Functional Characterization Of Important Pathogenicity Factors PstGSRE1 And PstCPK1 And Creation Of Wheat Durable Resistant Materials Using HIGS

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most damaging pathogens in wheat(Triticum aestivum)worldwide.Wheat stripe rust is an aeroborne disease.Planting resistant cultivars to control stripe rust.However,race-specific resistance can be overcome quickly due to the rapid variation of Pst population.In addition,breeding of new resistant cultivar is lagging.Therefore,studying the pathogenesis of Pst is critical for understanding how Pst virulence changes and how to create wheat cultivars with durable resistance to stripe rust.Pst,a biotrophic parasite fungus,uptakes nutrition from living cells and secrets proteins to host tissue to manipulate host immune system and promotes disease.During the progress of infection,two types of proteins play important roles.One type is pathogenicity and development-related gene which is conserved in pathogenic fungi,another is genes encoding secretes proteins.To abolish the infection and subsequent colonization,plant coevolved resistance(R)proteins to recognize certain pathogen secreted proteins,named as avirulence(AVR)effectors,to trigger a secondary wave of plant immune responses,this response always results in effector triggered immunity(ETI)which often associated with localized hypersensitive response(HR).Finding out R and Avr genes may contribute to resistance variety breeding,while studying of the mechanism of how importants effectors regulating hostimmunity is still important for controlling disease.Therefore,in this study,we identified and characterized a glycine-and serine-rich effector PstGSRE1.Additionally,we stably silenced an important pathogenicity factor PstCPK1 and created durable resistant wheat materials agaist Pst using host-induced gene silencing(HIGS)technique.The results were obtained as follows:1.A glycine-and serine-rich effector PstGSRE1 was cloned and identified in Pst.Sequence analysis indicated that it has a functional N-signal peptide,contains two cysteines and does not have any conserved domain.qRT-PCR assay showed that PstGSRE1 is up-regulated at the early stage of infection and up to peak at 24 hpi.PstGSRE1 can suppress Bax-induced PCD,EtHAn-induced callose deposition,and avirulence Pst-induced ROS accumulation.Subcellular localization showed that PstGSRE1 flows actively in plant cells;2.Transient silencing PstGSRE1 using BSMV-HIGS results in a reduction of urediniosppres production,indicating that PstGSRE1 positively contributes to the virulence of Pst.A PstGSRE1 RNAi construct stably expressed in three independent transgenic wheat lines(L33,L34,L43)confers strong resistance to Pst.Our results indicatied that PstGSRE1 plays an important role in the virulence of Pst;3.To fish for wheat interactors,we screened a Y2 H library using PstGSRE1 without signal peptide as the bait.We found a few candidates including TaLOL2、TaCAT1、TaCAT3、TaCIPK10.Y2 H analysis confirmed the interaction of PstGSRE1 with TaLOL2.Pull-down and Co-IP assay further confirmed the interaction in vitro and in vivo.Co-expressed PstGSRE1 and TaLOL2 as a variety of GFP or RFP fusion proteins by agro-infiltration in Nicotiana benthamiana showed PstGSRE1 and TaLOL2 interaction occurs in the nucleus and cytoplasm;4.TaLOL2 contains three zinc finger domains which have DNA binding activity.TaLOL2 located on 5A、5B、5D chromosome and these copies shown at least 96% identity.Knock down the expression of TaLOL2 by using BSMV-VIGS reduces resistance of wheat.ROS accumulation and HR also show a significantly reduction in TaLOL2-kncokdown plants compared with that in the control plants.Overexpression of TaLOL2 using the T3 SS assay in wheat could induce oxidative burst and showed a detectable chlorotic reaction phenotype on wheat plants which EtHAn could not trigger.These data indicated that TaLOL2 is a positive regulator of plant immunity.TaLOL2 can induce cell death in N.benthamiana.Subcellular localization indicated TaLOL2 has a strong signal in the nucleus although there is no conventional nuclear localization sequence.TaLOL2 and its deletion mutants with GFP recombinant protein expressed in wheat protoplasts confirmed that the first zinc finger domain of TaLOL2 elicits the nuclear localization;5.PstGSRE1 can suppress TaLOL2-induced cell death in N.benthamiana cells.modulated the nuclear translocation of TaLOL2,and suppressed ROS-mediated cell death induced by TaLOL2,thus compromising host immunity.This work reveals a novel strategy that rust fungi exploit to modulate host defense and facilitate pathogen infection;6.Protein kinase A(PKA)has been proved to play important roles in regulating the virulence of phytopathogenic fungi.PstCPK1,a PKA catalytic subunit gene from Pst,is highly induced at the early infection stage of Pst.The instantaneous silencing of PstCPK1 by barley stripe mosaic virus(BSMV)-mediated host-induced gene silencing(HIGS)results in a significant reduction in the length of infection hyphae and disease phenotype.These results indicate that PstCPK1 is an important pathogenicity factor by regulating Pst growth and development.Two transgenic lines expressing the RNA interference(RNAi)construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst both the T3 and T4 generations.The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting,and these RNAs were found to efficiently downregulate PstCPK1 expression in wheat.This study addresses important aspects for the development of fungal-derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.