System Construction Of Late Blight Resistance Signal Transduction And Resistance Resource Creation In Tomato

Late blight, caused by the oomycete Phytophthora infestans(Mont.)de Bary, is a devastating disease of two Solanaceous crops, tomato and potato. Over decades years efforts, five R genes have been cloned, such as R1 and R3a from Solanum demissum, RB (Rpi-blb1), Rpi-blb2 and Rpi-blb3 from S. bulbocastanum. But as new virulent races emerged rapidly, these race-specific R genes provided only short-lived resistance in the field. The similar situations have occurred in tomato, four resistance genes have been mapped and deployed in cultivars but the effectiveness is limited because of the fast evolving P. infestans pathogen in the field. In-depth understanding of signal transduction mechanisms underlying potato R gene resistance may help to generate effective measures to control late blight. R3a from potato and Avr3a from P. infestans are the first pair of R-AVR genes which have been cloned in potato and P. infestans which is the basic theory of our research.The results were as follows:1. Constructed the VIGS vectors contain key signal transduction gene EDS1, NDR1, RAR1, SGT1, HSP90, NPR1, NtWRKY1, SIPK1 respectively. Transient expressed R3a and Avr3a gene in Nicotiana benthamiana after the VIGS gene silencing. Hypersensitive cell death was interrupted in SGT1 and HSP90 silenced plants which indicated that SGT1 and HSP90 participated in R3a and Avr3a interaction and played a key role in R3a resistance sinal pathway.2. Transgenic tomatoes contain R3a gene displayed race-specific resistance to P. infestans which verified the conservation of resistance signal pathway in tomato and potato, so it is feasible to use tomato as a model plant to research tomato late blight gene R3a. In order to find new genes that participate in R3a and Avr3a interaction, a novel transgenic tomato plant conferred potato late blight resistance gene R3a and glucocorticoid-inducible avirulence gene Avr3a was constructed in our study. MM-R3a-Avr3a plants showed specific cell death response induced by DEX. This system will be used to large-scale screen the mutants which HR were interrupted.3. In order to screen homozygous MM-R3a-Avr3a lines to construct the mutant library, we use modified adaptor ligation PCR method amplified R3a and Avr3a gene flanking sequences in tomato genome. The flanking sequence of R3a had 100% similarity with clone SL_MboI-127L14 on chromosomeâ…£in NCBI database which indicated R3a gene integration in tomato genome on chromosomeâ…£. The flanking sequence of Avr3a had 100% similarity with a BAC end sequence LE_HBa0167K15_SP6₁3180 in SGN database, but until now we still didn't know this BAC end sequence located on which chromosome. By flanking sequence and border sequence we screened out homozygous MM-R3a-Avr3a lines in T1 transgenic generation.4. We analyzed Avr3a alleles of 22 tomato late blight isolates which demostrated that R3a gene can be used in tomato reisitance breeding in some areas. Other two potato late blight genes R1 and Rpi-blb1/RB were also introduced into tomato. Transgenic plants were inoculated by tomato widly distribute isolates and the high virulence isolates and showed resistance to some tomato late bligt isolates, especially RB, resist the high virulence isolates of tomato which offered a new way for tomato late blight transgene breeding.