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PLoS ONE. 2008; 3(8): e2875.
Published online Aug 6, 2008. doi:  10.1371/journal.pone.0002875
PMCID: PMC2483939

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

Hany A. El-Shemy, Editor

Abstract

Potato is the world's 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.

Introduction

Despite more than a century of resistance breeding [1], late blight remains a major constraint for potato cultivation resulting in multibillion dollar annual losses in most regions of the world. R genes that mediate resistance to P. infestans are common and diverse in wild Solanum germplasm but the identification and deployment of these genes faces a number of hurdles. First, the specificity spectrum of novel late blight R genes cannot be assessed in the absence of diagnostic pathogen races making it impossible to discriminate between genes with similar functional activities. Second, crossing barriers, linkage drag problems and the high quality trait demands of the potato crop severely slow down the laborious process of introgression breeding [2]. Nonetheless, the recent cloning of the R genes Rpi-blb1 (also known as RB) [3], [4] and Rpiblb2 [5] from the sexually incompatible species S. bulbocastanum enabled transgenic engineering of resistant potatoes. Plants carrying these genes have now entered the commercialization pipeline and are expected to be the first GM potatoes to be cultivated in Europe for consumption purposes [6]. In contrast to the S. demissum-derived race-specific R genes which were singly introduced and quickly overcome in the field [1], the combination of Rpi-blb1 and –blb2 is expected to remain effective to a broader spectrum of P. infestans isolates [4], [5]. Indeed, stacking of R genes that confer resistance to a broad and complementary set of isolates promises to deliver potatoes with durable late blight resistance using genetic modification, be it through transgenic or cisgenic approaches [2], [7]. However, knowledge of the pathogen targets of these R genes is essential for classifying them into functional and spectral categories, evaluating the likelihood of resistance durability, and devising scientifically sound stacking strategies.

Oomycete plant pathogens, such as P. infestans, secrete an arsenal of effector proteins that modulate host innate immunity and enable parasitic infection [8]. Although these effectors primarily function as virulence factors, specific effector molecules can also be recognized by plant R proteins in particular host genotypes resulting in activation of effector triggered immunity. In such cases, the effectors are said to have an avirulence (AVR) activity. The response induced by AVR proteins involves in most cases the hypersensitive response (HR), a form of programmed cell death, followed by restriction of the invading pathogen [9]. The AVR proteins of oomycete plant pathogens carry a N-terminal type II secretion signal peptide, followed by a conserved RXLR motif that characterizes a domain known to function in translocating the effectors into host cells [10][12]. Inside host cells, RXLR effectors are believed to contribute to virulence, but also to activate cognate cytosolic R proteins of the NB-LRR (nucleotide binding site and leucine-rich repeat) class resulting in hypersensitive cell death and resistance [10], [11]. We hypothesized that P. infestans RXLR effectors are candidate Avr genes that can be functionally profiled on Solanum to detect cognate R genes [13].

In this study, we expressed a set of 54 candidate RXLR effectors of P. infestans in late blight resistant Solanum plants and identified a variety of effector responses, some of which could be R-AVR interactions. For one of these interactions, we generated a population segregating for resistance and effector response, and determined the genetic position of the putative R gene in Solanum. We then took advantage of comparative genomics of an already cloned R gene to swiftly clone homologous cognate R genes from unrelated Solanum species. Effector-based identification of R genes combined with functional assays accelerated the cloning process of R genes compared to map-based or other approaches and provided insight into R gene redundancy within Solanum [14], [15].

