Actin Filaments-mediated Molecular Mechanisms Of Arabidopsis Immune Response And Wheat Against Puccinia Striiformis F.SP.Tritici

The actin filaments as a dynamically changing three-dimensional network structure in eukaryotic cells,play an important role in plant perception and resistance during the infection of pathogens.Actin filaments have been linked to plant immunity,while how it involves in plant immune signal transduction is still unknown.Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici,is a devastating disease in wheat production.However,the mechanism of wheat resistant to Puccinia striiformis f.sp.tritici remains largely undefined.Therefore,it is really necessary and important to full understand the actin mediated molecular mechanisms of Arabidopsis immunity and wheat resistant to Puccinia striiformis f.sp.tritici.In this research,ACT7 interaction proteins were screened and confirmed by yeast two hybrid,co-immunoprecipitation and confocal microscopy analyses.A combination of VIGS,LC/MS technique,and MAPK approaches was used to further study the functions of ACT7,actin binding protein CAP1 and wheat actin depolymerzing factor TaADF4 in plant immune response.Following the important results were obtained:1.A bait ACT7 protein was used to screen the Arabidopsis cDNA library,24 ACT7 interaction proteins were obtained,including actin binding proteins ADF1,ADF4 and ARPC3,plant defense related proteins RIN4,ROC4 and LOS2,and some unknow function proteins in plant immunity(e.g.,CAP1).The interactions between ACT7,RIN4 and CAP1 were confirmed by Y2 H and co-immunoprecipitation.2.ACT7 and CAP1 were found involved in DC3000 and DC3000AvrRpm1 induced Arabidopsis immune responses in Arabidopsis-Pseudomonas syringae pv.tomato interaction system.ACT7 negative regulated Arabidopsis resistance,however CAP1 positive regulated Arabidopsis resistant to Pst.Compare to wild type Col-0,act7-2 mutants inhibited DC3000 growth,on the contrary,cap1 mutants promoted bacterial growth.For the phenotype,cap1 mutants showed much more serious necrosis,act7-2 mutants showed less necrosis.After hand infiltrated with DC3000AvrRpm1,compare to wild type,act7-2 mutants restricted DC3000AvrRpm1 reproduction and showed less necrosis.Conversely,cap1 mutantsaccelerated DC3000AvrRpm1 reproduction,inhibited hypersensitive response and became more susceptible.3.Then,flg22 was infiltrated in plants to active MAPK signaling cascade.The accumulation of MAPK3/MAPK6 was significantly decreased in cap1 mutants after 10 min flg22 treatment,comparing with wild type,act7-2 mutants promoted MAPK3/MAPK6 accumulation.qRT-PCR analysis found that the expression of PTI signaling pathway marker gene FRK1 was induced increase in act7-2 mutants,while it was significant inhibited in cap1 mutants.These results showed us that ACT7 and CAP1 participate in flg22 induced MAPK signaling pathway in PTI.4.Phos-tag technique was used to detected the RIN4 phosphorylation in act7-2 and cap1 mutants induced by DC3000AvrRpm1.Compare to wild type,the RIN4 phosphorylation was accelerated and enhanced in act7-2 mutant,however it was inhibited in cap1 mutants.After inoculated with DC3000AvrRpm1,there was no difference between wild type and cap1 mutants for RIN4 accumulation.However,silencing ACT7 significantly inhibited RIN4 accumulation.qRT-PCR showed us that the expressions of ETI signaling related genes RPM1,PR1,WRKY70 were induced increase in act7-2 mutants after inoculated with DC3000AvrRpm1.However,these gene expressions were significantly inhibited in cap1 mutants.These data indicated that ACT7 and CAP1 play important roles in RIN4 related ETI signaling pathway.5.Six new ADF genes TaADF3,TaADF4,TaADF5,TaADF6,TaADF8 and TaADF11 were characterized and cloned form wheat cDNA library.Y2 H and co-immunoprecipitation results showed that TaADF4 interacted with TaACT1 and AtADF4 interacted with AtACT7,with additional reciprocal interaction between TaADF4 and AtACT7,AtADF4 and TaACT1.Homology modeling of the wheat and Arabidopsis ADF4 proteins showed that their predicted tertiary structures are highly similar.The positive interactions between TaADF4,TaADF3,TaADF5,TaADF6,TaADF7 and TaACT1 were identified,but no interaction was observed between TaADF8 and TaACT1.The data indicated that there is interaction specificity between wheat ADFs and TaACT1.In addition to identifying the interaction between TaADF4 and TaACT1,co-expression of TaADF4-RFP and TaACT1-GFP in N.benthamiana,it was observed that TaADF4-RFP co-localized to TaACT1-GFP filaments.For the activity of TaADF4 assays,it was found that TaADF4 and At ADF4 were phosphorylated by CPK3 in vitro.6.qRT-PCR analysis showed that the expression of TaADF4 is induced following abiotic and biotic stress perception.We observed a marked increase in TaADF4 mRNA accumulation following high temperature stress,MeJA and ABA treatment,and asignificantly inhibition expression in low temperature and salt stresses.A significantly increase in TaADF4 mRNA accumulation was observed in wheat and CYR23 incompatible interaction compared to wheat and CYR31 compatible interaction.VIGS inducing TaADF4 silence reduced wheat resistance to CYR23.LatB-treated induced TaADF4 expression increase,inhibited fungal growth,accelerated H2O2 accumulation and HR in wheat.Using LC/MS technique to test JA accumulation,TaADF4 silenced plants had significant reduced levels of JA,compared with what was detected in control plants.Taken together,we posit that TaADF4 is required for robust defense signaling in response to CYR23 infection.In conclusion,the results of this study have revealed the important role of actin filaments in plant immune response,demonstrated that ACT7 and CAP1 involved in flg22 induced MAPK signaling cascade in PTI and RIN4 mediated ETI signaling pathways,and found TaADF4 is required for wheat resistant to CYR23 by regulating JA signaling pathway.It advances our understanding of actin filaments mediated molecular mechanisms of Arabidopsis immune response and wheat-Puccinia striiformis f.sp.tritici interaction.