Study Of The Puccinia Striiformis F.sp.Tritici Sugar Absorption Mechanism In Infection Of Wheat

Puccinia striiformis f.sp.tritici(Pst)is the causal agent of wheat stripe rust which is one of the most threatening plant fungal diseases on wheat.It has been shown that growing resistant variety is the most economy and effective method to control wheat stripe rust.However,the virulence of Pst changes frequently.With new virulent strain appearance,the wheat cultivar resistance would be conquered in a few years.The interaction mechanism between Pst and wheat need to be studied further for proposing new strategy to control the disease.As Pst is a kind of obligate biotrophic fungus,it relies totally on its host for all the nutrients needed for growth and development.Although lots of studies had focused in the infection process of Pst and identification of virulence genes from Pst,little is known about the nutrition mechanism of Pst,especially what and how the fungus absorpts from its host.Sugars could serve as carbon source to support the growth and development of heterotrophic microbes,so we focused in Pst sugar absorpotion mechanism in this study.In this study,the sugar absorption mechanism of Pst has been shown for the first time.It could be a new theoretical basis for controlling wheat tripe rust,and moreover,it could be helpful for sustainable control of wheat stripe rust.The major results and conclusion are as below:1.To clarify which kind of sugar Pst absorbs in the interaction with wheat,the levels of all water-soluble carbohydrates in Pst-infected wheat leaves were investigated by HPLC.And the results revealed all of the water-soluble carbohydrates including glucose,fructose and sucrose could be detected in the infected leaves,while only sucrose level shows a significant change between the infected and uninfected wheat leaves.The sucrose levels of the infected leaves in both compatible and incompatible systems were tested for the early,middle and later stage of Pst infection process.And the results showed the sucrose levels in both compatible and incompatible systems were higher than those in the control.The test of the sucrose levels in the in vitro infected leaves indicated that the increased sucrose was synthesized by the infected leaves but not transported from the other part of the plant.Then the photosynthetic rates of the infected leaves and the expression profiles of genes related to photosynthesis were assayed both in compatible and incompatible systems during the Pst infection process.And the results indicated that Pst infection could up-regulate the expresstion of genes involved in photosynthesis to promote the photosynthetic rate,and ultimately induce sucrose accumulation in infected wheat leaves;2.With the analysis of the sequencing results of Pst,it was found that there is no sucrose transporter but only hexose transporters in this fungus.So Pst could not absorb sucrose from host directly,but it could uptake the decompositions of sucrose which are hexoses.Therefore,invertase,which could hydrolyze sucrose into glucose and fructose,is of great importance in Pst sugar absorption.In this study,an invertase gene,PsINV,was cloned from Pst showing great similarity to the invertase gene from Uromyces fabae.And a rust fungi specific structure was found in this invertase.Analysis of its expression profile and signal peptide function showed PsINV could be secreted into the host abundantly.The complementation experiment in Saccharomyces cerevisae and eukaryotic expression experiment confirmed that PsINV acts as invertase both in vivo and in vitro.Enzymatic characteristics of PsINV were determined with the active protein obtained by eukaryotic expression.Further research into the different domains of this protein indicated that the rust fungi specific sequence contributes to a higher efficiency of sucrose hydrolysis,which could be benefit for the fungus to compete with the host for sucrose.Finally,the biological function of PsINV was determined by HIGS.Silencing this gene during the Pst infection process resulted in reduced growth and only limited sporulation of Pst.As a result,the pathogenicity decreased with PsINV silenced.In conclusion,it has been shown that the pathogen invertase plays a pivotal role in the pathogen sugar absorption.And it has been proved that the obligate biotrophic fungus depends on its own invertase to hydrolyze host sucrose for carbon supplement.3.Only one highly expressed Pst hexose transporter gene in its infection process was found by former sequencing analysis.To elucidate how Pst absorbs hexoses from host the hexose transporter gene,PsHXT,was cloned from Pst in this study.Quantitative RT-PCR showed the expression of this gene is induced since the initial stage of infection process and decreased significantly at the late infection stage.The subcellular localization study of PsHXT showed it was a membrane protein.The function of PsHXT was verified in S.cerevisae,and the transport properties were further determined.The function of PsHXT in the interaction between Pst and wheat was analyzed with HIGS.And the result showed that the growth of Pst would be restricted and the infection type on the infected leaves would be decreased with PsHXT silenced.All these results indicate PsHXT plays an important part in Pst sugar absorption.And hexose was shown to be indispensable for biotrophic fungus normal growth and development as carbon source.