| Wheat stripe rust,which is caused by Puccinia striiformis f.sp.tritici(Pst),is a serious disease that devastingly threantens wheat yield and quality worldwide.As wheat stripe rustcould spread quickly,occur in a wide range,and pose a high frequency of happening,it threans the security of wheat production all over the world,and it has been a spot for study and research by scientists from different countries to control the disease.The use of disease-resistant wheat varieties is the most economic and sufficient method for controlling wheat stripe rust.However,wheat disease-resistant varieties are vulnerable to loss of resistance along with the frequent pathogenicity variation of wheat strip rust fungus and the appearance of new pathogenic races.Therefore,study of the interaction mechanism between wheat and Pst,espicially the pathogenic mechanism of Pst,is of great significance for the development of new sustainable disease resistant strategies.However,Pst is obligate biotrophic fungi and due to the lack of stable genetic transformation methods for Pst,the research of pathogenic mechanism of Pst faces serious obstacles.With genome sequencing of Pst and prediction by bioinformatics,a large number of pathogenicity-related effector molecules have been predicted.In addition,expression in heterologous systems has become useful methods for studying the function of Pst effectors,which provides an important foundation for the function identification of Pst effectors.Based on the whole genome sequencing of Pst,the function of some Pst effectors had been researched in this study.And the effectos played important parts in the pathogenicity of Pst on wheat via different mechanisms,and the targets of these effectors were also identified,suggesting the participation of them in the interaction between wheat and Pst during Pst infection of wheat.The main resuslts are as follows:1.Functional study and targets identification of Pst effector Pst＿8713Based on the genome sequencing results of CYR32 by our lab in advance,a candidate effector Pst＿8713 was cloned from Pst.The gene sequence analysis of Pst＿8713 showed it was a Pst specific gene,and highly conserved between different Pst races.Only a signal peptide was predicted from Pst＿8713 by Pfam,and the signal peptide was confirmed with the function of secretion.The expression characteristics showed that Pst＿8713 was highly induced for expression during Pst infection of wheat,and the highest expression peak was observed at the same period when haustoria were developed in great amounts.Pst＿8713 was confirmed to localize in plant cytoplasm and nuclei.Co-infiltrations of Pst＿8713 with either the mouse pro-apoptotic protein BAX or Phytophthora infestans PAMP-inducer INF1 in Nicotiana benthamiana demonsrated that Pst＿8713 was capable of suppressing cell death triggered by either of them.And expression of Pst＿8713 in plants suppressed pattern-triggered immunity(PTI)-associated callose deposition and expression of PTI-associated marker genes.Effector-triggered immunity(ETI)induced by an avirulent Pst isolate in wheat was dampened by overexpression of Pst＿8713 in wheat leaves,accompanied by the reduction of reactive oxygen species(ROS)accumulation and hypersensitive response(HR).In addition,knockdown the expression of Pst＿8713 by host induced gene silencing(HIGS)weakened the virulence of Pst to wheat.Taken together,the candidate effector Pst＿8713 was confirmed as an effector with the function of suppression plant defense responses and contribution to the successful invasion of wheat by Pst.Furthermore,the interaction targets of Pst＿8713 in wheat were identified as two subunits of photosynthesis-related ferredoxin-thioredoxin reductase,indicating that Pst＿8713 could facilitate the growth and development of Pst by influence on the host photosynthesis.2.Functional study and targets identification of Pst effector Pst＿8724Based on the genome sequencing results of CYR32 by our lab in advance,a rust-specific candidate effector Pst＿8724 was cloned from Pst.In spite of the functional signal peptide identified in Pst＿8724,a RING-finger domain was predicted in this protein.The gene sequence analysis of Pst＿8724 showed it was a rust-fungi specific gene.The expresstion characteristics showed that Pst＿8724 localized to cytoplasm in plant cells,and the expression was highly induced in both compatible and incompatible interactions of Pst and wheat.The highest expression peak was observed at the same time when haustoria were developed in great amounts.Expression of Pst＿8724 by either Agrobacterium or type III secretion system in plants could induce cell death in a variety of plants,such as N.benthamiana,Arabidopsis thalian,Solanum lycopersicum,Triticum aestivum.And the induction of cell death by Pst＿8724 depended on the RING-finger domain in Pst＿8724.Expression of Pst＿8724 in plants could also suppress the PTI-associated callose deposition and expression of PTI-associated marker genes in plants.With Pst＿8724 silenced by HIGS,the virulence of Pst decreased significantly in both compatible and incompatible interactions between Pst and wheat.And the development of Pst infection hyphae would be repressed with Pst＿8724silenced,while,the accumulation of ROS in the host would increase.Three targets of Pst＿8724 were screened out by yeast-two-hybrid system,and the interaction between two of them with Pst＿8724 was further confirmed.It could be concluded that Pst＿8724 influenced the interaction between Pst and wheat by the interaction with its targets.3.Functional study and targets identification of Pst effector PSTha2c7PSTha2c7,the rust-specific candidate effector which had been reported to be highly expressed in Pst haustoria cDNA library,was cloned by this study.PSTha2c7 had a functional signal peptide and there was another signal peptide for localization in peroxisomes on its C terminal.By subcellular localization experiment,PSTha2c7 was proved to localize to wheat peroxisomes.The expression of PSTha2c7 was up-regulated with the initiation of Pst infection of wheat,and reached the peak at the key moment when haustoria were formed.And then,the expression level of PSTha2c7 would come down,at the time of sporulation,the expression of PSTha2c7 was up-regulated again.Co-infiltrations of PSTha2c7 with either the mouse pro-apoptotic protein BAX or P.infestans PAMP-inducer INF1 in N.benthamiana demonsrated that PSTha2c7 was capable of suppressing cell death triggered by either of them,suggesting PSTha2c7 was a virulent factor.And expression of PSTha2c7 suppressed PTI-associated callose deposition and expression of PTI-associated marker genes.At the same time,overexpression of PSTha2c7 in host wheat enhanced the sporulation of CYR32,a virulent isolate of Pst.With PSTha2c7 silenced by HIGS,no obvious changes in phynotypes were observed on the wheat leaves challenged with Pst,and there was also no effects on the growth and development of Pst.But the accumulation of ROS in the host increased during Pst infection of wheat.An interaction target of PSTha2c7,ADF3(Actin depolymerizing factor 3),was screened out by yeast-two-hybrid system.And the interaction between PSTha2c7 and ADF3 was further confirmed by both Bi FC and CO-IP. |
