Cloning Of The Ph-3Gene Conferring Resistance To Late Blight(Phytophthora Infestans) In Tomato And Screening Of Its Effector

Late blight （LB）, caused by the Oomycetes Phytophthora infestans （Mont.） de Bary, is aworldwide disease, mainly affecting the production of potato and tomato. In the past decades, a numberof LB resistance genes have been cloned from potato. In contrast, much less work has been conductedon LB resistance in tomato. The late blight resistance gene Ph-3is widely used in tomato breeding forresistance to late blight. In the previous study, the Ph-3gene was mapped into a74kb region at the endof long arm of chromosome9. Eight open reading frames （ORFs） are present in the Ph-3region, amongwhich four ORFs encode CC-NBS-LRR class of plant resistance genes. In this study, a map-basedcloning approach was used to clone the Ph-3gene, which is the first cloned R gene for resistance to lateblight in tomato. Besides, the effector recognized by the Ph-3gene was identified through effectoromics.The results in this study will facilitate the molecular breeding for resistance to late blight in tomato aswell as the research on the interaction between the host and P. infestans. The main contents are asfollows:1. Based on the fine-mapping of the Ph-3gene, two conserved fragments between different R genehomologs （RGAs） in the Ph-3locus were selected for plasmid construction. The resulting plasmidswere used to transiently silence the candidate R gene family in the cultivar CLN2037B （containingthe Ph-3gene） through virus-induced gene silencing （VIGS）. After the treatment, CLN2037Bbecame susceptible to late blight, which suggested that the Ph-3gene belongs to CC-NBS-LRR classof R genes.2. Seven recombinants were obtained after screening about1900F2or F3plants. With the genotype andphenotype of these recombinants, Ph-3was furthrer narrowed down to a26kb region with flankingmarkers R2M1S and M67-3. According to the tomato genome annotation, there are three RGAs inthe target region.3. The BAC library was constructed with the Ph-3donor Solanum pimpinellifolium L3708, from whichthe single clone B25E21covering the Ph-3region was identified. The full length of this clone wassequenced and compared with the corresponding region in the reference genome Heinz1706. Thereis a28kb deletion in L3708, which leads to the missing of two RGAs. In the Ph-3region, fourRGAs are present in Heinz1706, while only two RGAs exist in L3708. There is only one RGA,named as SpRGA2, between the flanking markers R2M1S and M67-3in L3708. Then thecomplementary function assay showed that nine out of fourteen SpRGA2-transgenic Moneymakerplants were resistant to P. infestans. Therefore, SpRGA2is the Ph-3gene.4. The full length of late blight resistance gene Ph-3in tomato is2556nt, encoding a peptide of851aa.The predicted Ph-3protein belongs the CC-NBS-LRR class of plant R proteins.5. Among the cloned potato Rpi genes, Ph-3shares high identity ranging from74.7%-78.7%to threechromosome-9-derived potato Rpi （resistance to P. infestans） genes, Rpi-vnt1, Rpi-mcq1and R9a. As expected, the lowest identity among these Rpi proteins was found in the LRR domain. In addition,Ph-3also shares high amino acid identity to the tomato mosaic virus resistance gene Tm-22, which islocated near the centromere of chromosome9. All chromosome-9-derived Rpi genes （Ph-3,Rpi-vnt1.1, Rpi-vnt1.2, Rpi-vnt1.3, Rpi-mcq1and R9a） belong to the Tm-22family, which are distinctfrom other characterized Rpi genes in potato.6. To identify the effector recognized by the Ph-3gene, approximately240effectors were screenedthrough agro-infiltration in Nicotiana benthamiana. Among them, only one effector PITG₀9218was identified, which caused the hypersentitive response （HR） or chlorosis on the leaves. ThenPITG₀9218was analysed in the Ph-3donor S. pimpinellifolium L3708via PVX infection. Theresult showed that this effector triggered necrosis on the tomato leaves. The prelimitary conclusion isthat PITG₀9218is the candidate effector which interacts with the R gene Ph-3.7. The pathogenicity of51potato P. infestans isolates on two tomato accessions CLN2037B andMoneymaker was tested. Lesion size and production of sporangia were determined. Results showedthat CLN2037B and Moneymaker have non-host resistance to these potato P. infestans isolates.8. The Ph-3gene was transformed into the susceptible potato cultivar Desiree, resulting in11positivetransformants. Tested with detached-leaf assay, Ph-3-transgenic potato plants were susceptible tofour isolates （CA-65, IPO-C, EC-1and3928-A）, suggesting these isolates don’t contain the effectorrecognized by the Ph-3gene.