sequencing technology to explore the molecular mechanism of Bc AR156 to prevent and control root knot nematode by change the of root structure.The transcriptome results showed that Bc AR156 can affect a series of plant development related gene expression,and the most significant was associated with Abscisic acid,Ethylene and Heat shock protein.Based on all the above result,we can found that,the Bc AR156 can induce plant root structure change and accelerate the maturity of the root system to induced plant resistance to root knot nematode by regulate the genes expression,which was assocaited with Abscisic acid,Ethylene and Heat shock protein.5.Study on the mechanism of B.cereus AR156 against root knot nematode by suppressing the expression of nematode effectorsPreviously,we found that the root treated with Bc AR156 show more resistance to root knot nematode.In order to deeply explore the mechanism of its prevention and control,we employed molecular biological methods,and advanced sequencing technology in our study.The results of transcriptional sequencing shown that Bc AR156 treatment can significantly suppress the nematode effectorscoding gene expression.The Q-RT-PCR results shown that only 11 of predicted effector coding genes can be supressed sucessfully by Bc AR156.This study is the first to illustrate that one rhizobacterium can regulate the effector coding gene expression to effect the invasion process of root knot nemato,so that can sucessfully conrol the root knot nemaotde disease.