Genome-Wide Identification and Characterization of Homeobox Transcription Factors in Phoma sorghina var. saccharum Causing Sugarcane Twisted Leaf Disease.

A homeobox transcription factor is a conserved transcription factor, ubiquitous in eukaryotes, that regulates the tissue formation of structure, cell differentiation, proliferation, and cancer. This study identified the homeobox transcription factor family and its distribution in Phoma sorghina var. saccharum at the whole genome level. It elucidated the gene structures and evolutionary characteristics of this family. Additionally, knockout experiments were carried out and the preliminary function of these transcription factors was studied. Through bioinformatics approaches, nine homeobox transcription factors (PsHOX1-PsHOX9) were identified in P. sorghina var. saccharum, and these contained HOX-conserved domains and helix-turn-helix secondary structures. Nine homeobox gene deletion mutants were obtained using the homologous recombinant gene knockout technique. Protoplast transformation was mediated by polyethylene glycol (PEG) and the transformants were identified using PCR. The knockouts of PsHOX1, PsHOX2, PsHOX3, PsHOX4, PsHOX6, PsHOX8, and PsHOX9 genes resulted in a smaller growth diameter in P. sorghina var. saccharum. In contrast, the knockouts of the PsHOX3, PsHOX6, and PsHOX9 genes inhibited the formation of conidia and led to a significant decrease in the pathogenicity. This study's results will provide insights for understanding the growth and development of P. sorghina var. saccharum. The pathogenic mechanism of the affected sugarcane will provide an essential theoretical basis for preventing and controlling sugarcane twisted leaf disease.

Integration of Transcriptomic and Metabolomic Profiles Provides Insights into the Influence of Nitrogen on Secondary Metabolism in Fusarium sacchari.

Nitrogen availability might play an essential role in plant diseases by enhancing fungal cell growth and influencing the expression of genes required for successful pathogenesis. Nitrogen availability could modulate secondary metabolic pathways as evidenced by the significant differential expression of several core genes involved in mycotoxin biosynthesis and genes encoding polyketide synthase/nonribosomal peptide synthetases, cytochrome P450 and carbohydrate-active enzymes in Fusarium sacchari, grown on different nitrogen sources. A combined analysis was carried out on the transcript and metabolite profiles of regulatory metabolic processes and the virulence of Fusarium sacchari grown on various nitrogen sources. The nitrogen regulation of the gibberellin gene cluster included the metabolic flux and multiple steps of gibberellin synthesis. UHPLC-MS/MS-based metabolome analysis revealed the coordination of these related transcripts and the accumulation of gibberellin metabolites. This integrated analysis allowed us to uncover additional information for a more comprehensive understanding of biological events relevant to fungal secondary metabolic regulation in response to nitrogen availability.

Confirmation of 'Pollen- and Seed-Specific Gene Deletor' System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions.

The commercial application of genetically modified plants has been seriously impeded by public concern surrounding the potential risks posed by such plants to the ecosystem and human health. Previously, we have developed a 'pollen- and seed-specific Gene Deletor' system that automatically excised all transgenes from the pollen and seeds of greenhouse-grown transgenic Nicotiana tabacum. In this study, we conducted seven field experiments over three consecutive years to evaluate the stability of transgene excision under field conditions. Our results showed that transgenes were stably excised from transgenic Nicotiana tabacum under field conditions with 100% efficiency. The stability of transgene excision was confirmed based on PCR, as well as the GUS staining patterns of various organs (roots, leaves, petiole, stem, flower, fruit, and seeds) from transgenic N. tabacum. In six transgenic lines (D4, D10, D31, D56, and D43), the transgenes were stably deleted in the T0 and T1 generations. Thus, the 'Gene Deletor' system is an efficient and reliable method to reduce pollen- and seed-mediated unintentional gene flow. This system might help to alleviate the food safety concerns associated with transgenic crops.