Results

Solanum species recognize a diversity of P. infestans effectors

As part of a wide-ranging potato improvement program, we selected ten Solanum genotypes belonging to eight wild species representing a wide taxonomic and phylogenetic diversity [16][18], that exhibited late blight resistance to various P. infestans isolates in multi-year laboratory and field trials (Table 1, Table S1). To clarify the specificity spectra of these resistant plants, we assayed them with 54 putative effector genes predicted computationally from the P. infestans transcriptome to belong to the RXLR family of cytoplasmic effectors [19]. Functional high-throughput screening of effector gene elicitation of the HR in plants was performed using a Potato virus X (PVX) agroinfection assay optimized for Solanum [13]. Although this assay leads to false positives due to high expression levels of single effectors inside plant cells, PVX agroinfection results in comparable results to various low-throughput systems such as protein infiltrations [13], co-bombardment studies [20], A. tumefaciens co-infiltrations [20], [21], and ELISA (Figure S1). We expressed the 54 RXLR effector genes (Table S2) in the 10 late blight resistant Solanum genotypes, as well as in three susceptible potato genotypes (Table 1, Table 2). In 36 combinations, specific RXLR effectors triggered hypersensitivity in at least one resistant but not in the susceptible genotypes (Figure 1) suggesting that these effectors might exhibit avirulence activities and activate R genes that are absent in the three susceptible potatoes.

Figure 1
Specific responses to pGR106-IpiO in S. stoloniferum 17605-4 by PVX agroinfection.
Table 1
Resistance to P. infestans isolates in Solanum species.
Table 2
Functional profiling of candidate RXLR effector candidates for response in Solanum.

Genetic analysis indicates that Rpi-sto1 interacts with IpiO

To further investigate the genetic and physiological basis of the response to the candidate effectors in relation to late blight resistance in S. stoloniferum 17605-4 [22], we crossed this plant with the susceptible potato genotype RH89-039-16 (RH), which did not respond to any of the candidates (Table 2). We examined the parents and the F1 population for resistance to P. infestans and observed a near 1:1 (19 resistant : 14 susceptible) segregation ratio for all four tested isolates, suggesting inheritance of a dominant R gene which we designated Rpi-sto1 (Table 3A). Subsequent inoculations of Sto17605-4 with eight additional P. infestans isolates always resulted in resistance suggesting a potentially broad-spectrum resistance character of Rpi-sto1 (Table S1). To correlate the resistance to specific effector responses, we assayed the parents and F1 progeny for segregation of displaying hypersensitivity to the candidate effectors identified earlier (Table 2, Figure 1). A 100% correlation between resistance and response to members of the PexRD6 family effector was evident (Table 3B). PexRD6 corresponds to IpiO, a well-studied in planta induced gene, that is expressed at high levels in invading hyphae [23]. IpiO occurs as a small gene family consisting of at least two conserved genes IpiO1 and IpiO2 (PexRD6-2), which differ in only 4 amino acids at the protein level [24]. PexRD6-1 is identical in amino acid sequence to IpiO1, except for a K to N substitution at amino acid 143 [24] (Table S2). IpiO1-K143N and IpiO2 elicited cell death on the 19 resistant F1 plants but not on the 14 susceptible progeny, suggesting that these two are likely Avr candidates for the Rpi-sto1 gene. Sto17605-4 also responded amongst others to PexRD6-3, corresponding to an IpiO variant named IpiO4, which differs in 22 amino acids from IpiO1 (unpublished data, Table S2). However, no response to IpiO4 was observed in the segregating population (Table 3A, B), as was also the case for PexRD13-1, PexRD17-2, PexRD26-2, PexRD46 and PexRD50 underlining the tentative nature of the responses observed in the high throughput PVX-based effector screen, and thus the need for further confirmation using additional experimental systems. The lack of effector response in the F1 progeny could however reflect the presence of specific dominant HR suppressors in RH89-039-16, and further genetic studies are under way. The observed response to PexRD28-1 and PexRD46 did segregate in the population, indicative for a tentative receptor-effector interaction, but this response was as yet not related to resistance (data not shown), and we are further studying the biological relevance of this interaction.

Table 3
Segregation for resistance, ipiO response and genetic markers in the F1 population Sto1705-4×RH.

