Edwin van der Vossen

Effector Genomics Accelerates Discovery and Functional Profiling of Potato Disease Resistance and Phytophthora Infestans Avirulence Genes

Potato is the worlds fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.

In Planta Expression Screens of Phytophthora infestans RXLR Effectors Reveal Diverse Phenotypes, Including Activation of the Solanum bulbocastanum Disease Resistance Protein Rpi-blb2

The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD3645-1, suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34–amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2.

Exploiting Knowledge of R/Avr Genes to Rapidly Clone a New LZ-NBS-LRR Family of Late Blight Resistance Genes from Potato Linkage Group IV

In addition to the resistance to Phytophthora infestans (Rpi) genes Rpi-blb1 and Rpi-blb2, Solanum bulbocastanum appears to harbor Rpi-blb3 located at a major late blight resistance locus on LG IV, which also harbors Rpi-abpt, R2, R2-like, and Rpi-mcd1 in other Solanum spp. Here, we report the cloning and functional analyses of four Rpi genes, using a map-based cloning approach, allele-mining strategy, Gateway technology, and transient complementation assays in Nicotiana benthamiana. Rpi-blb3, Rpi-abpt, R2, and R2-like contain all signature sequences characteristic of leucine zipper nucleotide binding site leucine-rich repeat (LZ-NBS-LRR) proteins, and share amino-acid sequences 34.9% similar to RPP13 from Arabidopsis thaliana. The LRR domains of all four Rpi proteins are highly homologous whereas LZ and NBS domains are more polymorphic, those of R2 being the most divergent. Clear blocks of sequence affiliation between the four functional resistance proteins and those encoded by additional Rpi-blb3 gene homologs suggest exchange of LZ, NBS, and LRR domains, underlining the modular nature of these proteins. All four Rpi genes recognize the recently identified RXLR effector PiAVR2.

Rpi-vnt1.1, a Tm-2(2) homolog from Solanum venturii, confers resistance to potato late blight.

Despite the efforts of breeders and the extensive use of fungicide control measures, late blight still remains a major threat to potato cultivation worldwide. The introduction of genetic resistance into cultivated potato is considered a valuable method to achieve durable resistance to late blight. Here, we report the identification and cloning of Rpi-vnt1.1, a previously uncharacterized late-blight resistance gene from Solanum venturii. The gene was identified by a classical genetic and physical mapping approach and encodes a coiled-coil nucleotide-binding leucine-rich repeat protein with high similarity to Tm-2(2) from S. lycopersicum which confers resistance against Tomato mosaic virus. Transgenic potato and tomato plants carrying Rpi-vnt1.1 were shown to be resistant to Phytophthora infestans. Of 11 P. infestans isolates tested, only isolate EC1 from Ecuador was able to overcome Rpi-vnt1.1 and cause disease on the inoculated plants. Alleles of Rpi-vnt1.1 (Rpi-vnt1.2 and Rpi-vnt1.3) that differed by only a few nucleotides were found in other late-blight-resistant accessions of S. venturii. The late blight resistance gene Rpi-phu1 from S. phureja is shown here to be identical to Rpi-vnt1.1, suggesting either that this strong resistance gene has been maintained since a common ancestor, due to selection pressure for blight resistance, or that genetic exchange between S. venturii and S. phureja has occurred at some time.

Mapping and Cloning of Late Blight Resistance Genes from Solanum venturii Using an Interspecific Candidate Gene Approach

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases of potato. Resistance (R) genes from the wild species Solanum demissum have been used by breeders to generate late-blight-resistant cultivars but resistance was soon overcome by the pathogen. A more recent screening of a large number of wild species has led to the identification of novel sources of resistance, many of which are currently being characterized further. Here, we report on the cloning of dominant Rpi genes from S. venturii. Rpi-vnt1.1 and Rpi-vnt1.3 were mapped to chromosome 9 using nucleotide binding site (NBS) profiling. Subsequently, a Tm-2(2)-based allele mining strategy was used to clone both genes. Rpi-vnt1.1 and Rpi-vnt1.3 belong to the coiled-coil NBS leucine-rich repeat (LRR) class of plant R genes and encode predicted peptides of 891 and 905 amino acids (aa), respectively, which share 75% amino acid identity with the Tomato mosaic virus resistance protein Tm-2(2) from tomato. Compared with Rpi-vnt1.1, Rpi-vnt1.3 harbors a 14-aa insertion in the N-terminal region of the protein and two different amino acids in the LRR domain. Despite these differences, Rpi-vnt1.1 and Rpi-vnt1.3 genes have the same resistance spectrum.

