| Wheat leaf rust,caused by Puccinia triticina(Pt)is an airborne fungal disease in wheat worldwide,which can cause significant yield losses to the wheat production in the world.The disease happens in large scale area and the damage is serious,it has become a key factor affecting wheat production in China.However,due to the frequent pathogenic diversity and pathogenic types of wheat leaf rust,wheat leaf rust resistance is overcome continuously.It is of great significance to study the molecular mechanism of wheat leaf rust to understand the co-evolution of leaf rust and the resistance genes,to arrange the resistance genes reasonably,and to cultivate durable resistance products.In this paper,the means of bioinformatics,heterologous expression,bacteria type Ⅲ secretion system,HIGS,qReal-time PCR,Y2H technology were employed,to systematically analyze the ability of 20 candidate effector proteins to inhibit the BAX-induced PCD(proceeding cell death).the quantitative expression of effector proteins Pt13024,Pt18906,Pt20911 and Pt3863,and to identify their functional domains,analyze their functional sites,and preliminarily detect their virulence and avirulence functions.The main results are as follows:1.Clarified the sequence characteristics of 20 candidate effector proteinsFrom the 635 candidate effector proteins obtained in the previous stage,20 candidate effector proteins of small molecular weight and containing cysteine were selected and successfully cloned.Analysis of the structural characteristics of candidate effector proteins by the database revealed that 20 effector proteins were hypothetical proteins.Using Pfam software to analyze 20 effector proteins,it was found that none of the 20 effector proteins had any known Pfam motif.Using MEME 5.1.0 to predict effector proteins,it was found that 19 of the 20 candidate effector proteins had no known domain.Only Pt1 030 contains a known structural domain HARBI1_domain,which is found in IPR0015 84 related proteins and may be endonuclease of DDE super-family.Analysis of the primary structure of the secreted protein found that they all contained the cysteine,and Pt16411 contained 18 cysteine in 202 amino acids.2.Using heterologous expression to clarify that the effector protein has toxic effects.The toxicity of effector proteins have been analyzed though heterologous expression.Co-injecting 20 candidate effector proteins with BAX/INF1 into Nicotiana benthamiana showed that the selected 20 candidate effector proteins can effectively inhibit the PCD response induced by BAX and INF1,confirming that the 20 candidate effector proteins are confering virulence to wheat.3.Effector proteins can stimulate the expression of disease resistance of some disease resistance genes.20 effector proteins were transient expressed in a full set of near-isogenic lines in Thatcher background by agrobacterium carried the vector of pGR107 to survey the avirulence effector.Pt13024 was found can triggered the immunity in the near-isogenic lines TcLr30 with necrotic reaction.The effector Pt18906 inducde the HR on the near-isogenic lines TcLr27+31,the gene Pt20911 triggered the HR on the near-isogenic lines TcLr38,and Pt3863 induced HR on the near-isogenic lines TcLr10.4.Basic characteristics of effector proteins Pt13024,Pt18906,Pt20911 and Pt3863The secretion function of signal peptides of effector proteins Pt13024,Pt18906,Pt20911 and Pt3863 was verified,and it was concluded that Pt13024,Pt20911 and Pt3863 are secreted proteins.qRT-PCR was conducted to detecte the effector protein genes in Thatcher leaves at 0 h,6 h,12 h,18 h,24 h,36 h,48 h,72 h,96 h after inoculated by Pt strain 13-5-72.They were all up regulated in 6h-24hpi,and reached the highest peaks at 24 hpi.And 24 hpi was the stage in which the wheat leaf rust fungi produced haustorial mother cells and formed haustoria.It is also a stage in which the pathogen and the host intense interaction.Amino acid polymorphism analysis of 4 effector proteins revealed that there were 2 polymorphisms in Pt13024,3 polymorphisms in Pt18906,2 polymorphisms in Pt20911,and the sequence of Pt3863 was relatively conserved.Subcellular localization of four effector proteins showed that Pt13024,Pt18906,Pt20911,and Pt3863 GFP fusion-expressed proteins were localized in tobacco cells,indicating that effector proteins play a role in host cells.Analysis of deletion mutants of effector proteins revealed that the functional domain of Pt13024 was located at the N-terminal amino acids No.22 to 41,Pt18906 has a functional domain at the N-terminal amino acid 28-47,Pt20911 has a functional domain at the N-terminal amino acid 19-38,Pt3863 has a functional domain at the N-terminal amino acid 20-39 respectively.5.The effector protein stimulates the host’s dual-layer defense response.Bacterial TTSS(Type Ⅲ secretion system)-mediated transient transformation expresses was used to transfer the effector protein Pt13024 into TcLr30,Pt18906 into TcLr27+31,Pt20911 into TcLr38,and Pt3863 into TcLr10 respectively.It was found that the four effector proteins could stimulate the accumulation of host callose and the burst of reactive oxygen species on the hosts.The maximum amount of active oxygen reached 10 minutes after injection.6.The function of effector protein was analyzed using HIGS.The host-induced gene silencing technique(HIGS)was used to silence the effector protein Pt13024 in the near-isogenic line TcLr30,the effector protein Pt18906 in TcLr27+31,the effector protein Pt20911 in TcLr38 and,the effector protein Pt3863 in TcLr10 respectively.The silencing efficiency was detected using qPCR technology,the result showed that the expression levels of the four effector proteins were significantly reduced compared with the control,and the wheat leaf rust THSN successfully infected TcLr30,TcLr27+31,TcLr38 and TcLr10,respectively.It showed that these effector proteins were recognized by the corresponding disease-resistant proteins during the infection of the host by wheat leaf rust,which stimulated their corresponding disease-resistance reactions and inhibited the formation of pathogenic bacteria suction.Effector proteins had no toxic effects on Lr30,Lr27+31,Lr38 and Lr10,respectively.This study confirms that Pt are rich in effector proteins and lack a conserved domain.Effectors modulate plant defense responses in plant cells.Pt13024,Pt18906,Pt20911,and Pt3863 were initially identified as non-toxic effector proteins that infect TcLr30,TcLr27+31,TcLr38,and TcLr10 in Pt.The test results laid a foundation for studying the pathogenic mechanism of wheat leaf rust. |
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