IpiO is Avr-blb1

We noted that besides Sto17605-4, S. bulbocastanum genotype 8005-8 also responded to IpiO (Table 2). This genotype carries the previously cloned Rpi-blb1 gene [3]. To test whether Rpi-blb1 also recognizes IpiO, we co-expressed IpiO1, IpiO1-K143N, IpiO2, and IpiO4 with Rpi-blb1 in Nicotiana benthamiana leaves using an Agrobacterium tumefaciens transient assay (agroinfiltration). Three days later, hypersensitive cell death was observed in the leaf panels infiltrated with IpiO1, IpiO1-K143N, and IpiO2 (Figure 2A), and Rpi-blb1 but not in the controls. No cell death occurred after co-infiltration of A. tumefaciens strains containing Rpi-blb1 and IpiO4 (Figure S2). This indicates that IpiO1, IpiO1-K143N, or IpiO2 are specifically recognized by Rpi-blb1 suggesting that they have an Avr-blb1 activity. These data also suggest that Rpi-blb1 and Rpi-sto1 recognize the same P. infestans effectors IpiO1, IpiO1-K143N, and IpiO2, but not the sequence divergent IpiO4 (Table 3, Figure 2, Figure S2, Table S2).

Figure 2
Rpi-blb1, Rpi-sto1, and Rpi-pta1 functionally interact with the avirulence protein IpiO.

Exceptionally rapid cloning of Rpi-sto1 and Rpi-pta1 based on Rpi-blb1 orthology and functional equivalence

To further investigate the relationship between Rpi-blb1 and Rpi-sto1, we examined the segregation of the Rpi-blb1-linked marker CT88 [25] in the Sto17605-4×RH population. The CT88 marker co-segregated perfectly with late blight resistance and response to IpiO (Table 3C), suggesting that the two R genes might be orthologous. This prompted us to use PCR to amplify full-length copies of Rpi-blb1 homologues from genomic DNA of Sto17605-4 and S. papita (Pta) 17831-8, another IpiO (PexRD6) responding genotype (data not shown). Initially we used primers designed to amplify only the open reading frames (ORF) of putative Rpi-blb1 orthologues. Amplicons of the expected size were cloned into the binary vector pK7WG2 [26] behind a 35S promoter. For each genotype, cloning and restriction enzyme fingerprinting of 96 amplicons resulted in the identification of four classes of Rpi-blb1 homologues. From each class, one clone was transferred to A. tumefaciens, and co-expressed with IpiO2 in N. benthamiana. One homologue from both species responded to IpiO2, suggesting that Rpi-blb1, and the putative homologues Rpi-sto1, and a protein in Pta17831-8 (named Rpi-pta1) recognize the same P. infestans effector (data not shown).

Sequence analyses showed that the putatively functional homologues Rpi-sto1 and Rpi-pta1 are nearly identical to Rpi-blb1, with only 3 and 5 non-synonymous nucleotide substitutions, respectively (Figure S3). To exclude the possibility that the putative recognition was due to over-expression of the R genes by the 35S promoter, DNA fragments of 6.7 kb were PCR amplified from genomic DNA of Sto17605-4 and Pta17831-8 using PSRF1 and PSRR1 primers that were designed to amplify the functional Rpi-blb1 homologues including their natural transcriptional regulatory elements. Cloning of the larger amplicons into the binary vector pBINPLUS and subsequent sequence analysis, revealed that only one gene sequence per genotype had been amplified, corresponding to the putatively functional orthologues identified previously. Co-expression with IpiO2 in N. benthamiana showed the same response to IpiO2 as observed with the 35S constructs, indicating that Rpi-blb1, Rpi-sto1, and Rpi-pta1 are functionally equivalent (Figure 2A, B, C).

The high conservation of the three genes is remarkable considering that S. stoloniferum and S. papita are phylogenetically distant from S. bulbocastanum. This confirms that Rpi-blb1 is of ancient origin [3] and suggests an essential function of the Rpi-blb1 gene and its orthologs throughout Solanum evolution. Alternatively, interspecific gene transfer through hybridization may have occurred in Mexico, the natural area of origin for all three species [17].

