The Synchytrium endobioticum AvrSen1 Triggers a Hypersensitive Response in Sen1 Potatoes While Natural Variants Evade Detection.

Synchytrium endobioticum is an obligate biotrophic fungus of division Chytridiomycota. It causes potato wart disease, has a worldwide quarantine status and is included on the Health and Human Services and United States Department of Agriculture Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host resistance in a set of differential potato varieties. Thus far, 39 pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 (D1) resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum-specific secretome of 14 isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1 (D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response (HR), which cosegregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single-copy gene and encodes a 376-amino-acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.

The Solanum demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties.

KEY MESSAGE: The potato late blight resistance gene R8 has been cloned. R8 is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato. The broad spectrum late blight resistance gene R8 from Solanum demissum was cloned based on a previously published coarse map position on the lower arm of chromosome IX. Fine mapping in a recombinant population and bacterial artificial chromosome (BAC) library screening resulted in a BAC contig spanning 170 kb of the R8 haplotype. Sequencing revealed a cluster of at least ten R gene analogues (RGAs). The seven RGAs in the genetic window were subcloned for complementation analysis. Only one RGA provided late blight resistance and caused recognition of Avr8. From these results, it was concluded that the newly cloned resistance gene was indeed R8. R8 encodes a typical intracellular immune receptor with an N-terminal coiled coil, a central nucleotide binding site and 13 C-terminal leucine rich repeats. Phylogenetic analysis of a set of representative Solanaceae R proteins shows that R8 resides in a clearly distinct clade together with the Sw-5 tospovirus R protein from tomato. It was found that the R8 gene is present in late blight resistant potato varieties from Europe (Sarpo Mira), USA (Jacqueline Lee, Missaukee) and China (PB-06, S-60). Indeed, when tested under field conditions, R8 transgenic potato plants showed broad spectrum resistance to the current late blight population in the Netherlands, similar to Sarpo Mira.

Development of late blight resistant potatoes by cisgene stacking.

BACKGROUND: Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. RESULTS: We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. CONCLUSIONS: We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.

Cloning and characterization of r3b; members of the r3 superfamily of late blight resistance genes show sequence and functional divergence.

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.

Regeneration of pea (Pisum sativum L.) by a cyclic organogenic system.

In a five-step procedure, plants were regenerated from meristematic tissue initiated from nodal tissue in four pea cultivars ('Espace', 'Classic', 'Solara', and 'Puget'). In step 1, stem tissue with one node (1-cm size) was subcultured on medium containing thidiazuron. As a result multiple shoots were produced, appearing normal or swollen at their bases. The multiple shoots were subcultured in the same medium, resulting in the formation of a green hyperhydric tissue in the swollen bases of the multiple shoots, which is fully covered with small buds [bud-containing tissue (BCT)]. In step 2, BCT fragments were isolated and subcultured in the same medium and, as a result, they were able to reproduce themselves in a cyclic fashion. In step 3, subculture of BCT on medium supplemented with a combination of gibberelic acid, 6-benzyladenine and alpha-naphthalene acetic acid (NAA), resulted in the formation of shoots, which were rooted in step 4 on medium supplemented with 0.5 mg/l NAA, indole-3-acetic acid (IAA) or indole-3-butyric acid. In step 5, in vitro plants were transferred to the greenhouse for acclimatisation and further development. The four varieties tested were all able to produce meristematic tissue, suggesting that its production is genotype independent.

Isolation of a gene encoding a copper chaperone for the copper/zinc superoxide dismutase and characterization of its promoter in potato.

Gene expression during the potato (Solanum tuberosum) tuber lifecycle was monitored by cDNA-amplified fragment-length polymorphism, and several differentially expressed transcript-derived fragments were isolated. One fragment, named TDFL431, showed high homology to a copper (Cu) chaperone for Cu/zinc superoxide dismutase (CCS). The Ccs protein is responsible for the delivery of Cu to the Cu/zinc superoxide dismutase enzyme. The potato CCS (StCCS) full-length gene was isolated, and its sequence was compared with CCSs from other species. The promoter region of this gene was isolated, fused to the firefly luciferase coding sequence, and used for transformation of potato plants. The highest level of StCCS-luciferase expression was detected in the cortex of stem (like) tissues, such as stem nodes, stolons, and tubers; lower levels were detected in roots and flowers. The StCCS promoter contains regions highly homologous to several plant cis-acting elements. Three of them are related to auxin response, whereas four others are related to response to various stresses. Induction of the StCCS promoter was analyzed on 18 media, differing in hormone, sugar, and Cu content. StCCS expression was induced by auxin, gibberellins (GA4 + 7), fructose, sucrose, and glucose and was inhibited by relatively high concentrations of Cu.