Genetic Diversity and Pathogenicity of Fusarium fujikuroi Species Complex (FFSC) Causing Sugarcane Pokkah Boeng Disease (PBD) in China.

Pokkah boeng disease (PBD), a sugarcane foliar disease, is caused by various Fusarium spp. within the Fusarium fujikuroi species complex (FFSC). In the current study, we investigated the diversity of Fusarium spp. associated with PBD in China. In total, 320 leaf samples displaying PBD symptoms were collected over 10 consecutive years (2012 to 2021), during winter and summer, from six various sugarcane-growing regions (Guangxi, Yunnan, Guangdong, Zhejiang, Hainan, and Fujian) in China. Phylogenetic analysis of Fusarium spp. was reconstructed using translation elongation factor 1-α, and DNA-directed RNA polymerase II largest subunit and second-largest subunit multigene sequences. Evolutionary studies of these regions categorized the isolates into four FFSC species (F. sacchari, F. proliferatum, F. verticillioides, and F. andiyazi). The identified isolates, which developed irregular necrotic patches and rotting symptoms on the sugarcane plant after approximately 30 days were tested for their pathogenicity. Symptoms that appeared during pathogenicity testing were consistent with those observed under field conditions. Each strain of the pathogenic Fusarium spp. belonged to different vegetative compatibility groups (VCGs), and there was no affinity between VCGs. Our results contribute to understanding FFSC and accurately identifying Fusarium spp. associated with the sugarcane crop.

Comparative genome analysis unravels pathogenicity of Xanthomonas albilineans causing sugarcane leaf scald disease.

BACKGROUND: Xanthomonas is a genus of gram-negative bacterium containing more than 35 species. Among these pathogenic species, Xanthomonas albilineans (Xal) is of global interest, responsible for leaf scald disease in sugarcane. Another notable Xanthomonas species is Xanthomonas sachari (Xsa), a sugarcane-associated agent of chlorotic streak disease. RESULT: The virulence of 24 Xanthomonas strains was evaluated by disease index (DI) and Area Under Disease Progress Curve (AUDPC) in the susceptible inoculated plants (GT 46) and clustered into three groups of five highly potent, seven mild virulent, and twelve weak virulent strains. The highly potent strain (X. albilineans, Xal JG43) and its weak virulent related strain (X. sacchari, Xsa DD13) were sequenced, assembled, and annotated in the circular genomes. The genomic size of JG43 was smaller than that of DD13. Both strains (JG43 and DD13) lacked a Type III secretory system (T3SS) and T6SS. However, JG43 possessed Salmonella pathogenicity island-1 (SPI-1). More pathogen-host interaction (PHI) genes and virulent factors in 17 genomic islands (GIs) were detected in JG43, among which six were related to pathogenicity. Albicidin and a two-component system associated with virulence were also detected in JG43. Furthermore, 23 Xanthomonas strains were sequenced and classified into three categories based on Single Nucleotide Polymorphism (SNP) mutation loci and pathogenicity, using JG43 as a reference genome. Transitions were dominant SNP mutations, while structural variation (SV) is frequent intrachromosomal rearrangement (ITX). Two essential genes (rpfC/rpfG) of the two-component system and another gene related to SNP were mutated to understand their virulence effect. The mutation of rpfG resulted in a decrease in pathogenicity. CONCLUSION: These findings revealed virulence of 24 Xanthomonas strains and variations by 23 Xanthomonas strains. We sequenced, assembled, and annotated the circular genomes of Xal JG43 and Xsa DD13, identifying diversity detected by pathogenic factors and systems. Furthermore, complete genomic sequences and sequenced data will provide a theoretical basis for identifying pathogenic factors responsible for sugarcane leaf scald disease.

Functional characterization and analysis of transcriptional regulation of sugar transporter SWEET13c in sugarcane Saccharum spontaneum.