To test whether Rpi-sto1 and Rpi-pta1 confer resistance to P. infestans, we infiltrated entire leaves of N. benthamiana with A. tumefaciens strains carrying Rpi-sto1, Rpi-pta1, Rpi-blb1 and a non-functional R gene as a control, and then challenge-inoculated the leaves with the aggressive P. infestans isolate 90128. A distinct hypersensitive response, similar to the response obtained with Rpi-blb1, appeared at the inoculation sites in Rpi-sto1 and Rpi-pta1 treated leaves. In contrast, control leaves showed water-soaking symptoms followed by extensive P. infestans sporulation. To confirm these transient assay results, we generated stable transformants of potato cultivar Desiree using the pBINPLUS-Rpi-sto1 gene construct, which contains Rpi-sto1 in its natural transcriptional context. Inoculation with P. infestans isolate 90128 on in vitro transplants of primary transformants showed that Rpi-sto1 transformant plantlets remained resistant, whereas Desiree control plantlets started sporulating after 7 days, and turned completely diseased at 14–20 days (Figure 3). The results with the vitro assay were confirmed, and subsequently further expanded using mature greenhouse-grown plants [27]. A total of 40 independent primary transformants were selected and detached leaves were inoculated with P. infestans isolate 90128; leaves from 34 plants remained resistant, whereas those from wildtype Desiree control plants were fully susceptible. Although we did not generate stable transformants with Rpi-pta1, similar results are expected with Rpi-pta1. In summary, the complementation experiments confirm that Rpi-sto1 is a functional late blight R gene.

Figure 3
Complementation of Rpi-sto1 in potato.

Discussion

Our findings suggest that Rpi-sto1, Rpi-pta1, and Rpi-blb1 recognize the same RXLR effectors IpiO1 and IpiO2 and that this triggers resistance to P. infestans. With the identity of Avr-blb1 revealed as IpiO, it now becomes possible to monitor current and future field populations of P. infestans with advanced high throughput DNA fingerprinting techniques to detect the evolution of virulence to Rpi-blb1. Preliminary surveys indicate that IpiO1 and IpiO2 occur in the majority of European and North-American P. infestans isolates analyzed to date (data not shown) confirming the potentially broad nature of the resistance conferred by Rpi-blb1 and its orthologs, and arguing in favor of the deployment and usefulness of this R gene.

Stacking or polyculture of potato cultivars with functionally divergent R genes effective against the majority of current P. infestans isolates are promising strategies for management of late blight [28], [29]. Narrow-spectrum R genes can be classified on the basis of their differential response to pathogen races. However, the finding that three different Solanum species carry functionally equivalent potentially broad-spectrum R genes highlights the importance of profiling R genes for their recognition specificities and indicates that identifying R genes from unrelated Solanum species does not exclude redundancy. Effector profiling is essential for categorizing these potentially broad-spectrum R genes that are key to modern late blight resistance breeding, and enables judicious combinations of functionally complementary R genes. Resources will otherwise be wasted on the cloning and stacking of functionally similar R genes.

Our study demonstrates that Phytophthora effector genomics (effector-omics) greatly accelerates the identification of Avr genes and the cloning of the corresponding late blight R genes, the latter by simplifying the process of positional cloning and enabling rapid functional assays of candidate R genes. Compared to traditional inoculations with P. infestans isolates, the effectors also enabled the dissection of the activities of otherwise indistinguishable R genes into discrete recognition specificities, and moreover helped determine that Rpi-blb1 has two closely related orthologs in S. stoloniferum and S. papita. Of particular interest for plant breeding is that these two species are sexually more compatible with potato, and thus, in addition to the transgenics or cisgenics approaches, our findings enable traditional breeding strategies to introgress the Rpi-blb1 specificity into potato.

Materials and Methods

Solanum and Phytophthora experiments

Solanum plant material is listed in Table 1. Plants were clonally maintained in vitro, and cultured in greenhouses or climate chambers for P. infestans or A. tumefaciens assays, respectively [13]. Phytophthora infestans isolates used in this study are listed in Table S1, and disease testing was performed on detached leaves or in vitro plantlets [27], [30].

