Progress In Functional Genomics Research Articles

Major viral diseases in grain legumes: designing disease resistant legumes from plant breeding and OMICS integration.

Grain legumes play a crucial role in human nutrition and as a staple crop for low-income farmers in developing and underdeveloped nations, contributing to overall food security and agroecosystem services. Viral diseases are major biotic stresses that severely challenge global grain legume production. In this review, we discuss how exploring naturally resistant grain legume genotypes within germplasm, landraces, and crop wild relatives could be used as promising, economically viable, and eco-environmentally friendly solution to reduce yield losses. Studies based on Mendelian and classical genetics have enhanced our understanding of key genetic determinants that govern resistance to various viral diseases in grain legumes. Recent advances in molecular marker technology and genomic resources have enabled us to identify genomic regions controlling viral disease resistance in various grain legumes using techniques such as QTL mapping, genome-wide association studies, whole-genome resequencing, pangenome and 'omics' approaches. These comprehensive genomic resources have expedited the adoption of genomics-assisted breeding for developing virus-resistant grain legumes. Concurrently, progress in functional genomics, especially transcriptomics, has helped unravel underlying candidate gene(s) and their roles in viral disease resistance in legumes. This review also examines the progress in genetic engineering-based strategies, including RNA interference, and the potential of synthetic biology techniques, such as synthetic promoters and synthetic transcription factors, for creating viral-resistant grain legumes. It also elaborates on the prospects and limitations of cutting-edge breeding technologies and emerging biotechnological tools (e.g., genomic selection, rapid generation advances, and CRISPR/Cas9-based genome editing tool) in developing virus-disease-resistant grain legumes to ensure global food security.

Breeding and genomics approaches for improving phosphorus-use efficiency in grain legumes

Phosphorus (P) is an essential plant macronutrient, but P sources for plant growth are non-renewable, causing great concern for future sustainable agriculture and global food security. Thus, enhancing plant P-use efficiency (PUE) by improving P-acquisition and P-utilization efficiencies is urgently needed in various crops, including grain legumes, for intensive cropping systems. This review discusses how to harness the genetic variability in PUE traits across grain legume gene pools to improve PUE using various conventional breeding approaches and emerging breeding tools. The genetic architecture of PUE traits is complex, being quantitatively inherited and highly influenced by the environment. Thus, we discuss how the biparental QTL mapping approach has been used to dissect the genetic architecture of PUE traits. Unprecedented advances in legume genomics resources, especially high-throughput single nucleotide polymorphisms, have facilitated uncovering genomic regions related to PUE across the whole genome using a genome-wide association mapping approach. Likewise, the availability of complete genome sequence information, pangenome sequences, and the whole-genome resequencing approach have provided novel insights into structural variation, including presence/absence and copy number variations, underpinning PUE. Simultaneously, progress in functional genomics, including transcriptomics and computational biology, has facilitated the discovery of various underlying transcription factors and the corresponding downstream P signal perception genes and candidate gene(s) controlling PUE and conferring low-P tolerance in various legumes with putative function. We also discuss updated metabolomics and proteomics approaches that have improved our understanding of various gene networks, P-starvation signaling pathways, and P acquisition and utilization of molecular mechanisms important for PUE. Finally, we summarize how novel breeding schemes, including genomic selection, speed breeding, and emerging CRISPR/Cas9-based genome editing tools, will assist in designing P-use-efficient cultivars and adapting grain legumes to low-P environments. Thus, enhancing PUE by integrating various ‘omics’ approaches could improve P-acquisition and P-utilization efficiencies in various modern grain legume cultivars grown in intensive cropping systems to restrict P-fertilizer overuse and preserve the declining non-renewable global rock phosphate reservoir for securing an economical and sustainable future agriculture. • Phosphorus (P) is one of the most essential nutrients required for plant growth and development. • P fertilizers being non-renewable, judicious utilization of P fertilizer is urgently needed for sustaining legume yield for ensuring global food security. • Plants have evolved several molecular, physiological and biochemical mechanisms to sense, acquire and utilize soil P and adapt under P impoverished environments. • ‘Omics’ approach could greatly assist in unraveling the underlying genomic regions with their function contributing to improving phosphorus use efficiency in legume crops.

CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug

The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.

Revisiting Campylobacter jejuni Virulence and Fitness Factors: Role in Sensing, Adapting, and Competing.

Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis world wide and represents a major public health concern. Over the past two decades, significant progress in functional genomics, proteomics, enzymatic-based virulence profiling (EBVP), and the cellular biology of C. jejuni have improved our basic understanding of this important pathogen. We review key advances in our understanding of the multitude of emerging virulence factors that influence the outcome of C. jejuni–mediated infections. We highlight, the spatial and temporal dynamics of factors that promote C. jejuni to sense, adapt and survive in multiple hosts. Finally, we propose cohesive research directions to obtain a comprehensive understanding of C. jejuni virulence mechanisms.

ABC-GWAS: Functional Annotation of Estrogen Receptor-Positive Breast Cancer Genetic Variants.

Over the past decade, hundreds of genome-wide association studies (GWAS) have implicated genetic variants in various diseases, including cancer. However, only a few of these variants have been functionally characterized to date, mainly because the majority of the variants reside in non-coding regions of the human genome with unknown function. A comprehensive functional annotation of the candidate variants is thus necessary to fill the gap between the correlative findings of GWAS and the development of therapeutic strategies. By integrating large-scale multi-omics datasets such as the Cancer Genome Atlas (TCGA) and the Encyclopedia of DNA Elements (ENCODE), we performed multivariate linear regression analysis of expression quantitative trait loci, sequence permutation test of transcription factor binding perturbation, and modeling of three-dimensional chromatin interactions to analyze the potential molecular functions of 2,813 single nucleotide variants in 93 genomic loci associated with estrogen receptor-positive breast cancer. To facilitate rapid progress in functional genomics of breast cancer, we have created “Analysis of Breast Cancer GWAS” (ABC-GWAS), an interactive database of functional annotation of estrogen receptor-positive breast cancer GWAS variants. Our resource includes expression quantitative trait loci, long-range chromatin interaction predictions, and transcription factor binding motif analyses to prioritize putative target genes, causal variants, and transcription factors. An embedded genome browser also facilitates convenient visualization of the GWAS loci in genomic and epigenomic context. ABC-GWAS provides an interactive visual summary of comprehensive functional characterization of estrogen receptor-positive breast cancer variants. The web resource will be useful to both computational and experimental biologists who wish to generate and test their hypotheses regarding the genetic susceptibility, etiology, and carcinogenesis of breast cancer. ABC-GWAS can also be used as a user-friendly educational resource for teaching functional genomics. ABC-GWAS is available at http://education.knoweng.org/abc-gwas/.

Benzylisoquinoline alkaloid biosynthesis in opium poppy: an update

For nearly eight millennia, opium poppy (Papaver somniferum) has been bred and cultivated for therapeutic purposes. The medicinal properties of the plant are conferred by specialized metabolites known as benzylisoquinoline alkaloids (BIAs), comprising the narcotic analgesics morphine and codeine, the antimicrobial agent sanguinarine, and the potential anticancer drug noscapine. In addition, naturally occurring thebaine is used for the semi-synthesis of widely prescribed pain-relievers (e.g., oxycodone and hydrocodone), valuable drugs used in the treatment of opioid addiction (i.e., naltrexone), or antidotes for opioid overdose (i.e., naloxone). The complex stereochemistry of many opiates hinders their chemical synthesis and opium poppy remains the sole commercial source of these important pharmaceuticals. For decades, opium poppy has served as a model plant for research aimed at a comprehensive understanding of BIA metabolism. Recent progress in functional genomics has enabled the discovery of a nearly complete collection of BIA biosynthetic genes, many of which are clustered in the opium poppy genome. Advances in synthetic biology have facilitated the successful reconstitution of several BIA biosynthetic pathways in heterologous hosts such as Saccharomyces cerevisiae and Escherichia coli, although the initially low production levels suggest that commercial scale-up will present additional challenges. This review provides an update of key molecular and biochemical aspects of BIA metabolism in opium poppy, including recent biosynthetic gene discoveries, genomic organization, novel BIA transporters, metabolic regulation, and major efforts in the engineering of pathways in plants and microbes.

Genetic resources offer efficient tools for rice functional genomics research.

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T-DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene-rich regions, resulting in direct gene knockout or activation of genes within 20-30 kb up- and downstream of the T-DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T-DNA-tagged rice mutant population. We also discuss important features of T-DNA activation- and knockout-tagging and promoter-trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high-throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops.

