The phased Solanum okadae genome and Petota pangenome analysis of 23 other potato wild relatives and hybrids.

Potato is an important crop in the genus Solanum section Petota. Potatoes are susceptible to multiple abiotic and biotic stresses and have undergone constant improvement through breeding programs worldwide. Introgression of wild relatives from section Petota with potato is used as a strategy to enhance the diversity of potato germplasm. The current dataset contributes a phased genome assembly for diploid S. okadae, and short read sequences and de novo assemblies for the genomes of 16 additional wild diploid species in section Petota that were noted for stress resistance and were of interest to potato breeders. Genome sequence data for three additional genomes representing polyploid hybrids with cultivated potato, and an additional genome from non-tuberizing S. etuberosum, which is outside of section Petota, were also included. High quality short reads assemblies were achieved with genome sizes ranging from 575 to 795 Mbp and annotations were performed utilizing transcriptome sequence data. Genomes were compared for presence/absence of genes and phylogenetic analyses were carried out using plastome and nuclear sequences.

Diversity of the Rysto gene conferring resistance to potato virus Y in wild relatives of potato.

BACKGROUND: Potato virus Y (PVY) is among the economically most damaging viral pathogen in production of potato (Solanum tuberosum) worldwide. The gene Rysto derived from the wild potato relative Solanum stoloniferum confers extreme resistance to PVY. RESULTS: The presence and diversity of Rysto were investigated in wild relatives of potato (298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species) using PacBio amplicon sequencing. A total of 55 unique Rysto-like sequences were identified in 72 genotypes representing 12 accessions of 10 Solanum species and six resistant controls (potato cultivars Alicja, Bzura, Hinga, Nimfy, White Lady and breeding line PW363). The 55 Rysto-like sequences showed 89.87 to 99.98% nucleotide identity to the Rysto reference gene, and these encoded in total 45 unique protein sequences. While Rysto-like26 identified in Alicja, Bzura, White Lady and Rysto-like16 in PW363 encode a protein identical to the Rysto reference, the remaining 44 predicted Rysto-like proteins were 65.93 to 99.92% identical to the reference. Higher levels of diversity of the Rysto-like sequences were found in the wild relatives of potato than in the resistant control cultivars. The TIR and NB-ARC domains were the most conserved within the Rysto-like proteins, while the LRR and C-JID domains were more variable. Several Solanum species, including S. antipoviczii and S. hougasii, showed resistance to PVY. This study demonstrated Hyoscyamus niger, a Solanaceae species distantly related to Solanum, as a host of PVY. CONCLUSIONS: The new Rysto-like variants and the identified PVY resistant potato genotypes are potential resistance sources against PVY in potato breeding. Identification of H. niger as a host for PVY is important for cultivation of this plant, studies on the PVY management, its ecology, and migrations. The amplicon sequencing based on PacBio SMRT and the following data analysis pipeline described in our work may be applied to obtain the nucleotide sequences and analyze any full-length genes from any, even polyploid, organisms.

Characterisation and mapping of a Globodera pallida resistance derived from the wild potato species Solanum spegazzinii.

KEY MESSAGE: A new resistance locus acting against the potato cyst nematode Globodera pallida was mapped to chromosome VI in the diploid wild potato species Solanum spegazzinii CPC 7195. The potato cyst nematodes (PCN) Globodera pallida and Globodera rostochiensis are economically important potato pests in almost all regions where potato is grown. One important management strategy involves deployment through introgression breeding into modern cultivars of new sources of naturally occurring resistance from wild potato species. We describe a new source of resistance to G. pallida from wild potato germplasm. The diploid species Solanum spegazzinii Bitter accession CPC 7195 shows resistance to G. pallida pathotypes Pa1 and Pa2/3. A cross and first backcross of S. spegazzinii with Solanum tuberosum Group Phureja cultivar Mayan Gold were performed, and the level of resistance to G. pallida Pa2/3 was determined in progeny clones. Bulk-segregant analysis (BSA) using generic mapping enrichment sequencing (GenSeq) and genotyping-by-sequencing were performed to identify single-nucleotide polymorphisms (SNPs) that are genetically linked to the resistance, using S. tuberosum Group Phureja clone DM1-3 516 R44 as a reference genome. These SNPs were converted into allele-specific PCR assays, and the resistance was mapped to an interval of roughly 118 kb on chromosome VI. This newly identified resistance, which we call Gpa VIlspg, can be used in future efforts to produce modern cultivars with enhanced and broad-spectrum resistances to the major pests and pathogens of potato.