PexRD effectors

Candidate RXLR genes were mined mainly from a large collection of 80,000 ESTs covering several P. infestans developmental and infection stages [31]. A collection of 54 nonredundant RXLR effectors was identified and primer pairs based on the mature region of candidate RXLR effectors were designed and used to amplify total DNA from a panel of 9 P. infestans isolates (Table S2). The amplicons were cloned into the pGR106 [32] for intracellular targeting in the plant. Detailed sequences of individual clones are available in Table S2.

In planta expression of PexRD effectors

PVX agroinfection with the 54 PexRD effectors was performed using the binary PVX vector pGR106 [32] in A. tumefaciens strain GV3101 [33] as previously described [13]. Agroinfiltration was performed on N. benthamiana leaves of 4–5 week old plants. Agrobacterium tumefaciens strains GV3101 [33], COR308 [34] and AGL1 [35] containing the standard helper plasmids and, in addition, pGR106-empty, IpiO1-K143N, -IpiO2, -IpiO4, pCB302 [36]-empty, -IpiO1, -IpiO2, -IpiO4, pK7-empty, -IpiO1, -IpiO2, -IpiO4 [26] or pBINPLUS-Rpi-blb1, -sto1, -pta1 or -R3a were cultured, and co-infiltrations were performed as described previously [21].

Genetic mapping

The chromosome VIII specific PCR marker CT88, previously shown to be genetically linked to Rpi-blb1 [3] was tested on genomic DNA of the Sto17605-4×RH population. Following digestion with HinfI, the PCR products were separated on a 1.5% agarose gel and stained with ethidiumbromide. All amplification reactions were performed in a Biometra® T-Gradient or Biometra® Uno-II thermocycler (Westburg, Leusden, the Netherlands).

Cloning of Rpi-sto1 and Rpi-pta1

Primers GWBlb1F (5′-CACCATGTTGTAATTATTGGCGAAC) and ARO518 (5′-GTTGTTATAAGGGTATAAGTGAGC) were designed to amplify the coding region of Rpi-blb1. Primers PSRF1 (5′-TTGTTTCCTGCAGGCTTGCTAATTGAGTGTCTGTT) and PSRR1 (5′-TAATTGGCGCGCCTAGAGAATGACAGAGAATCGAA) were designed to amplify 6.7 kb genomic fragments of Rpi-sto1 or Rpi-pta1, including native transcriptional regulatory elements. Long range PCRs were performed on genomic DNA isolated from Sto17605-4 and Pta17831-8 using Pfu Turbo™ polymerase (Stratagene). Amplicons obtained with the former primer set were cloned into the pENTR directional TOPO® vector according to the manufacturer's instructions (Invitrogen) and subsequently into the binary Gateway® 35S overexpression vector pK7WG2 [26]. The larger amplicons obtained with the latter primer set were digested with SbfI and AscI and subsequently cloned into the binary plasmid pBINPLUS [37] also digested with SbfI and AscI. Based on restriction analysis with several four base cutters, the pENTR dTOPO clones derived from each species could be classified into 4 classes whereas the pBINPLUS clones displayed a single digestion pattern. Several clones from each class were sequenced using a primer walk strategy. Subsequently, all the clones with a unique sequence were transferred to the A. tumefaciens strain COR308 [34] and targeted for functional analysis.

Transient and stable complementation

For transient complementation assays on N. benthamiana, A. tumefaciens strains containing the appropriate plasmids were cultured as described above. Subsequently all fully expanded leaves of 4–5 week old plants were completely infiltrated with MMA culture at an OD of 0.1. Two days post infiltration with A. tumefaciens, detached leaves were inoculated with P. infestans isolate 90128 and infection phenotypes were assessed from 4 to 7 days post inoculation [30]. Inoculation sites displaying a clear hypersensitive response were regarded as incompatible reactions (resistance) whereas those displaying a characteristic water soaking phenotype in combination with significant sporulation of P. infestans were scored as compatible reactions (susceptible).

