Functional Characterization Of Actin Filaments Related Genes TaARPCs,TaPro And TaRop10 In The Interaction Between Wheat And Puccinia Striiformis F.sp.tritici

Wheat stripe rust caused by Puccinia striiformis f.sp.tritici（Pst）is one of the serious fungal diseases in wheat production areas around the world.Recently,the disease has occurred in new areas where the epidemic was infrequent.Development of the resistant cultivars was considered to be the most economic,effective and environmental friendly way to control the disease.Functional analysis of the resistant genes is an important way which provides the possibilities for the substantial advances in the development of resistant cultivars.However,the defense mechanism of wheat against Pst is largely undefined.The actin filaments which act as a dynamic network structure in eukaryotic cells,was proved to play an important role in the perception of the stimulus and resistance during the pathogen infection to the plant.The function of the actin cytoskeleton is dependent on the actin polymerization and depolymerization,while the dynamics of the actin filements is controlled by many actin-related regulators.Therefore,it is of great significance to study the actin-related regulators for further clarification of the molecular mechanism of wheat resisting against Pst.Wheat cultivar‘Suwon11’infected by Pst race CYR23 and CYR31,represents compatible and incompatible reactions,respectively.Pharmacology study was used to analyse the role of actin filements polymerization in wheat against Pst.Using updated biological techniques,including bioinformatic analysis,q RT-PCR,barley stripe mosic virus induced gene silencing,transient expression of genes,green-fluorescent protein marked,complementation of yeast mutant and yeast two-hybrid system,functional characterization of actin-related genes Ta ARPCs,Ta Pro and Ta Rop10 gene in the interaction of wheat and Pst has been achieved,and Ta Pro interaction proteins and Ta Rop10 interaction proteins were screened.The important results are as follows:1.Wheat leaves treated with cytochalasin E were inoculated with Pst race CYR23,resistant responses including hypersensitive response（HR）occurance and H₂O₂ accumulation were tested.The results showed that the resistance of wheat against Pst was weakened when actin cytoskeleton depolymerized,the occurance of the HR suppressed and the accumulation of H₂O₂ was reduced,suggesting that actin polymerization is involved in the process of wheat defense against Pst.2.Two actin polymerization factors Ta ARPC3、Ta ARPC4 were conserved by bio-informatic analysis.Knocking down Ta ARPC3/Ta ARPC4 expression by virus-induced gene silencing,the resistance of wheat against Pst weakened when inoculated with race CYR23 and limited sporulation around the necrotic spots was observed.When inoculated with CYR31,the susceptibility of wheat towards Pst infection was enhanced.Histological observation showed that hypha grew more freely in gene-silencing plant.Cloning the Ta ARPC3、Ta ARPC4 genes and fusing with PVX106 vector respectivily,transient transformation of the tobacco leaves via Agrobacterium was performed.The results showed that both Ta ARPC3 and Ta ARPC4 could not induce hypersensitive cell death.Subcellular localization of Ta ARPC3:GFP and Ta ARPC4:GFP fusion proteins in wheat protoplasts showed that Ta ARPC3 was found in nucleus and cytoplasm,while Ta ARPC4 was found in cytoplasm.Moreover,Ta ARPC3 complemented the ARPC3 mutation in yeast.The results showed that Ta ARPC3 partially rescued the yeast mutant by increasing the survival of stress and the ability of scavenging reactive oxygen in cells.3.Bioinformatic analysis of the amino acid sequence of actin polymerization factor Ta Pro showed that it shared a greater similarity with other Profilins.Transmembrane prediction and nucleus localization signals were excluded in Ta Pro.Knocking down Ta Pro expression by virus-induced gene silencing also weakened the resistance of wheat when inoculated with CYR23.The susceptibility of wheat was enhanced when inoculated with CYR31.The inhibition of hypha expansion was suppressed in Ta Pro-silencing by histological observation.Additionally,the expression levels of the defense response-related genes（PR1,PR2,PR3 and PR5）were reduced in Ta Pro knocking-down plants when compared with the control by q RT-PCR assay.Subcellular localization of Ta Pro:GFP was found in nucleus and membrane of protoplasts.Studies on the spatio-expression of Ta Pro gene showed that the higest expression level was found in wheat leaves,while the lowest in seeds.A bite Ta Pro was used to screen the wheat c DNA library,and the alternative Metallothionein-like protein was obtained.4.A gene Ta Rop10 with 696bp length was cloned from wheat cultivar Suwon 11.The encoded Ta Rop10 protein consisting of 231 amino acids belonged to the small G protein Ras superfamily,and shared 91%similarity with At Rop10.Characterization of Ta Rop10 in the compatible and incompatible interaction between wheat and Pst by q RT-PCR showed that the transcription level of Ta Rop10 in compatible interaction and incompatible interaction were significantly different at 12 hpi and 120 hpi.To further characterize the function,Ta Rop10were silenced with BSMV-VIGS system.The results showed that the susceptibility of wheat towards Pst weakened in Ta Rop10-knocking down plants when inoculated with compatible pathogen CYR31.The hypha expansion was suppressed in wheat leaves inoculated with CYR31 by histological observation.Moreover,the expression levels of PR1,PR2,PR3 and PR5 genes were increased differently in Ta Rop10 knocking-down plants by q RT-PCR analysis.Subcellular localization of Ta Rop10:GFP was found only in nucleus.Studies on the spatio-expression of Ta Pro gene indicated that the junction of stem showed the highest Ta Rop10 expression.A marked inhibition transcription of Ta Rop10 was observed following SA,ABA,high temperature and low temperature treatments;and a significantly increased transcription in Ta Rop10 was observed with ET and salt stress.Additionally,Ta Rop10 was down-regulated when wheat leaves were treated with cytochalasin E,indicating that Ta Rop10may be a negative regulator of actin polymerization.Three proteins were screened by two yeast-hybird systems,including resistance-related protein TRX,Ca²⁺signaling-related FK506-binding and IP5P9 protein.In conclusion,both actin-related regulators Ta ARPC3,Ta ARPC4,Ta Pro and small G protein gene Ta Rop10 take part in the interaction between wheat and Pst.Ta ARPC3,Ta ARPC4 and Ta Pro are related with the resistance of wheat against Pst by regulating actin filements polymerization,while Ta Rop10 plays role in susceptibility of wheat towards Pst infection by acting as a negative regulator of actin filements polymerization,or taking part in other signaling passway.Our study showed that actin cytoskeleton participates in the interaction of wheat and Pst,and actin filements polymerization play important role in wheat defense.