Identification Of Genes Involved In Resistance Of Tomato To Cladosporium Fulvum And Fuctional Analysis Of TRV 16K Gene

Gene-for-gene resistance is an important type of plant disease resistance. The pathosystem of tomato (Lycopersicon esculentum) and its leaf mould fungal pathogen Cladosporium fulvum is a model system to study gene-for-gene resistance. The recognition of C. fulvum avirulence gene product Avr by tomato resistance gene product Cf initiates downstream signal transduction, which activates defence responses, resulting in the Cf-dependent resistance to C. fulvum. Isolation and further functional analysis of key signalling components will provide great insights into the mechanism of Cf-dependent resistance. Virus induced gene silencing (VIGS) has recently emerged as a powerful method for gene function study. It has been broadly used in the function analysis of genes involved in disease resistance, growth and development, and metabolism. The TRV vector modified from the RNA virus Tobacco rattle virus (TRV) is the most frequently used VIGS vector. However, effect of the 16K open reading frame (ORF) of pTRV1 on VIGS is still unclear.This thesis study comprises two parts of contents. In the first part, six ACE (Avr/Cf elicited) gene fragments, which are the cDNA-AFLP fragments corresponding to genes specifically expressed during Cf/Avr-dependent hypersensitive response (HR), were chosen as representatives for VIGS analysis to elucidate their possible roles in Cf-4/Avr4-dependent HR, and thereby screened out the one(s) required for Cf-4/Avr4-dependent HR. While in the second part, modified pTRV1 expression construct in which the 16K ORF was deleted, was made. To investigate the role of the 16K ORF in gene silencing, efficiency of VIGS induced by the deletion mutant and the wild type vectors was compared. The main results are as follows:1,The role of the genes corresponding to the six ACE gene fragments in Cf-4/Avr4-dependent HR was identified. Function of the genes corresponding to the ACE fragments was predicted based on sequence homology analysis. Six ACE fragments, i.e. Fragments 5,14,39,85,95 and 111, were chosen for VIGS analysis. Function of genes ACE5 and ACE95 was unknown, while products of genes ACE14,ACE39,ACE85 and ACE111 have significant sequence homology to a calcineurin-like phosphatase, a 40S robosomal protein S13,a LRR-containing protein kinase and a HR marker HSR20 protein respectively. The recombinant pYL156 plasmids with the insertion of these ACE fragments were constructed. VIGS analysis using these constructs followed by analysis of Cf-4/Avr4-dependent HR was conducted. Plants in which genes ACE14,ACE85 and ACE111 were silenced respectively showed growth redardation and organ deformation, indicating that these genes may be involved in plant growth and development. Cf-4/Avr4-dependent HR was severely repressed in ACE95-silenced plants, revealing that ACE95 may be essential for Cf-4/Avr4-dependent HR and resistance.2,The effect of the 16K ORF of pTRV1 on gene silencing induction was analyzed. The pTRV1 expression construct with deletion of the 16K ORF was made using Spe I and Stu I sites. Efficiency of gene silencing induced by the deletion mutant and the wild type TRV vectors was comparatively studied. PDS gene silencing induced by the deletion mutant TRV vector was more complete compared with that by the wild type TRV vector. The percentage of completely silenced N. benthamiana plants out of total silenced plants induced by the deletion mutant vector was 16% higher than that by the wild type TRV vector. Neither the deletion mutant vector nor the wild type TRV vector induced efficient PDS gene silencing in N. tabacum cv. Samsun. However, efficiency of silencing induced by the deletion mutant vector was relatively higher than that by the wild type TRV vector. Additionally, TRV RNA accumulated at a significantly lower level in both inoculation and up uninoculated leaves of plants inoculated with Agrobacterium carrying the deletion mutant vector compared with plants inoculated with Agrobacterium carrying the wild type vector. These results demonstrate that the 16K ORF of pTRV1 plays an important role in TRV virus multiplication, and possibly acts as a weak suppressor of TRV-induced gene silencing in plants.