Vivianne G. A. A. Vleeshouwers

Resistance of Nicotiana benthamiana to Phytophthora infestans Is Mediated by the Recognition of the Elicitor Protein INF1

Phytophthora infestans, the agent of potato and tomato late blight disease, produces a 10-kD extracellular protein, INF1 elicitin. INF1 induces a hypersensitive response in a restricted number of plants, particularly those of the genus Nicotiana. In virulence assays with different P. infestans isolates, five Nicotiana species displayed resistance responses. In all of the interactions, after inoculation with P. infestans zoospores, penetration of an epidermal cell was observed, followed by localized necrosis typical of a hypersensitive response. To determine whether INF1 functions as an avirulence factor in these interactions, we adopted a gene-silencing strategy to inhibit INF1 production. Several transformants deficient in inf1 mRNA and INF1 protein were obtained. These strains remained pathogenic on host plants. However, in contrast to the wild-type and control transformant strains, INF1-deficient strains induced disease lesions when inoculated on N. benthamiana. These results demonstrate that the elicitin INF1 functions as an avirulence factor in the interaction between N. benthamiana and P. infestans.

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.

Understanding and Exploiting Late Blight Resistance in the Age of Effectors

Potato (Solanum tuberosum) is the worlds third-largest food crop. It severely suffers from late blight, a devastating disease caused by Phytophthora infestans. This oomycete pathogen secretes host-translocated RXLR effectors that include avirulence (AVR) proteins, which are targeted by resistance (R) proteins from wild Solanum species. Most Solanum R genes appear to have coevolved with P. infestans at its center of origin in central Mexico. Various R and Avr genes were recently cloned, and here we catalog characterized R-AVR pairs. We describe the mechanisms that P. infestans employs for evading R protein recognition and discuss partial resistance and partial virulence phenotypes in the context of our knowledge of effector diversity and activity. Genome-wide catalogs of P. infestans effectors are available, enabling effectoromics approaches that accelerate R gene cloning and specificity profiling. Engineering R genes with expanded pathogen recognition has also become possible. Importantly, monitoring effector allelic diversity in pathogen populations can assist in R gene deployment in agriculture.

The hypersensitive response is associated with host and nonhost resistance to Phytophthora infestans

Abstract. The interaction between Phytophthora infestans (Mont.) de Bary and Solanum was examined cytologically using a diverse set of wild Solanum species and potato (S. tuberosum L.) cultivars with various levels of resistance to late blight. In wild Solanum species, in potato cultivars carrying known resistance (R) genes and in nonhosts the major defense reaction appeared to be the hypersensitive response (HR). In fully resistant Solanum species and nonhosts, the HR was fast and occurred within 22 h. This resulted in the death of one to three cells. In partially resistant clones, the HR was induced between 16 and 46 h, and resulted in HR lesions consisting of five or more dead cells, from which hyphae were occasionally able to escape to establish a biotrophic interaction. These results demonstrate the quantitative nature of the resistance to P. infestans. The effectiveness of the HR in restricting growth of the pathogen differed considerably between clones and correlated with resistance levels. Other responses associated with the defense reaction were deposition of callose and extracellular globules containing phenolic compounds. These globules were deposited near cells showing the HR, and may function in cell wall strengthening.

Resistance to oomycetes: a general role for the hypersensitive response?

Oomycete plant pathogens, such as Phytophthora, downy mildews and Pythium, have devastating disease effects on numerous crop and ornamental plants. Various types of genetic resistance to oomycetes occur in plants, and can be determined at the subspecific or varietal level (race or cultivar-specific resistance), or at the species or genus level (nonhost resistance). In addition, resistance might be a quantitative phenotype (partial resistance). Resistance reactions are often associated with the hypersensitive response - a programed cell death pathway. Recent advances in the genetic, biochemical and cytological characterization of disease resistance suggests that the hypersensitive response is associated with all forms of resistance to Phytophthora and downy mildews. Identification of the resistance genes involved in nonhost and partial resistance to oomycetes remains an important challenge.

A gene encoding a protein elicitor of Phytophthora infestans is down-regulated during infection of potato.

Most species of the genus Phytophthora produce 10-kDa extracellular protein elicitors, collectively termed elicitins. Elicitins induce hypersensitive response in a restricted number of plants, particularly in the genus Nicotiana within the Solanaceae family. A cDNA encoding INF1, the major secreted elicitin of Phytophthora infestans, a pathogen of solanaceous plants, was isolated and characterized. The expression of the corresponding inf1 gene during the disease cycle of P. infestans was analyzed. inf1 was shown to be expressed in mycelium grown in various culture media, whereas it was not expressed in sporangiospores, zoospores, cysts, and germinating cysts. In planta, during infection of potato, particularly during the biotrophic stage, expression of inf1 was down-regulated compared to in vitro. The highest levels of expression of inf1 were observed in in vitro grown mycelium and in late stages of infection when profuse sporulation and leaf necrosis occur. The potential role of INF1 as an elicitor in interactions between P. infestans and Solanum species was investigated. Nineteen lines, representing nine solanaceous species with various levels of resistance to P. infestans, were tested for response to an Escherichia coli expressed INF1. Within the genus Solanum, resistance to P. infestans did not appear to be mediated by a defense response elicited by INF1. However, INF1 recognition could be a component of nonhost resistance of tobacco to P. infestans.

Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

Pest and pathogen losses jeopardise global food security and ever since the 19th century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

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.

A Laboratory Assay for Phytophthora infestans Resistance in Various Solanum Species Reflects the Field Situation

Physiological and molecular research on resistance responses of Solanum tuberosum cultivars and partially resistant Solanum species to Phytophthora infestans requires a reliable resistance test that can be used in the laboratory. Laboratory tests performed on detached leaves and intact plants were compared with field tests for similarity of late blight reactions. Detached leaves from field-grown plants were as resistant as detached leaves from climate chamber-grown plants when challenged with P. infestans. However, detached leaves incubated in covered trays at high relative humidity were more susceptible than detached leaves kept in open trays or leaves on intact plants. The incubation conditions of detached leaves in covered trays rather than detachment itself appeared to affect the resistance expression. Detached leaves of some wild Solanum genotypes became partially infected, whereas intact plants were completely resistant when inoculated. Inoculation of leaves on intact plants, however, resulted in lower infection efficiencies. These limitations should be taken into account when choosing the appropriate inoculation method for specific purposes. For resistance screening, laboratory tests proved to be a good alternative for field tests. The ranking of resistance levels for twenty plant genotypes was similar under laboratory and field conditions.

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.