BACKGROUND: Sugarcane is an important crop for sugar production worldwide. The Sugars Will Eventually be Exported Transporters (SWEETs) are a group of sugar transporters recently identified in sugarcane. In Saccharum spontaneum, SsSWEET13c played a role in the sucrose transportation from the source to the sink tissues, which was found to be mainly active in the mature leaf. However, the function and regulation of SWEETs in sugarcane remain elusive despite extensive studies performed on sugar metabolism. RESULTS: In this study, we showed that SsSWEET13c is a member of SWEET gene family in S. spontaneum, constituting highest circadian rhythm-dependent expression. It is a functional gene that facilitates plant root elongation and increase fresh weight of Arabidopsis thaliana, when overexpressed. Furthermore, yeast one-hybrid assays indicate that 20 potential transcription factors (TFs) could bind to the SsSWEET13c promoter in S. spontaneum. We combined transcriptome data from developmental gradient leaf with distinct times during circadian cycles and stems/leaves at different growth stages. We have uncovered that 14 out of 20 TFs exhibited positive/negative gene expression patterns relative to SsSWEET13c. In the source tissues, SsSWEET13c was mainly positively regulated by SsbHLH34, SsTFIIIA-a, SsMYR2, SsRAP2.4 and SsbHLH035, while negatively regulated by SsABS5, SsTFIIIA-b and SsERF4. During the circadian rhythm, it was noticed that SsSWEET13c was more active in the morning than in the afternoon. It was likely due to the high level of sugar accumulation at night, which was negatively regulated by SsbZIP44, and positively regulated by SsbHLH34. Furthermore, in the sink tissues, SsSWEET13c was also active for sugar accumulation, which was positively regulated by SsbZIP44, SsTFIIIA-b, SsbHLH34 and SsTFIIIA-a, and negatively regulated by SsERF4, SsHB36, SsDEL1 and SsABS5. Our results were further supported by one-to-one yeast hybridization assay which verified that 12 potential TFs could bind to the promoter of SsSWEET13c. CONCLUSIONS: A module of the regulatory network was proposed for the SsSWEET13c in the developmental gradient of leaf and circadian rhythm in S. spontaneum. These results provide a novel understanding of the function and regulation of SWEET13c during the sugar transport and biomass production in S. spontaneum.

RNA Interference: Promising Approach to Combat Plant Viruses.

Plant viruses are devastating plant pathogens that severely affect crop yield and quality. Plants have developed multiple lines of defense systems to combat viral infection. Gene silencing/RNA interference is the key defense system in plants that inhibits the virulence and multiplication of pathogens. The general mechanism of RNAi involves (i) the transcription and cleavage of dsRNA into small RNA molecules, such as microRNA (miRNA), or small interfering RNA (siRNA), (ii) the loading of siRNA/miRNA into an RNA Induced Silencing Complex (RISC), (iii) complementary base pairing between siRNA/miRNA with a targeted gene, and (iv) the cleavage or repression of a target gene with an Argonaute (AGO) protein. This natural RNAi pathway could introduce transgenes targeting various viral genes to induce gene silencing. Different RNAi pathways are reported for the artificial silencing of viral genes. These include Host-Induced Gene Silencing (HIGS), Virus-Induced Gene Silencing (VIGS), and Spray-Induced Gene Silencing (SIGS). There are significant limitations in HIGS and VIGS technology, such as lengthy and time-consuming processes, off-target effects, and public concerns regarding genetically modified (GM) transgenic plants. Here, we provide in-depth knowledge regarding SIGS, which efficiently provides RNAi resistance development against targeted genes without the need for GM transgenic plants. We give an overview of the defense system of plants against viral infection, including a detailed mechanism of RNAi, small RNA molecules and their types, and various kinds of RNAi pathways. This review will describe how RNA interference provides the antiviral defense, recent improvements, and their limitations.

Hearing improvement among children with cholesteatoma who underwent canal wall up and canal wall down surgical management.