Stable transformation of cv. Desiree with the binary construct pBINPLUS-Rpi-sto1 was carried out as described previously [38], [39]. A total of 40 independent primary transformants were selected and cultured in the greenhouse and subsequently tested for resistance to P. infestans isolate 90128 using the detached leaf assay [30].

Data deposition

The sequences of Rpi-sto1 and Rpi-pta1 are available in the Genbank nucleotide sequence database, accession EU884421 and EU884422, respectively.

Supporting Information

Figure S1

Quantification of PVX replication upon agroinfection with pGR106-Avr3a in a broad variation of Solanum genotypes. R3a-containing plants include two S. demissum genotypes 17810-01 and -06 from the R3a donor accession CGN17810, the Mastenbroek R3a differential [1], [2], three R3a recombinants SW8537-033, SW8539-004, SW8540-025 [3], and the R3a transformant T68.3-005 in the susceptible S. tuberosum 1029-31 [4], and R3a-transformants T68.4-002 and T68.4-006 in potato cultivar Desirée [4]. R3a-lacking genotypes include the susceptible S. tuberosum cv. Bintje, Desirée, and RH89-039-16. Two-week old potato plantlets were toothpick-inoculated with A. tumefaciens strains containing the empty pGR106, pGR106-Avr3a, or pGR106-Crn2, and a control group was left uninoculated. At 18 dpi young leaves were collected and PVX titers were quantified by ELISA. The results shown are from one experiment. The experiments were repeated several times with the same and with other effectors and plant material and the results were comparable.

(0.11 MB PDF)

Figure S2

Co-expression of Rpi-blb1 with ipiO1, IpiO2 and IpiO4. The complete amino acid sequence of Rpi-blb1 is shown and amino acid residues from Rpi-sto1 or -pta1 that differ from the corresponding residue in Rpi-blb1. The coiled-coil domain is underlined with a dotted line. Conserved motifs in the NBS domain are indicated in lowercase. The regions of the LRRs that correspond to the β-strand/β-turn motif xxLxLxxxx are underlined. An asterisk indicates codons that harbour synonymous nucleotide subsitutions.

(0.32 MB PDF)

Figure S3

Amino acid sequence alignment of Rpi-blb1, Rpi-sto1 and Rpi-pta1

(0.06 MB PDF)

Table S1

Phytophthora infestans isolates used in this study. The virulence spectrum of the P. infestans isolates on Sto17605-4 (Sto), RH89-039-16 (RH), and the R gene differential set R1-R11 [1], [2], the geographic origin, collection year, and provider of the isolates, and the experiment described in this study are presented.

(0.01 MB PDF)

Table S2

RXLR effector candidates used in this study. PexRD, PexRD family members (nr), Agrobacterium tumefaciens clones, known genes, the Phytopthora infestans strain and amino acid sequences are presented. PexRD and A. tumefaciens clones correspond to Table 2.

(0.07 MB PDF)

Acknowledgments

We thank David Baulcombe for providing pGR106, Dirk Budding for disease testing, Estelle Verzaux for genetic mapping, Marcel Visser and Henk Smid for taking care of Solanum plants, Joe Win for bioinformatic predictions of the RXLR sequences, Minkyoung Lee and Thirumala Kanneganti for assistance with the RXLR library, Diane Kinney and Jose Cuervo for support, and Peter van de Vondervoort for ipiO constructs.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This research was financed by WUR Plant Breeding, Wageningen. The Czech University of Agriculture has financially supported PK, and the Center of Biosystems Genomics (CBSG) has financially supported the retrieving of resistant germplasm. CY and SKO were funded by NSF Plant Genome Research Program grant DBI-0211659. SK was supported by the Gatsby Charitable Foundation.

