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SUMMARY: Phytophthora infestans is a pathogenic oomycete that causes the infamous potato late blight disease. Resistance (R) genes from diverse Solanum species encode intracellular receptors that trigger effective defense responses upon the recognition of cognate RXLR avirulence (Avr) effector proteins. To deploy these R genes in a durable fashion in agriculture, we need to understand the mechanism of effector recognition and the way the pathogen evades recognition. In this study, we cloned 16 allelic variants of the Rpi‐chc1 gene from Solanum chacoense and other Solanum species, and identified the cognate P. infestans RXLR effectors. These tools were used to study effector recognition and co‐evolution. Functional and non‐functional alleles of Rpi‐chc1 encode coiled‐coil nucleotide‐binding leucine‐rich repeat (CNL) proteins, being the first described representatives of the CNL16 family. These alleles have distinct patterns of RXLR effector recognition. While Rpi‐chc1.1 recognized multiple PexRD12 (Avrchc1.1) proteins, Rpi‐chc1.2 recognized multiple PexRD31 (Avrchc1.2) proteins, both belonging to the PexRD12/31 effector superfamily. Domain swaps between Rpi‐chc1.1 and Rpi‐chc1.2 revealed that overlapping subdomains in the leucine‐rich repeat (LRR) domain are responsible for the difference in effector recognition. This study showed that Rpi‐chc1.1 and Rpi‐chc1.2 evolved to recognize distinct members of the same PexRD12/31 effector family via the LRR domain. The biased distribution of polymorphisms suggests that exchange of LRRs during host–pathogen co‐evolution can lead to novel recognition specificities. These insights will guide future strategies to breed durable resistant varieties. Significance statement: In this manuscript the cloning of novel late blight resistance genes and their cognate effectors is described. The mechanism of recognition, host pathogen co‐evolution, and implications for deployment of the knowledge in agriculture are discussed. [ABSTRACT FROM AUTHOR]

Insufficient dietary intake of micronutrients, known as “hidden hunger”, is a devastating global burden, affecting two billion people. Deficiency of folates (vitamin B9), which are known to play a central role in C 1 metabolism, causes birth defects in at least a quarter million people annually. Biofortification to enhance the level of naturally occurring folates in crop plants, proves to be an efficient and cost-effective tool in fighting folate deficiency. Previously, introduction of folate biosynthesis genes GTPCHI and ADCS , proven to be a successful biofortification strategy in rice and tomato, turned out to be insufficient to adequately increase folate levels in potato tubers. Here, we provide a proof of concept that additional introduction of HPPK/DHPS and/or FPGS , downstream genes in mitochondrial folate biosynthesis, enables augmentation of folates to satisfactory levels (12-fold) and ensures folate stability upon long-term storage of tubers. In conclusion, this engineering strategy can serve as a model in the creation of folate-accumulating potato cultivars, readily applicable in potato-consuming populations suffering from folate deficiency. [ABSTRACT FROM AUTHOR]

Late blight, caused by Phytophthora infestans , remains the most devastating disease in potato resulting in economic costs that sum up 5.2 billion euros, globally. The use of resistant varieties is a powerful, viable and environmentally friendly alternative or supplement for the current, commonly deployed chemical control strategies. In this study, we set out to improve the susceptible potato variety Desiree by transformation with single or multiple late blight ( R ) resistance genes. Rpi-sto1 , Rpi-vnt1.1 and a stack of Rpi-sto1 : Rpi-vnt1.1 : Rpi-blb3, were transformed and eight, eight, and ten independent transformants (events) respectively, were selected because of absence of vector backbone, low T-DNA copy number, responsiveness to the cognate Avr effectors, P. infestans resistance in detached leaf assays (DLAs) and preliminary field experiments. The performance of the selected events was studied under field conditions in The Netherlands and Belgium, after P. infestans inoculation and/or to natural late blight infection during two consecutive growing seasons. All selected events were more resistant than the non-transformed susceptible reference clone. The different individual R genes, however, contributed to different levels of resistance. The selected events were also compared to conventionally bred late blight resistant varieties with (partially) known R gene content. Generally, it was found that plants with single R genes showed a lower level of resistance than plants with R gene stacks. Only the events harbouring three late blight R genes remained unaffected until the end of the growing season, in both locations and in both growing seasons. [ABSTRACT FROM AUTHOR]

Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure-function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating. [ABSTRACT FROM AUTHOR]