StoMYB41 positively regulates the Solanum torvum response to Verticillium dahliae in an ABA dependent manner.

MYB transcription factor despite their solid involvement in growth are potent regulator of plant stress response. Herein, we identified a MYB gene named as StoMYB41 in a wild eggplant species Solanum torvum. The expression level of StoMYB41 was higher in root than the tissues including stem, leaf, and seed. It induced significantly by Verticillium dahliae inoculation. StoMYB41 was localized in the nucleus and exhibited transcriptional activation activity. Silencing of StoMYB41 enhanced susceptibility of Solanum torvum against Verticillium dahliae, accompanied by higher disease index. The significant down-regulation of resistance marker gene StoABR1 comparing to the control plants was recorded in the silenced plants. Moreover, transient expression of StoMYB41 could trigger intense hypersensitive reaction mimic cell death, darker DAB and trypan blue staining, higher ion leakage, and induced the expression levels of StoABR1 and NbDEF1 in the leaves of Solanum torvum and Nicotiana benthamiana. Taken together, our data indicate that StoMYB41 acts as a positive regulator in Solanum torvum against Verticillium wilt.

Chromosome-level genome assembly and annotation of the prickly nightshade Solanum rostratum Dunal.

The prickly nightshade Solanum rostratum, an annual malignant weed, is native to North America and has globally invaded 34 countries, causing serious threats to ecosystems, agriculture, animal husbandry, and human health. In this study, we constructed a chromosome-level genome assembly and annotation of S. rostratum. The contig-level genome was initially assembled in 898.42 Mb with a contig N50 of 62.00 Mb from PacBio high-fidelity reads. With Hi-C sequencing data scaffolding, 96.80% of the initially assembled sequences were anchored and orientated onto 12 pseudo-chromosomes, generating a genome of 869.69 Mb with a contig N50 of 72.15 Mb. We identified 649.92 Mb (72.26%) of repetitive sequences and 3,588 non-coding RNAs in the genome. A total of 29,694 protein-coding genes were predicted, with 28,154 (94.81%) functionally annotated genes. We found 99.5% and 91.3% complete embryophyta_odb10 genes in the pseudo-chromosomes genome and predicted gene datasets by BUSCO assessment. The present genomic resource provides essential information for subsequent research on the mechanisms of environmental adaptation of S. rostratum and host shift in Colorado potato beetles.

Transcriptome-Assisted SNP Marker Discovery for Phytophthora infestans Resistance in Solanum lycopersicum L.

Late blight, caused by oomycetes Phytophthora infestans is one of the most challenging fungal diseases to manage in tomato plants (Solanum lycopersicum L.). Toward managing the disease, conventional breeding has successfully introgressed genetic loci conferring disease resistance from various wild relatives of tomato into commercial varieties. The cataloging of disease-associated SNP markers and a deeper understanding of disease-resistance mechanisms are needed to keep up with the demand for commercial varieties resistant against emerging pathogen strains. To this end, we performed transcriptome sequencing to evaluate the gene expression dynamics of tomato varieties, resistant and susceptible to Phytophthora infection. Further integrating the transcriptome dataset with large-scale public genomic data of varieties with known disease phenotypes, a panel of single nucleotide polymorphism (SNP) markers correlated with disease resistance was identified. These SNPs were then validated on 31 lines with contrasting phenotypes for late blight. The identified SNPs are located on genes coding for a putative cysteine-rich transmembrane module (CYSTM), Solyc09g098310, and a nucleotide-binding site-leucine-rich repeat protein, Solyc09g098100, close to the well-studied Ph-3 resistance locus known to have a role in plant immunity against fungal infections. The panel of SNPs generated by this study using transcriptome sequencing showing correlation with disease resistance across a broad set of plant material can be used as markers for molecular screening in tomato breeding.

