Function Characterization Of Avr1b And GMSGT1 In The Interactions Of Phytophthora Sojae And Glycine Max

Soybean (Glycine max (L.) Merr.) is a commercially important crop across the world. Root and stem rot disease caused by Pytophthora sojae is one of the most destructive diseases and is responsible for $1-2 billion losses per year. The most effective method to reduce damage would be to grow Phytophthora-resistant cultivars, and two types of host resistance have been described. The complete resistance conditioned by single dominant Rps genes and quantitatively inherited partial resistance conferred by multiple genes could both provide protection from the pathogen. In this thesis, Agrobacterium mediated soybean hairy root and cotyledon transformation approaches are used to characterize the function of Avr1b and GmSGT1 in the interaction between soybean and P. sojae. This research resulted in the identification of the defence pathway disturbed by Avr1b and the positive regulation of GmSGTl in soybean resistance to P. sojae.Avr1b is the first Avr gene cloned from P. sojae, it can enhance the virulence of P. sojae when over expresses and also can suppress programmed cell death triggered in soybean, Nicotiana benthamiana, and Saccharomyces cerevisiae cells by the pro-apoptotic protein BAX. However, the mechanism of how Avr1b disturbs host defenses signal pathway is remain poorly understood. In this study, we overexpressed Avr1b in soybean hairy root, and then reached on the influence of the host defence system by Avr1b, the main results are as below:1. Avr1b promote the infection of P. sojae in hairy rootsThe inoculation results show that, the extent of the lesion length in Avrlb overexpressed hairy roots were faster than that in control roots. The biomass of P. sojae in the transgenic hairy roots were also much higher.2. Avrlb accelerate the host cell death in the late stage of the interaction The electrolyte leakage assay was used to investigate the cell death response of soybean hairy roots infected with P. sojae zoospores. At the late stage (36 h,48 h), the conductivity of the Avr1b overexpressed hairy roots were much higher.After 48h inoculated with P. sojae zoospores suspension, the hairy roots which overexpressed Avrl b were hardly seen the GFP fluorescence, nearly all the tissues showed the yellow autofluorescence. At the meanwhile, the yellow autofluorescence only can be seen at the top end of the roots.3. Avr1b suppress the host basal resistanceAniline blue staining assay was used to investigate whether Avr1b overexpression affect callose accumulation induced by P. sojae infection. In Avr1b overexpresses hairy roots, the level of callose accumulation was only 20% compared with control.The 3,30-diaminobenzidine (DAB) staining was used to detect H2O2 accumulation after inoculated with P. sojae zoospores suspension. The roots harbored GFP control vector were significantly showed reddish brown at 6h after inoculation, but the phenotype can’t be see in Avr1b overexpressed hairy roots. So the callose deposition and H2O2 accumulation were both strongly suppressed in the Avr1b overexpressed roots.4. Avr1b didn’t have a clearly subcellular localization in plant cellsThe Avrlb haven’t a clearly subcellular localization in both Nicotiana tabacum leaves and soybean hairy roots. Our result shows that, Avrlb can both localizated on the cell membrane and nucleolus. And in next work, we will try to characterize the relationship between the virulence function and sub-localization.5. The influence of the whole genome transcription by Avr1bUsing Digital Gene Expression Tag profiling (DGE), we analyzed expression profiles in soybean hairy root between over-expressed Avr1b and GFP control to identify the transcriptome response induced by Avr1b. The Pearson correlation coefficient implied that Avrlb only has a limited influence. A total of 355 differentially expressed genes were detected, of which 143 were up-regulated and 212 were down-regulated. The fold change (log2 ratio) distribution of differentially expressed genes among -10.5～11, and more than 90% genes among -5.0～5.0.In order to characterize the main pathway signal,158 of the 355 differentially expressed genes are enriched in 68 pathways. We next annotated the genes function of which the fold changes |log2Ratio|≥5. There are 4 candidate genes have a function annotation in other plants, two of them encode Peroxidase (Gma#S53087018, Gma#S39318532), one encode amine oxidase (Gma#S 19075998), the last encode 60S ribosomal protein (Gma#S23062251).This study provides a global view of transcriptome response and gene expression profiling of soybean hairy root in response to Avr1b. The results can help us to improve our current understanding the mechanisms of the Avr genes to pathogenicity.The SGT1 protein is essential for disease resistance in many plant species. Here we analyzed and characterized functions of GmSGT1 gene family in R protein-mediated resistance and basal defense in this important crop. And the main results are as below.1. GmSGT1 genes are induced expression upon P. sojae infectionWe totally found five candidates located on the different chromosomes of soybean. Phylogenetic tree shows they were grouped into three clades. Transcriptional levels of all the tested genes were highly induced upon P. sojae infection in three soybean cultivars that confer different resistant levels2. The pathways of Rps-mediated disease resistance are diverse in soybeanUsing a gene silencing system in soybean cotyledons, we demonstrated that silencing GmSGT1 genes comprised race-specific resistance in soybean lines carrying genes at the following loci for race-specific resistance to P. sojae:Rps1a, Rps1c, Rps1d, Rps1k, and Rps8. In contrast, the resistance mediated by Rps2 or Rps3a was not affected.3. GmSGT1 confers the basal resistance to P. sojae in soybean, and P. capsici in N. benthamianaSilencing GmSGTl in cotyledons can reduce the resistance to P. sojae, over-expressed GmSGT1-1 in hairy roots can enhance the resistance to the pathogen, so we conclude that GmSGT1 may function in soybean basal resistance to this pathogen.We further showed that transient over-expression of GmSGT1-1 in Nicotiana benthamiana could enhance the resistance to P. capsici. The results imply that the function of GmSGT1-1 may be conserved.4. Over express GmSGT1-1 in hairy roots activated the JA signal pathway, suppress the H2O2 scavenging and SA signal pathwayWe analyzed the expression patterns of a subset of genes related to producing or scavenging H2O2, as well as genes related to SA- and JA-mediated signaling. Before inoculation, the expression of all tested genes did’t have difference between GmSGT1-1 overexpressed and control hairy roots. After inoculation, The H2O2 producing gene, NADPHOX, was not regulated by SGT1. The H2O2 scavenging gene, APX and CAT1 were significantly down-regulated in GmSGT1-1 overexpressed hairy roots after inoculation with P. sojae. The expression of marker genes of the SA pathway, such as NPR1, PR1a and PR5, were all down-regulated after inoculation with P. sojae. But the expression levels of genes related to the JA-signaling pathway (ERF) were substantially higher in GmSGT1-1 overexpressed hairy roots. We presumed that the over-expression of GmSGT1-1 in hairy roots activated the JA signal pathway, suppress the H2O2 scavenging and SA signal pathway.