Regulation Of The Hypersensitive Response Mediated By Tomato Cf-4 And Cf-9 Genes And Improvement Of The Gene Silencing Technique

Gene-for-gene resistance is an important type of plant disease resistance. It is governed by plant resistance (R) genes and their matching pathogen avirulence (Avr) genes. This resistance is characterized by a hypersensitive response (HR) resulting from the interaction between products of a complementary R and Avr gene pair. The pathosystem of tomato (Lycopersicon esculentum) and its leaf mould fungal pathogen Cladosporium fulvum is a model system to study gene-for-gene resistance. Several R/Avr gene pairs, including for example Cf-4/Avr4 and Cf-9/Avr9, have been cloned in this pathosystem. Content of this thesis research consists of two parts. The first part is on characterization of Cf-4/Avr4- and Cf-9/Avr9-dependent HR employing Cf/Avr tomato seedlings that both express a Cf gene and the matching Avr gene, mainly includes comparison of transcription regulatory activity of the Cf-4 and the Cf-9 promoters, and investigation of effect of temperature and humidity on Cf-4/Avr4- and Cf-9/Avr9-dependent HR, whereas the second part is focused on exploitation and optimisation of virus-induced gene silencing (VIGS) in model plants tomato and Arabidopsis thaliana. The main results are as follows:1. Difference was found in transcriptional regulatory activity of the Cf-4 and the Cf-9 promoters. The 3 kb untranscribing regions upstream the open reading frames (ORF) of the Cf-4 and the Cf-9 genes (simplified as pCf4 and pCf9, respectively) were ligated to the ORFs of the Cf-9 gene and the gene encoding green fluorescence protein (GFP), respectively, to release the four chimeric constructs pCf4::Cf-9, pCf9::Cf-9, pCf4::GFP and pCf4::GFP. The construct 35S::GFP was also made in which the ORF of the GFP was driven by the 35S promoter of Cauliflower mosaic virus. The transgenic tobacco plants of the five constructs were obtained by Agrobacterium-medisted leaf disc system. Homozygous 35S::Avr9 plants were crossed to the TO plants of pCf4::Cf-9 and pCf9::C/-9 transgenic lines. The seeds germinated normally at 25℃. HR occurred in half of the obtained F₁ progeny due to carrying the Cf-9/Avr9 gene pair, and finally these seedlings died. It was observed that the pCf4::Cf-9/Avr9 seedlings differed from the pCf9::Cf-9/Avr9 seedlings in the severity of the resulting HR, and its effect on seedling development and the sensitivity to high temperature. When grown at 25°C, the pCf9::Cf-9/Avr9 seedlings shown less cotyledon expansion, retained the testa, lower total fresh weight, when compared with the pCf4::Cf-9/Avr9 seedlings. However, when grown at 32℃, the two genotypic seedlings shown the similar growth and HR development, and died at the similar time. Results of RT-PCR revealed that the transcripts of pCf9::Cf-9 and pCf9::GFPaccumulated to a higher level than that of pCf4::Cf-9 and pCf4::GFP. These results demonstrate that compared with the pCf4, the pCf9 promoter is more active in transcriptional regulation, and more sensitive to high temperature.2. The effect of the humidity on the Cf-4/Avr4- and Cf-9/Avr9-dependent HR was made clear. Employing the Cf/Avr seedlings, it was found that both Cf-4/Avr4- and Cf-9/Avr9-dependent HR was delayed and reduced under high humidity (95%), and that Cf-9/Avr9-dependent HR was more sensitive to high humidity when compared to Cf-4/Avr4-dependent HR. Furthermore, high humidity acts synergistically with high temperature on HR suppression, resulting in complete blocking of the HR. The transcript profile of over 60 genes, related to HR, signalling and defence, in Cf/Avr seedlings grown under high humidity and thus showing no HR, significantly differed from that of the same seedlings grown under normal humidity and thus showing HR. These results demonstrate that high humidity probably acts at a very early point of the Cf downstream signalling pathway, or alternatively influences the interaction between the Cf and the Avr proteins.3. The VIGS system in Arabidopsis thaliana was optimised. Employing the improved TRV vector system, the effect of the concentration and recovery-culture of Agrobacterium...