References

1. Müller KO, Black W. Potato breeding for resistance to blight and virus diseases during the last hundred years. Zeitschrift für Pflanzenzüchtung. 1952;31:305–318.
2. Jacobsen E, Schouten HJ. Cisgenesis strongly improves introgression breeding and induced translocation breeding of plants. Trends in Biotechnology. 2007;25:219–223. [PubMed]
3. van der Vossen E, Sikkema A, Hekkert BL, Gros J, Stevens P, et al. An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato. Plant Journal. 2003;36:867–882. [PubMed]
4. Song J, Bradeen JM, Naess SK, Raasch JA, Wielgus SM, et al. Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight. Proc Natl Acad Sci U S A. 2003;100:9128–9133. [PMC free article] [PubMed]
5. van der Vossen EAG, Gros J, Sikkema A, Muskens M, Wouters D, et al. The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring broad-spectrum late blight resistance in potato. Plant Journal. 2005;44:208–222. [PubMed]
6. Application GM field trial. Potato with improved resistance to Phytophthora infestans. BASF Plant Science GmbH 2005
7. Schouten HJ, Krens FA, Jacobsen E. Do cisgenic plants warrant less stringent oversight? Nature biotechnology. 2006;24:753. [PubMed]
8. Kamoun S. Groovy times: filamentous pathogen effectors revealed. Current Opinion in Plant Biology. 2007;10:358–365. [PubMed]
9. Jones JDG, Dangl The plant immune system. Nature. 2006;444:323–329. [PubMed]
10. Birch PRJ, Rehmany AP, Pritchard L, Kamoun S, Beynon JL. Trafficking arms: oomycete effectors enter host plant cells. Trends in Microbiology. 2006;14:8–11. [PubMed]
11. Kamoun S. A catalogue of the effector secretome of plant pathogenic oomycetes. Annu Rev Phytopathol. 2006;44:1–20. [PubMed]
12. Whisson SC, Boevink PC, Moleleki L, Avrova AO, Morales JG, et al. A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature. 2007;450:115–118. [PubMed]
13. Vleeshouwers VGAA, Driesprong JD, Kamphuis LG, Torto-Alalibo T, van 't Slot KAE, et al. Agroinfection-based high throughput screening reveals specific recognition of INF elicitins in Solanum. Molecular Plant Pathology. 2006;7:499–510. [PubMed]
14. Bendahmane A, Querci M, Kanyuka K, Baulcombe DC. Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. Plant Journal. 2000;21:73–81. [PubMed]
15. Riely BK, Martin GB. Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto. Proc Natl Acad Sci U S A. 2001;98:2059–2064. [PMC free article] [PubMed]
16. Hawkes J. London: Belhaven Press; 1990. The potato: evolution, biodiversity, and genetic resources.
17. Spooner DM, Van Den Berg RG, Rodriguez A, Bamberg J, Hijmans RJ, et al. Systemic Botany Monographs. Wild potatoes (Solanum section Petota; Solanaceae) of North and Central America; In: Anderson C, editor. Ann Arbor, Michigan: The American Society of Plant Taxonomists; 2004. p. 209.
18. Spooner DM, Castillo TR. Reexamination of series relationships of South American wild potatoes (Solanaceae: Solanum sect. Petota): evidence from chloroplast DNA restriction site variation. American Journal of Botany. 1997;84:671–685. [PubMed]
19. Bhattacharjee S, Hiller NL, Liolios K, Win J, Kanneganti T-D, et al. The Malarial Host-Targeting Signal Is Conserved in the Irish Potato Famine Pathogen. PLoS Pathogens. 2006;2:e50. [PMC free article] [PubMed]
20. Armstrong MR, Whisson SC, Pritchard L, Bos JIB, Venter E, et al. An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm. Proc Natl Acad Sci U S A. 2005;102:7766–7771. [PMC free article] [PubMed]
21. Bos JIB, Kanneganti T-D, Young C, Cakir C, Huitema E, et al. The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana. Plant Journal. 2006;48:165–176. [PubMed]
22. McKee RK. Identification of R-genes in Solanum stoloniferum. Euphytica. 1962;11:42–46.