Systematic Investigation of TCP Gene Family: Genome-Wide Identification and Light-Regulated Gene Expression Analysis in Pepino (Solanum Muricatum).

Plant-specific transcription factors such as the TCP family play crucial roles in light responses and lateral branching. The commercial development of S. muricatum has been influenced by the ease with which its lateral branches can be germinated, especially under greenhouse cultivation during the winter with supplemented LED light. The present study examined the TCP family genes in S. muricatum using bioinformatics analysis (whole-genome sequencing and RNA-seq) to explore the response of this family to different light treatments. Forty-one TCP genes were identified through a genome-wide search; phylogenetic analysis revealed that the CYC/TB1, CIN and Class I subclusters contained 16 SmTCP, 11 SmTCP and 14 SmTCP proteins, respectively. Structural and conserved sequence analysis of SmTCPs indicated that the motifs in the same subcluster were highly similar in structure and the gene structure of SmTCPs was simpler than that in Arabidopsis thaliana; 40 of the 41 SmTCPs were localized to 12 chromosomes. In S. muricatum, 17 tandem repeat sequences and 17 pairs of SmTCP genes were found. We identified eight TCPs that were significantly differentially expressed (DETCPs) under blue light (B) and red light (R), using RNA-seq. The regulatory network of eight DETCPs was preliminarily constructed. All three subclusters responded to red and blue light treatment. To explore the implications of regulatory TCPs in different light treatments for each species, the TCP regulatory gene networks and GO annotations for A. thaliana and S. muricatum were compared. The regulatory mechanisms suggest that the signaling pathways downstream of the TCPs may be partially conserved between the two species. In addition to the response to light, functional regulation was mostly enriched with auxin response, hypocotyl elongation, and lateral branch genesis. In summary, our findings provide a basis for further analysis of the TCP gene family in other crops and broaden the functional insights into TCP genes regarding light responses.

Contribution of a WRKY Transcription Factor, ShWRKY81, to Powdery Mildew Resistance in Wild Tomato.

Tomato powdery mildew, caused by Oidium neolycopersici, is a destructive fungal disease that damages almost all of the aerial parts of tomato, causing devastating losses in tomato production worldwide. WRKY transcription factors are key regulators of plant immunity, but the roles of ShWRKYs in wild tomato Solanum habrochaites LA1777 against O. neolycopersici still remain to be uncovered. Here, we show that ShWRKY81 is an important WRKY transcription factor from wild tomato Solanum habrochaites LA1777, contributing to plant resistance against O. neolycopersici. ShWRKY81 was isolated and identified to positively modulate tomato resistance against On-Lz. The transient overexpression of the ShWRKY81-GFP (green fluorescent protein) fusion protein in Nicotiana benthamiana cells revealed that ShWRKY81 was localized in the nucleus. ShWRKY81 responded differentially to abiotic and biotic stimuli, with ShWRKY81 mRNA accumulation in LA1777 seedlings upon On-Lz infection. The virus-induced gene silencing of ShWRKY81 led to host susceptibility to On-Lz in LA1777, and a loss of H2O2 formation and hypersensitive response (HR) induction. Furthermore, the transcripts of ShWRKY81 were induced by salicylic acid (SA), and ShWRKY81-silenced LA1777 seedlings displayed decreased levels of the defense hormone SA and SA-dependent PRs gene expression upon On-Lz infection. Together, these results demonstrate that ShWRKY81 acts as a positive player in tomato powdery mildew resistance.

Evidence for Cardiac Glycosides in Foliage of Colorado Potato Beetle-Resistant Solanum okadae.