Objectives: The most frequently used surgical methods for treating cholesteatoma include canal wall up and canal wall down procedures. The objective of the study was to compare the hearing improvement among children with cholesteatoma who underwent canal wall up and canal wall down surgical management. Methods: The cross-sectional analytical study design was used. The study was conducted at the ENT Department of Nishtar Medical University & Hospital Multan from 15th June to 15th Nov 2020.. Forty six patients with cholesteatoma were enrolled in the study after taking informed consent. Inclusion and exclusion criteria were followed. The participants were categorized into two groups. Group-A was treated with canal wall-up surgery while Group-B was treated with canal wall down Mastoidectomy. A 12-month post-operative follow-up and the audiometry assessment were compared with pre-surgical values. Additionally, a COMOT-15 survey was administered to analyze self-perceived hearing functions. The Chi-square test was used for comparative analysis of the surgical outcome and hearing improvement among the two groups. P-value (p value<0.05) was considered statistically significant. Results: Forty six patients were included in the study with 23 participants in each group. Among 46, 26 were male and 20 were female. The pre and post-operative mean Pure-tone average values were significantly different in (Group-A) who underwent canal wall up Mastoidectomy (p<0.05) than in Group-B, who underwent canal wall down Mastoidectomy. Similarly, hearing sub-section responses of the COMOT-15 survey favored the Canal wall technique. However, the survey showed no significant differences in the mental health status of the two groups (p<0.05). Conclusion: Our data collected after a one-year follow-up of patients suggests canal wall up as a preferred technique for hearing improvement than canal wall down technique.

ScGAIL, a sugarcane N-terminal truncated DELLA-like protein, participates in gibberellin signaling in Arabidopsis.

The hormone gibberellin (GA) is crucial for internode elongation in sugarcane. DELLA proteins are critical negative regulators of the GA signaling pathway. ScGAI encodes a DELLA protein that was previously implicated in the regulation of sugarcane culm development. Here, we characterized ScGAI-like (ScGAIL) in sugarcane, which lacked the N-terminal region but was otherwise homologous to ScGAI. ScGAIL differed from ScGAI in its chromosomal location, expression patterns, and cellular localization. Although transgenic Arabidopsis overexpressing ScGAIL were insensitive to GAs, GA synthesis was affected in these plants, suggesting that ScGAIL disrupted the GA signaling pathway. After GA treatment, the expression patterns of GA-associated genes differed between ScGAIL-overexpressing and wild-type Arabidopsis, and the degradation of AtDELLA proteins in transgenic lines was significantly inhibited compared with wild-type lines. A sugarcane GID1 gene (ScGID1) encoding a putative GA receptor was isolated and interacted with ScGAIL in a GA-independent manner. Five ScGAIL-interacting proteins were verified by yeast two-hybrid assays, and only one interacted with ScGAI. Therefore, ScGAIL may inhibit plant growth by modulating the GA signaling pathway.

Comparative Analysis of Sugar Metabolites and Their Transporters in Sugarcane Following Sugarcane mosaic virus (SCMV) Infection.

Sugarcane mosaic virus (SCMV) is one of the major pathogens of sugarcane. SCMV infection causes dynamic changes in plant cells, including decreased photosynthetic rate, respiration, and sugar metabolism. To understand the basics of pathogenicity mechanism, we performed transcriptome and proteomics analysis in two sugarcane genotypes (Badila: susceptible to SCMV and B-48: SCMV resistant). Using Saccharum spontaneum L. genome as a reference, we identified the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) that participate in sugar metabolism, transport of their metabolites, and Carbohydrate Activating enZYmes (CAZymes). Sequencing data revealed 287 DEGs directly or indirectly involved in sugar metabolism, transport, and storage, while 323 DEGs are associated with CAZymes. Significant upregulation of glucose, sucrose, fructose, starch, and SWEET-related transcripts was observed in the Badila after infection of SCMV. B-48 showed resistance against SCMV with a limited number of sugar transcripts up-regulation at the post-infection stage. For CAZymes, only glycosyltransferase (GT)1 and glycosyl hydrolase (GH)17 were upregulated in B-48. Regulation of DEGs was analyzed at the proteomics level as well. Starch, fructose, glucose, GT1, and GH17 transcripts were expressed at the post-translational level. We verified our transcriptomic results with proteomics and qPCR data. Comprehensively, this study proved that Badila upregulated sugar metabolizing and transporting transcripts and proteins, which enhance virus multiplication and infectionl.