Comparative genomics provides a tool to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. In this report, we adopted a local RGA approach using genomic information from the model Solanaceous plant tomato to isolateR3a, a potato gene that confers race-specific resistance to the late blight pathogenPhytophthora infestans.R3ais a member of theR3complex locus on chromosome 11. Comparative analyses of theR3complex locus with the correspondingI2complex locus in tomato suggest that this is an ancient locus involved in plant innate immunity against oomycete and fungal pathogens. However, theR3complex locus has evolved after divergence from tomato and the locus has experienced a significant expansion in potato without disruption of the flanking colinearity. This expansion has resulted in an increase in the number ofRgenes and in functional diversification, which has probably been driven by the co-evolutionary history betweenP. infestansand its host potato. Constitutive expression was observed for theR3agene, as well as some of its paralogues whose functions remain unknown. [ABSTRACT FROM AUTHOR]

Potato (Solanum tuberosum) is an important food crop consumed all over the world, but it is generally sensitive to drought conditions. One of the major physiological processes affected by drought stress is carbon partitioning: the plant's choice of where to allocate its photoassimilates. Our aim was to investigate the molecular factors and possible bottlenecks affecting carbon partitioning during drought. We studied potato cultivars with contrasting drought responses in the greenhouse in the years 2013–2015, and further investigated the expression of genes involved in carbon partitioning and metabolite levels. Our results indicate that one of the most severe effects of drought stress on potato is the arrest of stolon differentiation and formation of tubers. We also identified some physiological traits like stomatal conductance and chlorophyll content as affecting carbon assimilation, partitioning and eventual tuber yield. The gene expressions and biochemical analyses highlight the various tissues prioritized by the plant for assimilate transport during drought stress, and give indications of what distinguishes drought tolerance and sensitivity of cultivated potato. Some of the key genes studied (like Sucrose synthase and Sucrose transporters) may be inclusive breeding targets for drought tolerance in potato. • Sugar export from source leaves, not its availability, is key in drought tolerance. • Tuber initiation arrest can alter carbon transport to underground tissues. • Starch storage in leaves is a limitation to potato tuber yield under drought. [ABSTRACT FROM AUTHOR]

Highlights • Increasing the daily light integral (DLI) accelerates flower bud appearance in potato. • Increasing DLI decreases the number of leaves formed before the inflorescence. • Faster flowering under high DLI is not controlled by flowering time gene StSP3D. Abstract Little is known on the environmental control of potato flowering. With recent developments in potato breeding, and the growing interest in true potato seed production, more knowledge on potato flowering is required. This research aims to elucidate the effect of the daily light integral (DLI: the accumulated light throughout a day) on potato flower initiation time and investigate which mechanisms underlie this control. We grew potato plants in climate chambers to compare flower initiation under different DLIs in short and long days. We measured the time until the first appearance of the flower buds and the number of leaves formed before the inflorescence. Furthermore, gene expression changes of the potato flowering time gene StSP3D were measured, and potato plants silenced in StSP3D were used to determine whether DLI mediated flowering time was regulated through StSP3D. Additionally, we determined sucrose and starch concentrations and measured the transcription levels of StTPS1 , a gene involved in sugar mediated flowering control. Increasing DLI clearly accelerated flowering in potato. The role of carbohydrates (sucrose and starch) and StTPS1 in DLI-accelerated flowering was inconclusive. Although StSP3D was upregulated under high DLI, transgenic lines silenced in StSP3D also showed accelerated flowering under higher DLIs. We therefore conclude that high DLI accelerates potato flowering and this acceleration happens independently of StSP3D upregulation. [ABSTRACT FROM AUTHOR]

Abstract: GA biosynthesis and catabolism has been shown to play an important role in regulating tuberization in potato. Active GAs are inactivated in the stolon tips shortly after induction to tuberization. Overexpression of a GA inactivation gene results in an earlier tuberization phenotype, while reducing expression of the same gene results in delayed tuberization. In addition, overexpression of genes involved in GA biosynthesis results in delayed tuberization, while decreased expression of those genes results in earlied tuberization. The final step in GA biosynthesis is catalysed by StGA3ox1 and StGA3ox2 activity, that convert inactive forms of GA into active GA1 and GA4. In this study we cloned StGA3ox2 gene in an RNAi construct and used this construct to transform potato plants. The StGA3ox2 silenced plants were smaller and had shorter internodes. In addition, we assayed the concentrations of various GAs in the transgenic plants and showed an altered GA content. No difference was observed on the time point of tuber initiation. However, the transgenic clones had increased number of tubers with the same yield, resulting in smaller average tuber weight. In addition, we cloned the promoter of StGA3ox2 to direct expression of the GUS reporter gene to visualize the sites of GA biosynthesis in the potato plant. Finally, we discuss how changes of several GA levels can have an impact on shoot, stolon and tuber development, as well as the possible mechanisms that mediate feed-forward and feed-back regulation loops in the GA biosynthetic pathway in potato. [Copyright &y& Elsevier]