23. van West P, de Jong AJd, Judelson HS, Emons AMC, Govers F. The ipiO gene of Phytophthora infestans is highly expressed in invading hyphae during infection. Fungal Genetics and Biology. 1998;23:126–138. [PubMed]
24. Pieterse CMJ, Van West P, Verbakel HM, Brasse PWHM, Van Den Berg Velthuis GCM, et al. Structure and genomic organization of the ipiB and ipiO gene clusters of Phytophthora infestans. Gene. 1994;138:67–77. [PubMed]
25. Wang M, Allefs S, van den Berg RG, Vleeshouwers VGAA, van der Vossen EAG, et al. Allele mining in Solanum: conserved homologues of Rpi-blb1 are identified in Solanum stoloniferum. Theoretical and applied genetics. 2008 DOI 10.1007/s00122-008-0725-3. [PubMed]
26. Karimi M, Inze D, Depicker A. Gateway vectors for Agrobacterium-mediated plant transformation. . Trends in Plant Science. 2002;7:193–195. [PubMed]
27. Huang S, Vleeshouwers VGAA, Visser RGF, Jacobsen E. An accurate in vitro assay for high-throughput disease testing to Phytophthora infestans in potato. Plant Disease. 2005;89:1263–1267.
28. Jones JD. Putting knowledge of plant disease resistance genes to work. Curr Opin Plant Biol. 2001;4:281–287. [PubMed]
29. Jacobsen E, Hutten R. Haase NU, Haverkort AJ, editors. Stacking resistance genes in potato by cisgenesis instead of introgression breeding. Potato developments in a changing Europe: Wageningen Academic Publishers. 2006. pp. 46–57.
30. Vleeshouwers VGAA, van Dooijeweert W, Keizer LCP, Sijpkes L, Govers F, et al. A laboratory assay for Phytophthora infestans resistance in various Solanum species reflects the field situation. European Journal of Plant Pathology. 1999;105:241–250.
31. Randall TA, Dwyer RA, Huitema E, Beyer K, Cvitanich C, et al. Large-scale gene discovery in the oomycete Phytophthora infestans reveals likely components of phytopathogenicity shared with true fungi. Molecular plant microbe interactions. 2005;18:229–243. [PubMed]
32. Lu R, Martin Hernandez AM, Peart JR, Malcuit I, Baulcombe DC. Virus-induced gene silencing in plants. Methods. 2003;30:296–303. [PubMed]
33. Holsters M, Silva B, Van Vliet F, Genetello C, De Block M, et al. The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid. 1980;3:212–230. [PubMed]
34. Hamilton CM, Frary A, Lewis C, Tanksley SD. Stable transfer of intact high molecular weight DNA into plant chromosomes. Proceedings of National Academy of Science USA. 1996;93:9975–9979. [PMC free article] [PubMed]
35. Whitham S, Dinesh-Kumar SP, Choi D, Hehl R, Corr C, et al. The product of the tobacco mosaic virus resistance gene N: similarity to toll and the interleukin-1 receptor. Cell. 1994;78:1101–1115. [PubMed]
36. Xiang C, Han P, Lutziger I, Wang K, Oliver DJ. A mini binary vector series for plant transformation. Plant Mol Biol. 1999;40:711–717. [PubMed]
37. van Engelen FA, Molthoff JW, Conner AJ, Nap J-P, Pereira A, et al. pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Research. 1995;4:288–290. [PubMed]
38. Filati J, Kiser J, Rose R, Comai L. Efficient transfer of a glyphosphate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Biotechnology. 1987;5:726–730.
39. Hoekema A, Huisman MJ, Molendijk L, van den Elzen PJM, Cornelissen BJC. The genetic engineering of two commercial potato cultivars for resistance to potato virus X. Biotechnology. 1989;7:273–278.
40. Torto TA, Li SA, Styer A, Huitema E, Testa A, et al. EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. Genome Research. 2003;13:1675–1685. [PMC free article] [PubMed]
41. Huang S, van der Vossen EAG, Kuang H, Vleeshouwers VGAA, Zhang N, et al. Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato. The Plant Journal. 2005;42:251–261. [PubMed]
42. Jones L, Hamilton AJ, Voinnet O, Thomas CL, Maule AJ, et al. RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell. 1999;11:2291–2301. [PMC free article] [PubMed]

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