Photosynthetic characterization and expression profiles of sugarcane infected by Sugarcane mosaic virus (SCMV).

Sugarcane mosaic virus (SCMV), belonging to genus Potyvirus, family Potyviridae, is a severe pathogen of several agricultural important crops, mainly sugarcane. Due to complex nature of sugarcane, the effect of SCMV pathogenicity on sugarcane photosynthetic systems remains to be explored. In this study, we investigated the alterations occurring in the photosynthetic system in the sugarcane genotypes at the cytopathological, physiological and biological, transcriptome and proteome level. We generated the transcriptome assembly of two genotypes (susceptible Badila and resistant B-48) using Saccharum spontaneum L. as a reference genome. RNA-sequencing data revealed the significant upregulation of NAD(P)H, RubisCO, oxygen-evolving complex, chlorophyll a and b binding protein, Psb protein family, PSI reaction center subunit II, and IVgenes in B-48, as compared to its counterparts. Upregulated genes in B-48 are associated with various processes such as stability and assembly of photosystem, protection against photoinhibition and antiviral defense. The expression pattern of differentially abundant genes were further verified at the proteomics level. Overall, differentially expressed genes/proteins (DEGs/DEPs) showed the consistency of expression at both transcriptome and proteome level in B-48 genotype. Comprehensively, these data supported the efficiency of B-48 genotype under virus infection conditions and provided a better understanding of the expression pattern of photosynthesis-related genes in sugarcane.

Gene expression profiling of reactive oxygen species (ROS) and antioxidant defense system following Sugarcane mosaic virus (SCMV) infection.

BACKGROUND: Viruses are infectious pathogens, and plant virus epidemics can have devastating consequences to crop yield and quality. Sugarcane mosaic virus (SCMV, belonging to family Potyviridae) is one of the leading pathogens that affect the sugarcane crop every year. To combat the pathogens' attack, plants generate reactive oxygen species (ROS) as the first line of defense whose sophisticated balance is achieved through well-organized antioxidant scavenging pathways. RESULTS: In this study, we investigated the changes occurring at the transcriptomic level of ROS associated and ROS detoxification pathways of SCMV resistant (B-48) and susceptible (Badila) sugarcane genotypes, using Saccharum spontaneum L. genome assembly as a reference genome. Transcriptomic data highlighted the significant upregulation of ROS producing genes such as NADH oxidase, malate dehydrogenase and flavin-binding monooxygenase, in Badila genotype after SCMV pathogenicity. To scavenge the ROS, the Badila genotype illustrated a substantial enhancement of antioxidants i.e. glutathione s-transferase (GST), as compared to its resistant counterpart. GST is supposed to be a key indicator of pathogen attacks on the plant. A remarkably lower GST expression in B-48, as compared to Badila, indicated the development of resistance in this genotype. Additionally, we characterized the critical transcription factors (TFs) involved in endowing resistance to B-48. Among these, WRKY, AP2, NAC, bZIP, and bHLH showed enhanced expression in the B-48 genotype. Our results also confirmed the linkage of transcriptomic data with the enzymatic and qPCR data. The estimation of enzymatic activities for superoxide dismutase, catalase, ascorbate peroxidase, and phenylalanine ammonia-lyase supported the transcriptomic data and evinced higher resistance in B-48 genotype. CONCLUSION: The current study supported the efficiency of the B-48 genotype under SCMV infection. Moreover, comparative transcriptomic data has been presented to highlight the role of significant transcription factors conferring resistance to this genotype. This study provides an in-depth knowledge of the expression profiling of defense mechanisms in sugarcane.