Leptinotarsa decemlineata, the Colorado potato beetle (CPB), is a herbivore that primarily feeds on Solanum foliage and is a global pest of the potato agricultural industry. Potato breeding through cross-hybridization with CPB-resistant wild relatives is used for genetic improvement. The wild species Solanum okadae was demonstrated to deter CPB feeding in choice and no choice feeding assays. Liquid chromatography-mass spectrometry (LC-MS) was used for comparative metabolite profiling between S. okadae and CPB-susceptible domesticated potato variety, Solanum tuberosum cv. Shepody. Major foliar metabolites detected were steroidal glycoalkaloids (SGAs) with tomatine and dehydrotomatine produced in S. okadae and solanine and chaconine in S. tuberosum cv. Shepody. Cardiac glycosides were also detected in the foliar metabolite profile of S. okadae but not S. tuberosum cv. Shepody. This class of plant compounds have known insecticidal activity through inhibition of animal Na+/K+ ATPase. Thin-layer chromatography (TLC) separation of foliar extracts also provided evidence for cardiac glycosides in S. okadae. Cardiac glycosides are known inhibitors of Na+/K+ ATPase, and foliar extracts from S. okadae (OKA15), but not S. tuberosum cv. Shepody, were able to inhibit the Na+/K+ ATPase of CPB. These findings suggest a novel mechanism of plant resistance against CPB involving production of cardiac glycosides in S. okadae.

A potato late blight resistance gene protects against multiple Phytophthora species by recognizing a broadly conserved RXLR-WY effector.

Species of the genus Phytophthora, the plant killer, cause disease and reduce yields in many crop plants. Although many Resistance to Phytophthora infestans (Rpi) genes effective against potato late blight have been cloned, few have been cloned against other Phytophthora species. Most Rpi genes encode nucleotide-binding domain, leucine-rich repeat-containing (NLR) immune receptor proteins that recognize RXLR (Arg-X-Leu-Arg) effectors. However, whether NLR proteins can recognize RXLR effectors from multiple Phytophthora species has rarely been investigated. Here, we identified a new RXLR-WY effector AVRamr3 from P. infestans that is recognized by Rpi-amr3 from a wild Solanaceae species Solanum americanum. Rpi-amr3 associates with AVRamr3 in planta. AVRamr3 is broadly conserved in many different Phytophthora species, and the recognition of AVRamr3 homologs by Rpi-amr3 activates resistance against multiple Phytophthora pathogens, including the tobacco black shank disease and cacao black pod disease pathogens P. parasitica and P. palmivora. Rpi-amr3 is thus the first characterized resistance gene that acts against P. parasitica or P. palmivora. These findings suggest a novel path to redeploy known R genes against different important plant pathogens.

A de novo genome assembly of Solanum verrucosum Schlechtendal, a Mexican diploid species geographically isolated from other diploid A-genome species of potato relatives.

There are over 100 known species of cultivated potatoes and their wild relatives. Many of these species, including cultivated potatoes, share the A genome; these species are mainly distributed in South America and are reproductively isolated from Mexican diploid species. The only diploid A-genome species distributed in Mexico is Solanum verrucosum Schlechtendal, which is also a maternal progenitor of Mexican polyploid species. In this study, we constructed a high-quality de novo assembly of the S. verrucosum genome using PacBio long-read sequencing and Hi-C scaffolding technologies. A monohaploid clone (2n = x = 12) of S. verrucosum was used to reduce assembly difficulty due to the heterozygous nature of the species. The final sequence assembly consisted of 780.2 Mb of sequence, 684.0 Mb of which were anchored to the 12 chromosomes, with a scaffold N50 of 55.2 Mb. Putative centromeres were identified using publicly available data obtained via chromatin immunoprecipitation sequencing against a centromere-specific histone 3 protein. Transposable elements accounted for approximately 61.8% (482.1 Mb) of the genome, and 46,904 genes were functionally annotated. High gene synteny and similarity were revealed among the genomes of S. verrucosum, Solanum commersonii, Solanum chacoense, Solanum phureja, Solanum tuberosum, and Solanum lycopersicum. The reference-quality S. verrucosum genome will provide new insights into the evolution of Mexican polyploid species and contribute to potato breeding programs.

