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-β-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

A Hitchhiker’s guide to the potato wart disease resistance galaxy

International audienceTwo 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 Solanum demissumR8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties

<p> The potato late blight resistance geneR8has been cloned.R8is 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.Abstract: 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.</p

The ROSEA1 and DELILA transcription factors control anthocyanin biosynthesis in Nicotiana benthamiana and Lilium flowers

The activity of anthocyanin biosynthesis genes is regulated at the transcriptional level, thus manipulation of transcription factors (TFs) is an ideal strategy to alter the expression of multiple target genes. In this study, we investigated the effect of introducing ROSEA1 (ROS1, a MYB-type) and DELILA (DEL, a bHLH-type) TFs from snapdragon under control of a flower specific promoter, Floral Binding Protein 1 (FBP1) from petunia into Nicotiana benthamiana flowers and Lilium tepals. The usefulness of the FBP1 promoter was demonstrated by the generation of purplish flowers in otherwise normal-growing plants of N. benthamiana, while the expression by the 35S promoter led to the development of stunted plants with anthocyanins in all parts. N. benthamiana was successfully transformed by ROS1 alone and by a combination of ROS1 + DEL. The observed accumulation of delphinidin corresponded to the expression of NbCHS, NbF3H, NbDFR and NbANS. The effect of ROS1 + DEL on Lilium flower colour was investigated using agroinfiltration. A higher cyanidin accumulation was observed in tepals of the Oriental hybrid lily cv. ‘Perth’ resulting in deeper pink colouration at the infiltrated area. Nevertheless, the introduction of ROS1 + DEL did not produce any phenotypic changes to the white-flowered L. longiflorum cv. ‘Lincoln’ and the white Oriental hybrid lily cv. ‘Rialto’ due to other deficiencies in their anthocyanin biosynthetic pathway. Co-expression of ROS1 + DEL under control of the FBP1 promoter together with active structural anthocyanin biosynthetic genes can result in modification of Lilium flower colour.</p

The Synchytrium endobioticum AvrSen1 triggers a Hypersensitive Response in Sen1 potatoes while natural variants evade detection

Synchytrium endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota. It causes potato wart disease, has a world-wide quarantine status and is included on the HHS and USDA 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. So far, thirty-nine pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 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 fourteen isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1(D1) isolates but absent in others. We discovered a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response, which co-segregated 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. 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

The Synchytrium endobioticum AvrSen1 triggers a Hypersensitive Response in Sen1 potatoes while natural variants evade detection

Synchytrium endobioticum is an obligate biotrophic fungus of the phylum Chytridiomycota. It causes potato wart disease, has a world-wide quarantine status and is included on the HHS and USDA 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. So far, thirty-nine pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 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 fourteen isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1(D1) isolates but absent in others. We discovered a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response, which co-segregated 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. 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

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 PiAVR