A Synergistic Genetic Engineering Strategy Induced Triacylglycerol Accumulation in Potato (Solanum tuberosum) Leaf.

Potato is the 4th largest staple food in the world currently. As a high biomass crop, potato harbors excellent potential to produce energy-rich compounds such as triacylglycerol as a valuable co-product. We have previously reported that transgenic potato tubers overexpressing WRINKLED1, DIACYLGLYCEROL ACYLTRANSFERASE 1, and OLEOSIN genes produced considerable levels of triacylglycerol. In this study, the same genetic engineering strategy was employed on potato leaves. The overexpression of Arabidopsis thaliana WRINKED1 under the transcriptional control of a senescence-inducible promoter together with Arabidopsis thaliana DIACYLGLYCEROL ACYLTRANSFERASE 1 and Sesamum indicum OLEOSIN driven by the Cauliflower Mosaic Virus 35S promoter and small subunit of Rubisco promoter respectively, resulted in an approximately 30- fold enhancement of triacylglycerols in the senescent transgenic potato leaves compared to the wild type. The increase of triacylglycerol in the transgenic potato leaves was accompanied by perturbations of carbohydrate accumulation, apparent in a reduction in starch content and increased total soluble sugars, as well as changes of polar membrane lipids at different developmental stages. Microscopic and biochemical analysis further indicated that triacylglycerols and lipid droplets could not be produced in chloroplasts, despite the increase and enlargement of plastoglobuli at the senescent stage. Possibly enhanced accumulation of fatty acid phytyl esters in the plastoglobuli were reflected in transgenic potato leaves relative to wild type. It is likely that the plastoglobuli may have hijacked some of the carbon as the result of WRINKED1 expression, which could be a potential factor restricting the effective accumulation of triacylglycerols in potato leaves. Increased lipid production was also observed in potato tubers, which may have affected the tuberization to a certain extent. The expression of transgenes in potato leaf not only altered the carbon partitioning in the photosynthetic source tissue, but also the underground sink organs which highly relies on the leaves in development and energy deposition.

Upregulated Lipid Biosynthesis at the Expense of Starch Production in Potato (Solanum tuberosum) Vegetative Tissues via Simultaneous Downregulation of ADP-Glucose Pyrophosphorylase and Sugar Dependent1 Expressions.

Triacylglycerol is a major component of vegetable oil in seeds and fruits of many plants, but its production in vegetative tissues is rather limited. It would be intriguing and important to explore any possibility to expand current oil production platforms, for example from the plant vegetative tissues. By expressing a suite of transgenes involved in the triacylglycerol biosynthesis, we have previously observed substantial accumulation of triacylglycerol in tobacco (Nicotiana tabacum) leaf and potato (Solanum tuberosum) tuber. In this study, simultaneous RNA interference (RNAi) downregulation of ADP-glucose pyrophosphorylase (AGPase) and Sugar-dependent1 (SDP1), was able to increase the accumulation of triacylglycerol and other lipids in both wild type potato and the previously generated high oil potato line 69. Particularly, a 16-fold enhancement of triacylglycerol production was observed in the mature transgenic tubers derived from the wild type potato, and a two-fold increase in triacylglycerol was observed in the high oil potato line 69, accounting for about 7% of tuber dry weight, which is the highest triacylglycerol accumulation ever reported in potato. In addition to the alterations of lipid content and fatty acid composition, sugar accumulation, starch content of the RNAi potato lines in both tuber and leaf tissues were also substantially changed, as well as the tuber starch properties. Microscopic analysis further revealed variation of lipid droplet distribution and starch granule morphology in the mature transgenic tubers compared to their parent lines. This study reflects that the carbon partitioning between lipid and starch in both leaves and non-photosynthetic tuber tissues, respectively, are highly orchestrated in potato, and it is promising to convert low-energy starch to storage lipids via genetic manipulation of the carbon metabolism pathways.

Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant.

Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the ß-glucuronidase gene (GUS) at the 3' end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21-24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.