Application of proteomics to investigate barley-Fusarium graminearum interaction

Due to the great loss of barley grain yield and quality in addition to mycotoxins contamination caused by Fusarium head blight (FHB), it is essential to understand the molecular interaction between barley and Fusarium graminearum, one of the primary Fusarium species causing FHB, in order to control the disease. Due to the advantages of gel-based proteomics that differentially expressed proteins involved in the interaction can be directly detected by comparing protein profiles displayed on 2-D gels, it is used as a tool for studying the barley- Fusarium graminearum interaction form three different aspects in this thesis shown in Chapter 2, 3 and 4. In Chapter 2, the effect of nitrogen on FHB in a susceptible barley cultivar was investigated with using two levels of nitrogen fertilizers (15 and 100 kg ha-1). Albumin proteome analysis of the infected and control kernels under two N levels showed that i) spots increasing in intensity in the infected plants included fungal proteins and proteolytic fragments of plant proteins, ii) spots decreasing in intensity contained plant proteins possibly degraded by fungal proteases, iii) greater spot volume changes in response to the fungus were observed in plants under low N and iv) proteomes of uninfected plants were similar under two N levels. Correlation of level of proteolysis induced by the fungus with measurement of Fusarium-damaged kernels, fungal biomass and mycotoxin levels indicated that FHB was more severe in barley with low N. In Chapter 3, the molecular mechanisms of barley defense to Fusarium graminearum at the early infection stage were studied. Antibodies against barley β-amylases were shown to be the markers for infection at proteome level and for selection of the time for proteome analysis before extensive degradation caused by the fungus. Pathogenesis-related (PR) proteins and proteins involved in energy metabolism were induced and protein involved in the secondary metabolism and protein synthesis changed in abundance in the infected barley. qRTPCR analysis showed the upregulation of several PR genes and expression of two fungal genes encoding proteases which could be responsible for proteolysis of β-amylases in the infected barley. In Chapter 4, the in vitro secretome of F. graminearum on the 2-D gels in the presence of substrates of barley or wheat grain was studied. Totally 69 unique fungal proteins identified were mainly cell-wall-degrading enzymes and proteases. Besides Tri5 gene, ten selected genes encoding protein expressed in vitro were also expressed in the F. graminearum-infected wheat and barley from 2-6 day after inoculation (dai), suggesting the in vitro proteome approach may be an ideal strategy to discover pathogenicity factors. In addition, sharper increase in fungal biomass was observed in barley than in wheat and fungal induced proteolytic fragments of - amylases were only observed in barley not in wheat. Furthermore, a barley PR17 protein and a fungal hypothetical protein were expressed in E. coli and purified in Chapter 5. The functional characterization of two proteins is undergoing. In Chapter 6, microarray data of F. graminearum during interaction with barley and wheat was analysed. The expression patterns of 11fungal genes in microarray analysis were different from qRT-PCR results in Chapter 4. Overall, our results will give some insights into the cellular activities during the interaction between barley and Fusarium graminearum for designing new efficient strategies for the control of FHB disease.