The Ph-3 gene from Solanum pimpinellifolium encodes CC-NBS-LRR protein conferring resistance to Phytophthora infestans

Abstract

Ph-3 is the first cloned tomato gene for resistance to late blight and encodes a CC-NBS-LRR protein. Late blight, caused by Phytophthora infestans, is one of the most destructive diseases in tomato. The resistance (R) gene Ph-3, derived from Solanum pimpinellifolium L3708, provides resistance to multiple P. infestans isolates and has been widely used in tomato breeding programmes. In our previous study, Ph-3 was mapped into a region harbouring R gene analogues (RGA) at the distal part of long arm of chromosome 9. To further narrow down the Ph-3 interval, more recombinants were identified using the flanking markers G2-4 and M8-2, which defined the Ph-3 gene to a 26 kb region according to the Heinz1706 reference genome. To clone the Ph-3 gene, a bacterial artificial chromosome (BAC) library was constructed using L3708 and one BAC clone B25E21 containing the Ph-3 region was identified. The sequence of the BAC clone B25E21 showed that only one RGA was present in the target region. A subsequent complementation analysis demonstrated that this RGA, encoding a CC-NBS-LRR protein, was able to complement the susceptible phenotype in cultivar Moneymaker. Thus this RGA was considered the Ph-3 gene. The predicted Ph-3 protein shares high amino acid identity with the chromosome-9-derived potato resistance proteins against P. infestans (Rpi proteins).

Fig. 1

Physical map of the Ph-3 genomic region from S. pimpinellifolium L3708. a Positions of markers in BAC clone B25E21. Indel-3 and P-55 were previously identified Ph-3 flanking markers (Zhang et al. 2013). b The predicted ORFs between markers G2-4 and M8-2

Fig. 2

Comparison of the Ph-3-containing BAC sequence with the Heinz1706 reference sequence. The X-axis shows the sequence of BAC B25E21, and the Y-axis shows the corresponding Heinz1706 reference sequence. The sequences were analysed using dottup (v6.0.1) with a window size 10. Arrow points to the large picture of the variable region carrying RGAs

Fig. 3

Schematic of the microsynteny between the R gene clusters at the Ph-3 locus in S. pimpinellifolium L3708 and S. lycopersicum Heinz1706. The green arrows at the top show the R gene homologs in the L3708 BAC sequence, and the yellow arrows at the bottom indicate RGAs at the corresponding locus of Heinz1706. The transcriptional orientations are indicated by the direction of arrows. The orange, purple and blue lines linking the L3708 and Heinz1706 sequences indicate an identity above 95, 90–95, and 85–90 %, respectively

Fig. 4

The recombination point in the key recombinant 8-25. The ORF3 fragments from the Ph-3 donor plant L3708, the susceptible parent LA4084 and the susceptible recombinant 8-25 were aligned. Based on two SNPs (residues 434 and 900) in this region, the recombination site of 8-25 was located within the ORF3. The numbers above the arrows indicate the positions of nucleotides in the ORF3

Fig. 5

Expression of the resistant allele of Ph-3 in susceptible Moneymaker resulted in resistance to P. infestans. Transgenic lines were tested in two independent experiments. a Non-transformed Moneymaker showed mycelium growing on the infected leaf areas; b transformed Moneymaker expressing Ph-3 showed no symptom and c transformed Moneymaker not expressing Ph-3 showed mycelium growing on the infected leaf areas. It is worthwhile to note that non-transformed Moneymaker plants were grown from seeds and that transformed Moneymaker were from cuttings. Three leaflets in a, b or c were taken from one inoculated leaf and three leaves per plant were tested. Photographs were taken 7 days post-inoculation

Fig. 6

The domain structure of the predicted Ph-3 protein. The predicted coiled coil in the CC domain was underlined. Boxes indicate positions of conserved NB-ARC motifs. The 16 imperfect LRRs were aligned according to the consensus sequence xxLxLxx (where L represents leucine or other aliphatic amino acid, and x is any residue)

Fig. 7

Phylogenetic analysis of Tm-2 ²-like resistance proteins and all cloned potato Rpi proteins. The resistance protein sequences were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). The phylogenetic tree was performed using MEGA4 with Bootstrap test. Numbers at the branches are confidence values