A Hitchhiker's guide to the potato wart disease resistance galaxy.

KEY MESSAGE: Two novel major effect loci (Sen4 and Sen5) and several minor effect QTLs for potato wart disease resistance have been mapped. The importance of minor effect loci to bring full resistance to wart disease was investigated. Using the newly identified and known wart disease resistances, a panel of potato breeding germplasm and Solanum wild species was screened. This provided a state-of-the-art "hitch-hikers-guide" of complementary wart disease resistance sources. Potato wart disease, caused by the obligate biotrophic soil-born fungus Synchytrium endobioticum, is the most important quarantine disease of potato. Because of its huge impact on yield, the lack of chemical control and the formation of resting spores with long viability, breeding for resistant varieties combined with strict quarantine measures are the only way to efficiently and durably manage the disease. In this study, we set out to make an inventory of the different resistance sources. Using a Genome-Wide Association Study (GWAS) in the potato breeding genepool, we identified Sen4, associated with pathotypes 2, 6 and 18 resistance. Associated SNPs mapped to the south arm of chromosome 12 and were validated to be linked to resistance in one full-sib population. Also, a bulked segregant analysis combined with a Comparative Subsequence Sets Analysis (CoSSA) resulted in the identification of Sen5, associated with pathotypes 2, 6 and 18 resistance, on the south arm of chromosome 5. In addition to these two major effect loci, the GWAS and CoSSA allowed the identification of several quantitative trait loci necessary to bring full resistance to certain pathotypes. Panels of varieties and Solanum accessions were screened for the presence of Sen1, Sen2, Sen3, Sen4 and Sen5. Combined with pedigree analysis, we could trace back some of these genes to the ancestral resistance donors. This analysis revealed complementary resistance sources and allows elimination of redundancy in wart resistance breeding programs.

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.

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.

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 SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis.

The interface between plants and the environment plays a dual role as a protective barrier as well as a medium for the exchange of gases, water, and nutrients. The primary aerial plant surfaces are covered by a cuticle, acting as the essential permeability barrier toward the atmosphere. It is a heterogeneous layer composed mainly of lipids, namely cutin and intracuticular wax with epicuticular waxes deposited on the surface. We identified an Arabidopsis thaliana activation tag gain-of-function mutant shine (shn) that displayed a brilliant, shiny green leaf surface with increased cuticular wax compared with the leaves of wild-type plants. The gene responsible for the phenotype encodes one member of a clade of three proteins of undisclosed function, belonging to the plant-specific family of AP2/EREBP transcription factors. Overexpression of all three SHN clade genes conferred a phenotype similar to that of the original shn mutant. Biochemically, such plants were altered in wax composition (very long fatty acid derivatives). Total cuticular wax levels were increased sixfold in shn compared with the wild type, mainly because of a ninefold increase in alkanes that comprised approximately half of the total waxes in the mutant. Chlorophyll leaching assays and fresh weight loss experiments indicated that overexpression of the SHN genes increased cuticle permeability, probably because of changes in its ultrastructure. Likewise, SHN gene overexpression altered leaf and petal epidermal cell structure, trichome number, and branching as well as the stomatal index. Interestingly, SHN overexpressors displayed significant drought tolerance and recovery, probably related to the reduced stomatal density. Expression analysis using promoter-beta-glucuronidase fusions of the SHN genes provides evidence for the role of the SHN clade in plant protective layers, such as those formed during abscission, dehiscence, wounding, tissue strengthening, and the cuticle. We propose that these diverse functions are mediated by regulating metabolism of lipid and/or cell wall components.

Activation tagging using the En-I maize transposon system in Arabidopsis.

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays) En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant called thread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that the En-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.