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Size-dependent sex allocation in Solanum lycocarpum St. Hil. (Solanaceae) / Alocação de sexo dependente de tamanho em Solanum lycocarpum St. Hil. (Solanaceae)

Resource allocation to reproduction can change depending on size, as predicted by the size-dependent sex allocation. This theory is based on the fact that small individuals will invest in the allocation of sex with lower cost of production, usually male gender. In plants, there are some andromonoecy species, presence of hermaphrodite and male flowers in the same individual. Andromonoecy provides a strategy to optimally allocate resources to male and female function, evolving a reproductive energy-saving strategy. Thus, our objective was to investigate the size-dependent sex allocation in Solanum lycocarpum St. Hil. We tested the hypothesis that plants with larger size will invest in the production of hermaphrodite flowers, because higher individuals have greater availability of resources to invest in more complex structures involving greater energy expenditure. The studied species was S. lycocarpum, an andromonoecious species. From June 2016 to March 2017 the data were collected in 38 individuals, divided in two groups: the larger plant group (n=18; height=3-5 m) and the smaller plant group (n=20; height=1-2 m).Our data show that there was effect of plant size on the flower production and the sexual gender allocation. The larger plants showed more flowers and higher production of hermaphrodite flowers. Furthermore, in the flower scale, we observed allometric relationship among the flower's traits with proportional investments in biomass, anther size and gynoecium size. Our results are in agreement with size-dependent sex allocation theory and andromonoecy hypothesis related to mechanisms for optimal resource allocation to male and female function.

A alocação de recursos para reprodução pode mudar dependendo do tamanho, conforme previsto pela alocação sexual dependente do tamanho. Essa teoria é baseada no fato de que indivíduos pequenos investirão na alocação sexual com menor custo de produção, geralmente do sexo masculino. Nas plantas, existem algumas espécies andromonoicas, presença de hermafrodita e flores masculinas no mesmo indivíduo. A andromonoicia fornece uma estratégia para alocar recursos de maneira ideal às funções masculina e feminina, desenvolvendo uma estratégia reprodutiva de economia de energia. Assim, nosso objetivo foi investigar a alocação sexual dependente do tamanho em Solanum lycocarpum St. Hil. Testamos a hipótese de que plantas de maior tamanho investirão na produção de flores hermafroditas, pois indivíduos mais altos economizam mais disponibilidade de recursos para investir em estruturas mais complexas que envolvem maior gasto de energia. A espécie estudada foi S. lycocarpum, uma espécie andromonoica. De junho de 2016 a março de 2017, os dados foram coletados em 38 indivíduos, divididos em dois grupos: o maior grupo de plantas (n = 18; altura = 3-5 m) e o menor grupo de plantas (n = 20; altura = 1-2 m). Nossos dados mostram que houve efeito do tamanho da planta na produção de flores e na alocação sexual. As plantas maiores apresentaram mais flores e maior produção de flores hermafroditas. Além disso, observamos uma relação alométrica entre as características da flor, com investimentos proporcionais em biomassa, tamanho da antera e tamanho do gineceu. Nossos resultados estão de acordo com a teoria de alocação de sexo dependente de tamanho e a hipótese de andromonoicia relacionada a mecanismos para a alocação ótima de recursos para a função masculina e feminina.

A Cysteine-Rich Protein, SpDIR1L, Implicated in S-RNase-Independent Pollen Rejection in the Tomato (Solanum Section Lycopersicon) Clade.

