Cloning And Functional Analysis Of PsCS1 Gene From Puccinia Striiformis F.sp. Tritici

TCA cycle is the ubiquitous metabolic pathway in aerobic organisms and the most effective way for the body to obtain energy by oxidation of sugar or other substances.Citrate synthase,a rate-limiting enzyme for TCA cycle,is involved in a variety of intracellular physiological processes such as mitochondrial energy metabolism,seed germination and stress resistance.Also,it has a wide range of application value in medicine,agriculture.Puccinia striiformis f.sp.Tritici(Pst),the causal fungus of wheat stripe rust,is an obligate biotrophic basidiomycete,mainly rely on the infection structure-haustorium to obtain nutrition from the host.Sheds light on the Pst nutrient metabolism is of great significance for the prevention and control of wheat stripe rust.A large number of studies have shown that citrate synthase plays an important role in the growth and development of pathogens.However,little is currently known regarding the metabolic requirements of Pst during infection.Therefore,we cloned an CS gene,PsCS1,and identified its function.The main results are as follows:We searched the cDNA libraries constructed in our lab and a complete ORF wheat stripe rust citrate synthase gene was successfully cloned,named PsCS1,the open reading frame(ORF)of PsCS1 consists of 1407 nucleotides and is predicted to encode a polypeptide of 491 amino acids with a citrate synthase domain;The PsCS1 transcript levels were measured during Pst infection by quantitative reverse transcription PCR(qRT-PCR),which showed PsCS1 was obviously up-regulated at early stage during Pst infection;The prokaryotic expression demonstrates that it has the function of citrate synthase;Biochemical characterization of PsCS1 showed that the enzyme activity was higher at low temperature,the pH optimum was determined to be approximately pH 8.In addition,K + could significantly increase the activity of citrate synthase;The bimolecular fluorescence complementation(BiFC)experiment showed that PsCS1 worked as a multimer;Overexpression of PsCS1 in E.coli can increase its resistance to salt stress;Transient expression of PsCS1 in Nicotiana benthamiana suggest it may located in mitochondria;Moreover,knockdown of PsCS1 using a host-induced gene silencing(HIGS)system significantly reduced the area of colony at 120 h and when the silence of plants compared with the control,the bacteria biomass decreased by 33% at 7 days,which reveal that the knockdown of PsCS1 leads to the restriction of thegrowth and development of the wheat stripe rust.The results will help clarify the role of citrate synthase in wheat stripe rust during the infection and disease processes,which laid the foundation for revealing the molecular mechanism of energy metabolism and also provided the theoretical basis for the prevention and control strategy of wheat stripe rust.