The Study On Biological Functions Of Nudix Effectors From Potato Late Blight Fa Mine Pathogen Phytophthora Infestans

Potato late blight,the agent of Phytophthora infestans.It is easy to lead to disease spread and epidemic under suitable conditions,and can be infected for several times,causing the potato to be reduced or even to be produced,which is one of the most destructive oomycete diseases.The most effective measure for controlling the disease is to use resistant cultivars in the agricultural production,however,the virulence diversity of P.infestanS,the increasing lack of potato resistance resources often leads cultivars to lost resistance.the existing control measures of potato late blight disease are also faced by A serious challenge.In the process of infecting host plants,which involve a complex network of cross-linked signalling and regulatory processes:The first line of inducible defence in plants involves the recognition of conserved microbial molecules?Pathogen associated molecular patterns;PAMPs?triggering?PATP triggered immunity,PTI?defense reactions and non-toxic effectors?effector triggered immunity,ETI?triggering immune response.At the same time,Phytophthora infestans can secret effectors that inhibit the host immune response and thus decreased the host suscepbility.However,there are many mechanisms for how the P.infestans effectors to induce the susceptibility is unclear.The study of the mechanism of AVR effectors minipulates plant susceptibility not only can analyze the pathogenic mechanism of Phytophthora infestans and to provide a new strategy for the controlling methord of potato late blight,but also it can provide some reference for the pathogenesis of other phytophthora diseases.In previously work in our lab,Avr3b is a firstly reported Nudix effector from Phytophthora sojae to manipulates the plant immunity,which can recruit the Cyclophilin protein of soybean to activate its enzymatic activity.However,the whole sequencing of genome from Phytophthora ramorum,Phytophthora infestans,Phytophthora capsici and Phytophthora parasicita find that there respectively contain 3,5,1 and 5 RXLR-Nudix effectors and these effectors have highly sequence variation,suggesting that it may have complex biological functions.In this thesis,Nudix effectors were used as the main research object,and the biological function of Nudix effector of Phytophthora infestans was systematically studied by means of bioinformatics,molecular biology,cell biology and biochemistry.Bioinformatics studies have found that there are five Nudix effectors homologous sequences in the genome of Phytophthora infestans,but the two gene sequences are thought to be pseudogenes because of sequence mutations or have never been detected.Three Nudix effector genes were cloned from the cDNA samples of Phytophthora infestans T30-4 infection the tomato at different stages,named NUD1?PITG₁5679?,NUD2?PITG₀5846?and NUD3?PITG₀6308?,all of which contained two intros,whereares nud3 can undergoes two transcriptions NUD3.1 and NUD3.2 by variable shear.NUD1,NUD2 and NUD3.2 have a complete Nudix hydrolytic enzyme conserved domain,and NUD3.1 loss of Nudix hydrolase domain due to variable splicing differences leading to the appearance of the stop codon.Real-time reverse transcription PCR showed that these genes were significantly up-regulated in the early infection stage,and the expression of NUD3.2 was the highest at 6 hours of infection,NUD2 followed,and the expression of nudl presented very low in all stages,suggesting that NUD2 and NUD3.2 may play an important role in the process during the infection stage.While the Nudix effectors were overexpressed in the tobacco,and found that only NUD3.2 could significantly enhaced the suscepbility to plant,suggesting that NUD3.2 may be may play an important role in the process during the infection stage,and shows that the three Nudix effectors may have some different function during their evolution.To make clear that the Nudix hydrolase substrate is also important for the further analysis of the biological functions of the effectors.We have found that the Nudix hydrolase domain of these effectors is clustered with the known hydrolase of AP6A by sequence phylogenetic tree analysis.it is presumed that the substrate is more preferred for AP6A and its analogues.Since the three Nudix proteins of Phytophthora infestans do not contain conserved a mino acid sites that interact with Cyclophilin,it is presumed that their enzyme activity may not need to be activated by plants and can produce functional recombinant proteins by prokaryotic expression.Using UPLC-ESI-MS/MS methord combined with in vitro biochemical experiments confirmed that the recombinant Nudix effector protein had the activity of hydrolyzing Ap6A.To further verify its hydrolase activity,we mutated four key points related to enzyme activity in the Nudix functional domain A mino acid catalytic sites and prepare NUD2QQQQ and NUD3.2QQQQ mutants.Biochemical measurements showed that the mutants significantly lost the ability to hydrolyze AP6A.In order to study the role of Nudix effectors in late blight and host interaction,the Nudix effectors was identified to localized the cell membrane and cytoplasm by microscopic observation technique.And then expressed the Nudix effectors on the tobacco,to detect its inhibition function on suppress the Phytophthora elicitor induced cell death.A preli minary study found that three Nudix effectors have the ability to inhibit the extracellular elicitor INF 1-induced cell death in Phytophthora infestans,but only NUD3.2 can inhibit the cell death induced by the new elicitor PiXEGl.NUD2QQQQ and NUD3.2QQQQ did not alter their localization in the plant cell,but it could not suppress INF 1-induced cell death.NUD3.2QQQQ could not suppress cell death induced by PiXEG1,suggesting that the suppress function of Nudix effectors does not depend on its hydrolase activity.To further investigate whether the activity of Nudix effectors is related to its subcellular localization,we have added the nuclear localization signal at the C-ter minus of the Nudix effectors to construct the NUD2-NLS and NUD3.2-NLS mutants and found that the mutants NUD2-NLS and NUD3.2-NLS was expressed in tobacco were mainly localized in nuclear,and NUD2-NLS and NUD3.2-NLS were found have lost the function of suppressed the cell death triggered by INF 1 and PiXEG1,suggesting that the cytoplasmic localization of Nudix effectors play an important role for their virulence function.The results of this study firstly reveal the genetic structure of Nudix effectors and its gene expression in the early stage of infection,and report the subcellular localization of Nudix effectors and a novel substrate Ap6A,It is clear that the Nudix effectorse can suppress the two Phytophthora elicitors triggered cell death and promote the infection ability of Phytophthora infestans,and these functions mainly depend on its enzymatic activity and cytoplasmic localization.These function mainly rely on their hydrolase activity to suppress the plant immune response and promote the pathogenicity of pathogen infection is relatively conservative,and their function between the evolution of the process also obvious differentiation.