Analysis On Differentially Expressed Proteins Of Leaf And Grain Influenced By Powdery Mildew In Wheat (Tricticum Aestivum L.)

Powdery mildew of wheat is one of the most severe fungal diseases which occurred in the major wheat producing areas of the world,leading to a decrease in yield and quality in wheat agriculture.At present,chemical control and breeding resistance cultivars are effective methods to control powdery mildew of wheat.However,a rapid coevolution of the pathogen with its host and the emergence of new pathogen species results in loss of the resistant ability of numerous drugs and wheat cultivars.Therefore,studying the mechanism of compatible interaction of wheat and powdery mildew,analyzing the impact of the pathogen on the development of wheat grain and exploring further the changes of wheat source-sink on a molecular level are significant.In this study,we used wheat cultivar Xinong 979 as experimental material which is susceptible for powdery mildew,and the susceptibility level was estimated at 6 on a scale rang from 0(immune)to 9(highly susceptible)identified by our lab.Wheat powdery mildew physiological race E20 with strong pathogenicity was epidemic in field.Combination with laboratory and field experiments,the changes of profiles of proteome wheat leaves and grains during powdery mildew infection were studied respectively by proteomic techniques.In addition,we analyzed the impacts of the pathogen on the expression of key enzymes of starch synthesis using q RT-PCR.Our results might provide an insight on the plant primary resistance,and theoretical references for the effective control of the disease and the evaluation of the crop loss.The main results obtained in this study were as follows:1.Proteome characteristics of wheat leaves with the powdery mildew infection were investigated by two-dimensional electrophoresis and tandem MALDI-TOF/TOF-MS.We identified 46 unique proteins which were differentially expressed at 24,48,and 72 hours post-inoculation.The functional classification of these proteins showed that most of them were involved in photosynthesis,carbohydrate and nitrogen metabolism,defense responses,and signal transduction.Upregulated proteins included primary metabolism pathways and defense responses,while proteins related to photosynthesis and signal transduction were mostly downregulated.As expected,more antioxidative proteins were activated at the later infection stage than the earlier stage,suggesting that the antioxidative system of host plays a role in maintaining the compatible interaction between wheat and powdery mildew.A high accumulation of 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase in infected leaves indicated the regulation of the TCA cycle and pentose phosphate pathway in parallel to the activation of host defenses.The downregulation of MAPK5 could be facilitated for the compatible interaction of wheat plants and Bgt.In order to validate observed protein modulations,we performed an analysis of gene expression to further investigate gene expression profiles along the entire time course of infection.The results showed that the transcriptional expression patterns of 13 of 15 candidates were consistent with the observed protein expression at the corresponding time points.q RT-PCR analysis supported the data of protein expression profiles.Our results reveal the relevance of primary plant metabolism and defense responses during compatible interaction,and provide new insights into the biology of susceptible wheat in response to Bgt infection.2.Proteome characterization during grain development in susceptible wheat cultivar Xinong 979 with powdery mildew infection was investigated by 2-DE and tandem MALDI-TOF/TOF-MS.Identification of 111 differentially accumulated protein spots representing 85 unique proteins,and six expression patterns showed a chronological description of wheat grain formation.Significant proteome expression differences between control and infected grains were found.Comparative proteomic characterization demonstrated 36 infection induced protein spots that co-accumulated in the two samples with different expression patterns,and 34 spots displayed significant change in abundance,which mainly related to stress/defense responses,primary metabolism and storage protein.The down-regulation of defense responses-related proteins including alpha-purothionin,lactoylglutathione lyase and alpha-amylase inhibitor CM16 in infected grains compared to control during filling stages might be related to the susceptibility of wheat to Bgt,while the enhanced expression of beta-amylase and glyceraldehyde-3-phosphate dehydrogenase and the down-regulation of ADP glucose pyrophosphorylase in infected grains probably resulted in negative effects on yield formation.The profiles of 10 proteins were verified at the transcriptional level by q RT-PCR.The results indicated that the transcriptional expression profiles of 7 candidates matched the observed protein expression trends at four development stages.q RT-PCR analysis generally corresponded to the data of protein expression profiles.Our data reveal the complex grain metabolism mechanisms and defense responses during compatible interactions,and provide valuable information for dissecting the underlying molecular processes which possibly yield novel strategies for breeding resistant cultivars and protection strategies in the field.3.To determine the impact of powdery mildew on starch synthesis of wheat grain,the susceptible wheat variety Xinong 979 to powdery mildew was used as experiment material.The expression levels of five starch synthase genes were investigated by quantitative real-time PCR,including ADP-glucose pyrophosphorylase gene(AGPase),granule bound starch synthase I(GBSS I)gene,soluble starch synthase(SSS)gene,starch branching enzyme(SBE)gene and starch debranching enzyme(DBE)gene.The change of starch synthase(AGPase,GBSS I,SSS,SBE and DBE)activity was conducted during grain development stages as well.The results indicated that comparison with control,the expression of AGPase,SBE and SSS was depressed in infected wheat during grain development.However,the gene expression level and enzyme activity of GBSS I were higher in infected than in control.The expression of DBE had no significant difference in infected and control grains,but enzyme activity of DBE was higher in infected grains than in control.In addition,amylopectin content and total starch content were significant reduced in infected grains compared with control with the development of infection,but the content of amylase was increased in infected grains.These results suggested that powdery mildew could inhibit the accumulation of starch by impacting the expression levels and enzyme activities of starch synthases during grain filling stage.In summary,three main conclusions of this study could be drawn:(1)During powdery mildew infection,compared with uninfected leaves,in infected leaves the metabolism was enhanced,the rate of photosynthesis was reduced,and the expression of many antioxidative proteins occurred at the late stage of infection.The decline of the expression of MAPK5 at the early stage of infection suggested that MAP kinase signal transduction pathway was suppressed.(2)With the comparation of uninfection wheat,in infected wheat grains,the respiration rate was increased,starch breakdown metabolism was enhanced and starch synthesis was depressed,and the abundance of storage proteins was increased.Moreover,the decreased levels of several allergens implied that the infection of powdery mildew restrained the expression of defense response-related proteins in susceptible wheat.(3)The reduced photosynthesis assimilation in infected leaves resulted in the decrease of carbon nutrient sources which were transported to grainsfor the synthesis of storage materials.Furthermore,the decrease of key enzymes for starch synthesis leaded to the decline of sink organ activity.These results indicated that in infected leaves,the decline of production capacity of photosynthate and the transformed ability of reserve materials in sink organ might be critical limiting factors for grain development.Furthermore,the delayed expression of defense response-related proteins in infected leaves and the decline of disease-resistant proteins in grains might be the prominent characteristics of susceptible wheat for powdery mildew.