Abstract: In the post-genomic era, high-throughput technologies have led to data collection in fields like transcriptomics, metabolomics and proteomics and, as a result, large amounts of data have become available. However, the integration of these ∼omics data sets in relation to phenotypic traits is still problematic in order to advance crop breeding. We have obtained population-wide gene expression and metabolite (LC–MS) data from tubers of a diploid potato population and present a novel approach to study the various ∼omics datasets to allow the construction of networks integrating gene expression, metabolites and phenotypic traits. We used Random Forest regression to select subsets of the metabolites and transcripts which show association with potato tuber flesh color and enzymatic discoloration. Network reconstruction has led to the integration of known and uncharacterized metabolites with genes associated with the carotenoid biosynthesis pathway. We show that this approach enables the construction of meaningful networks with regard to known and unknown components and metabolite pathways. [Copyright &y& Elsevier]

Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis. [Copyright &y& Elsevier]

The potato tuber life cycle involves several developmental stages including tuberisation, tuber growth, dormancy and sprouting. Gene expression during the potato tuber life cycle has been monitored using a RNA fingerprinting technique termed cDNA-AFLP. The expression profile of the nearly 2000 transcript derived fragments (TDFs) was analysed and general conclusions concerning the different stages of tuber life cycle and tissue specificity were drawn. Comparison of 116 TDFs isolated and sequenced to the NCBI databases indicated that most of the genes expressed during tuber life cycle are involved in defence, stress, storage and signal transduction pathways. In order to identify time and tissue specific regulatory elements, the TDFs were further screened for their time and tissue specific expression and the putative promoters corresponding to the twelve differentially expressed TDFs were isolated. Comparison of the 12 upstream sequences to all described cis-acting elements resulted in the identification of 12 know cis-acting elements: two general cis-elements, one enhancer and nine specific boxes. These specific cis-elements have been characterised in other plant species and they respond to light, sugars and hormones such as gibberellins, auxins and abscisic acid (ABA). All these environmental factors play an important role during the potato tuber life cycle. [Copyright &y& Elsevier]

An RNA fingerprinting study of potato leaf protoplasts was performed to explore its suitability for identifying candidate genes involved in primary cell wall biosynthesis. Microscopic analysis, using calcofluor white to stain cellulose, showed that the protoplasts generated a new cell wall in the first 18 h after transfer to a culture medium. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) was used to visualise differential gene expression at five distinct time-points within these first 18 h. In vitro plants (with and without exposure to severe physical damage) served as controls. Around 8500 transcript derived fragments (TDFs) were visualised which showed varying expression patterns in the protoplasts and controls. In total 156 TDFs were isolated, sequenced and used to search for homologies. Over 50% of these TDFs showed homology to described genes, involved in several general plant processes. However, only one cell wall related TDF (a pectin esterase) was found. Our results showed that even though the protoplasts actively regenerate a new cell wall, this did not result in highly increased expression of genes involved in cell wall biosynthesis or modification. [Copyright &y& Elsevier]

Summary Potato plants form tuberous storage organs on underground modified stems called stolons. Tubers are rich in starch, proteins, and other important nutrients, making potato one of the most important staple food crops. The timing of tuber development in wild potato is regulated by day length through a mechanism that is closely related to floral transition [ 1, 2 ]. Tuberization is also known to be regulated by the availability of assimilates, in particular sucrose, the transported form of sugar, required for starch synthesis. During the onset of tuber development, the mode of sucrose unloading switches from apoplastic to symplastic [ 3 ]. Here, we show that this switch may be mediated by the interaction between the tuberization-specific FT homolog StSP6A and the sucrose efflux transporter StSWEET11 [ 4 ]. The binding of StSP6A to StSWEET11 blocked the leakage of sucrose to the apoplast, and is therefore likely to promote symplastic sucrose transport. The direct physical interaction between StSWEET11 and StSP6A proteins represents a link between the sugar and photoperiodic pathways for the regulation of potato tuber formation. Our data suggest that a previously undiscovered function for the FT family of proteins extends their role as mobile signals to mediators of source-sink partitioning, opening the possibility for modifying source-sink interactions. Graphical Abstract Highlights • Tuber formation is dependent on the link between photoperiod and sugar transport • Potato FT-like tuberigen interacts with a sugar transporter to block sucrose leakage • StSWEET11-StSP6A interaction promotes symplastic transport of sucrose Abelenda et al. show that a tuber-specific, FT-like protein interacts with a SWEET sugar efflux transporter to block sucrose leakage into the apoplast. This work helps to explain the redirection of assimilate flow to developing sink organs and opens the possibility of engineering source-sink relationships. [ABSTRACT FROM AUTHOR]