Tomato clade species (Solanum sect. Lycopersicon) display multiple interspecific reproductive barriers (IRBs). Some IRBs conform to the SI x SC rule, which describes unilateral incompatibility (UI) where pollen from SC species is rejected on SI species' pistils, but reciprocal pollinations are successful. However, SC x SC UI also exists, offering opportunities to identify factors that contribute to S-RNase-independent IRBs. For instance, SC Solanum pennellii LA0716 pistils only permit SC Solanum lycopersicum pollen tubes to penetrate to the top third of the pistil, while S. pennellii pollen penetrates to S. lycopersicum ovaries. We identified candidate S. pennellii LA0716 pistil barrier genes based on expression profiles and published results. CRISPR/Cas9 mutants were created in eight candidate genes, and mutants were assessed for changes in S. lycopersicum pollen tube growth. Mutants in a gene designated Defective in Induced Resistance 1-like (SpDIR1L), which encodes a small cysteine-rich protein, permitted S. lycopersicum pollen tubes to grow to the bottom third of the style. We show that SpDIR1L protein accumulation correlates with IRB strength and that species with weak or no IRBs toward S. lycopersicum pollen share a 150 bp deletion in the upstream region of SpDIR1L. These results suggest that SpDIR1L contributes to an S-RNase-independent IRB.

A single cytochrome P450 oxidase from Solanum habrochaites sequentially oxidizes 7-epi-zingiberene to derivatives toxic to whiteflies and various microorganisms.

Secretions from glandular trichomes potentially protect plants against a variety of aggressors. In the tomato clade of the Solanum genus, glandular trichomes of wild species produce a rich source of chemical diversity at the leaf surface. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we report the identification and characterisation of 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxyzingiberene (9H10epoZ), two derivatives of 7-epi-zingiberene produced in glandular trichomes of S. habrochaites LA2167. Using a combination of transcriptomics and genetics, we identified a gene coding for a cytochrome P450 oxygenase, ShCYP71D184, that is highly expressed in trichomes and co-segregates with the presence of the zingiberene derivatives. Transient expression assays in Nicotiana benthamiana showed that ShCYP71D184 carries out two successive oxidations to generate 9HZ and 9H10epoZ. Bioactivity assays showed that 9-hydroxy-10,11-epoxyzingiberene in particular exhibits substantial toxicity against B. tabaci and various microorganisms including Phytophthora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.

Functional analysis of MYB alleles from Solanum chilense and Solanum lycopersicum in controlling anthocyanin levels in heterologous tobacco plants.

Flavonoids are natural pigments occurring in plants and are present in fruits, leaves, stems, roots, and flowers. Tobacco plants transformed with an MYB regulatory gene from either Solanum chilense (Sc) or S. lycopersicum (Sl) demonstrate that ScANT1 induces a higher level of anthocyanin accumulation in comparison to SlANT1 and that this gene is sufficient to promote increased anthocyanin levels. We compared the aptitude of ScANT1 protein to induce anthocyanin accumulation to that of SlANT1 protein in tobacco plants. We also tested the effect of amino acid substitutions in ScANT1 and SlANT1. We examined these synthetic alleles' effect following the over-expression of additional anthocyanin synthesis regulators, such as the tomato bHLH (SlJAF13) protein. Our results show that the amino acid changes that differentiate ScANT1 from SlANT1 are the main contributors to the advantage that ScANT1 has over SlANT1 in anthocyanin accumulation per transcript unit. We further demonstrated that altering the amino acid composition of SlANT1 could increase anthocyanin accumulation, while reciprocally modifying ScANT1 lowers the anthocyanin level. These results confirm the increased anthocyanin level in tobacco is attributed to the amino acid differences between ScANT1 and SlANT1. We also show that the co-expression of SlJAF13 with SlANT1 in tobacco plants represses the anthocyanin production.

Developing a new model system for potato genetics by androgenesis.