The Late Blight Resistance Locus Rpi-blb3 from Solanum bulbocastanum Belongs to a Major Late Blight R Gene Cluster on Chromosome 4 of Potato

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpi-abpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.

Phytophthora infestans Isolates Lacking Class I ipiO Variants Are Virulent on Rpi-blb1 Potato

A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of P. infestans and related species resulted in 16 ipiO variants in three distinct classes. Class I and class II but not class III ipiO variants induce cell death when coinfiltrated with Rpi-blb1 in Nicotiana benthamiana. Class I is highly diverse and is represented in all analyzed P. infestans isolates except two Mexican P. infestans isolates, and these were found virulent on Rpi-blb1 plants. In its C-terminal domain, IPI-O contains a W motif that is essential for triggering Rpi-blb1-mediated cell death and is under positive selection. This study shows that profiling the variation of Avr-blb1 within a P. infestans population is instrumental for predicting the effectiveness of Rpi-blb1-mediated resistance in potato.

Cross-Species Bacterial Artificial Chromosome–Fluorescence in Situ Hybridization Painting of the Tomato and Potato Chromosome 6 Reveals Undescribed Chromosomal Rearrangements

Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC–FISH as a unique tool for comparative genetic studies across Solanum species.

Allele mining in Solanum: conserved homologues of Rpi-blb1 are identified in Solanum stoloniferum

Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of R genes. Here we focus on two R genes, Rpi-blb1 and Rpi-blb2, originally derived from Solanum bulbocastanum. The Rpi-blb1 gene is part of a cluster of four paralogues and is flanked by RGA1-blb and RGA3-blb. Highly conserved RGA1-blb homologues were discovered in all the tested tuber-bearing (TB) and non-tuber-bearing (NTB) Solanum species, suggesting RGA1-blb was present before the divergence of TB and NTB Solanum species. The frequency of the RGA3-blb gene was much lower. Interestingly, highly conserved Rpi-blb1 homologues were discovered not only in S. bulbocastanum but also in Solanum stoloniferum that is part of the series Longipedicellata. Resistance assays and genetic analyses in several F1 populations derived from the relevant late blight resistant parental genotypes harbouring the conserved Rpi-blb1 homologues, indicated the presence of four dominant R genes, designated as Rpi-sto1, Rpi-plt1, Rpi-pta1 and Rpi-pta2. Furthermore, Rpi-sto1 and Rpi-plt1 resided at the same position on chromosome VIII as Rpi-blb1 in S. bulbocastanum. Segregation data also indicated that an additional unknown late blight resistance gene was present in three populations. In contrast to Rpi-blb1, no homologues of Rpi-blb2 were detected in any material examined. Hypotheses are proposed to explain the presence of conserved Rpi-blb1 homologues in S. stoloniferum. The discovery of conserved homologues of Rpi-blb1 in EBN 2 tetraploid species offers the possibility to more easily transfer the late blight resistance genes to potato varieties by classical breeding.

Elicitin recognition confers enhanced resistance to Phytophthora infestans in potato

Potato late blight, caused by the destructive Irish famine pathogen Phytophthora infestans, is a major threat to global food security1,2. All late blight resistance genes identified to date belong to the coiled-coil, nucleotide-binding, leucine-rich repeat class of intracellular immune receptors3. However, virulent races of the pathogen quickly evolved to evade recognition by these cytoplasmic immune receptors4. Here we demonstrate that the receptor-like protein ELR (elicitin response) from the wild potato Solanum microdontum mediates extracellular recognition of the elicitin domain, a molecular pattern that is conserved in Phytophthora species. ELR associates with the immune co-receptor BAK1/SERK3 and mediates broad-spectrum recognition of elicitin proteins from several Phytophthora species, including four diverse elicitins from P. infestans. Transfer of ELR into cultivated potato resulted in enhanced resistance to P. infestans. Pyramiding cell surface pattern recognition receptors with intracellular immune receptors could maximize the potential of generating a broader and potentially more durable resistance to this devastating plant pathogen.