High heterozygosity and tetrasomic inheritance complicate studies of asexually propagated polyploids, such as potato. Reverse genetics approaches, especially mutant library construction, can be an ideal choice if a proper mutagenesis genotype is available. Here, we aimed to generate a model system for potato research using anther cultures of Solanum verrucosum, a self-compatible diploid potato with strong late blight resistance. Six of the 23 regenerants obtained (SVA4, SVA7, SVA22, SVA23, SVA32, and SVA33) were diploids, and their homozygosity was estimated to be >99.99% with 22 polymorphic InDel makers. Two lines-SVA4 and SVA32-had reduced stature (plant height ≤80 cm), high seed yield (>1,000 seeds/plant), and good tuber set (>30 tubers/plant). We further confirmed the full homozygosity of SVA4 and SVA32 using whole-genome resequencing. These two regenerants possess all the characteristics of a model plant: diploidy, 100% homozygosity, self-compatibility, and amenability to transgenesis. Thus, we have successfully generated two lines, SVA4 and SVA32, which can potentially be used for mutagenesis and as model plants to rejuvenate current methods of conducting potato research.

Evolution of a guarded decoy protease and its receptor in solanaceous plants.

Rcr3 is a secreted protease of tomato that is targeted by fungal effector Avr2, a secreted protease inhibitor of the fungal pathogen Cladosporium fulvum. The Avr2-Rcr3 complex is recognized by receptor-like protein Cf-2, triggering hypersensitive cell death (HR) and disease resistance. Avr2 also targets Rcr3 paralog Pip1, which is not required for Avr2 recognition but contributes to basal resistance. Thus, Rcr3 acts as a guarded decoy in this interaction, trapping the fungus into a recognition event. Here we show that Rcr3 evolved > 50 million years ago (Mya), whereas Cf-2 evolved <6Mya by co-opting the pre-existing Rcr3 in the Solanum genus. Ancient Rcr3 homologs present in tomato, potato, eggplants, pepper, petunia and tobacco can be inhibited by Avr2 with the exception of tobacco Rcr3. Four variant residues in Rcr3 promote Avr2 inhibition, but the Rcr3 that co-evolved with Cf-2 lacks three of these residues, indicating that the Rcr3 co-receptor is suboptimal for Avr2 binding. Pepper Rcr3 triggers HR with Cf-2 and Avr2 when engineered for enhanced inhibition by Avr2. Nicotiana benthamiana (Nb) is a natural null mutant carrying Rcr3 and Pip1 alleles with deleterious frame-shift mutations. Resurrected NbRcr3 and NbPip1 alleles were active proteases and further NbRcr3 engineering facilitated Avr2 inhibition, uncoupled from HR signalling. The evolution of a receptor co-opting a conserved pathogen target contrasts with other indirect pathogen recognition mechanisms.

Effects of reciprocal grafting on cadmium accumulation in post-grafting generations of two ecotypes of Solanum photeinocarpum.

Farmland and mining ecotypes of the potential cadmium (Cd)-hyperaccumulator Solanum photeinocarpum were collected to study the effects of reciprocal grafting on the growth of, and Cd accumulation in, the post-grafting generations. The post generations of the following plant materials were evaluated in a pot experiment: the un-grafted farmland ecotype, grafted plants with the farmland ecotype as the scion or the rootstock, the un-grafted mining ecotype, and grafted plants with the mining ecotype as the scion or the rootstock. The results showed that reciprocal grafting increased the biomass, the activities of superoxide dismutase, peroxidase, and catalase, and the soluble protein content in the post-grafting generations of both ecotypes S. photeinocarpum. Reciprocal grafting also increased the Cd content in, and amount of Cd extracted by, the post-grafting generations of both ecotypes S. photeinocarpum as a result of lower soil pH and higher soil available Cd concentrations. Additionally, grafting affected the DNA methylation levels by inducing hypermethylation or demethylation in the post-grafting generation. Therefore, reciprocal grafting can enhance the Cd accumulation (phytoremediation) capacity of post-grafting generations of both ecotypes S. photeinocarpum by affecting DNA methylation levels.

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species.

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide-binding leucine-rich repeat protein (NLR)-encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA-Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.