Expanded microbial genome coverage and improved protein family annotation in the COG database.

Microbial genome sequencing projects produce numerous sequences of deduced proteins, only a small fraction of which have been or will ever be studied experimentally. This leaves sequence analysis as the only feasible way to annotate these proteins and assign to them tentative functions. The Clusters of Orthologous Groups of proteins (COGs) database (http://www.ncbi.nlm.nih.gov/COG/), first created in 1997, has been a popular tool for functional annotation. Its success was largely based on (i) its reliance on complete microbial genomes, which allowed reliable assignment of orthologs and paralogs for most genes; (ii) orthology-based approach, which used the function(s) of the characterized member(s) of the protein family (COG) to assign function(s) to the entire set of carefully identified orthologs and describe the range of potential functions when there were more than one; and (iii) careful manual curation of the annotation of the COGs, aimed at detailed prediction of the biological function(s) for each COG while avoiding annotation errors and overprediction. Here we present an update of the COGs, the first since 2003, and a comprehensive revision of the COG annotations and expansion of the genome coverage to include representative complete genomes from all bacterial and archaeal lineages down to the genus level. This re-analysis of the COGs shows that the original COG assignments had an error rate below 0.5% and allows an assessment of the progress in functional genomics in the past 12 years. During this time, functions of many previously uncharacterized COGs have been elucidated and tentative functional assignments of many COGs have been validated, either by targeted experiments or through the use of high-throughput methods. A particularly important development is the assignment of functions to several widespread, conserved proteins many of which turned out to participate in translation, in particular rRNA maturation and tRNA modification. The new version of the COGs is expected to become an important tool for microbial genomics.

Diabetic nephropathy--emerging epigenetic mechanisms.

Diabetic nephropathy (DN), a severe microvascular complication frequently associated with both type 1 and type 2 diabetes mellitus, is a leading cause of renal failure. The condition can also lead to accelerated cardiovascular disease and macrovascular complications. Currently available therapies have not been fully efficacious in the treatment of DN, suggesting that further understanding of the molecular mechanisms underlying the pathogenesis of DN is necessary for the improved management of this disease. Although key signal transduction and gene regulation mechanisms have been identified, especially those related to the effects of hyperglycaemia, transforming growth factor β1 and angiotensin II, progress in functional genomics, high-throughput sequencing technology, epigenetics and systems biology approaches have greatly expanded our knowledge and uncovered new molecular mechanisms and factors involved in DN. These mechanisms include DNA methylation, chromatin histone modifications, novel transcripts and functional noncoding RNAs, such as microRNAs and long noncoding RNAs. In this Review, we discuss the significance of these emerging mechanisms, how they mediate the actions of growth factors to augment the expression of extracellular matrix and inflammatory genes associated with DN and their potential usefulness as diagnostic biomarkers or novel therapeutic targets for DN.

Nodular diagnosis for ecological engineering of the symbiotic nitrogen fixation with legumes

As a major contributor to the reduced nitrogen pool in the biosphere, symbiotic nitrogen fixation by legumes plays a critical role in a sustainable production system. However this legume contribution varies with the physico-chemical and biological conditions of the nodulated-root rhizosphere. In order to assess the abiotic and biotic constrains that might limit this symbiosis at the agroecosystem level, a nodular diagnosis is proposed with common bean as a model grain-legume, and a major source of plant proteins for world human nutrition. The engineering of the legume symbiosis is addressed by participatory assessment of bean recombinant inbred lines contrasting for their efficiency in use of phosphorous for symbiotic nitrogen fixation. With this methodology, in field-sites chosen with farmers of an area of cereal-cropping in the Mediterranean basin, a large spatial and temporal variation in the legume nodulation was found. Soil P availability was a major limiting factor of the rhizobial symbiosis. In order to relate the field measurements with progress in functional genomics of the symbiosis, in situ RT-PCR on nodule sections has been implemented showing that the phytase gene is expressed in the cortex with significantly higher number of transcripts in P-efficient RILs. It is concluded that various tools and indicators are available for developing the ecological engineering of the rhizobial symbiosis, in particular for its beneficial contribution to the bio-geochemical cycle of N, and also P and C.

Anatomic Demarcation of Cells: Genes to Patterns

An organizing principle of the diverse cell types in multicellular organisms is their anatomic location. In turn, anatomic location is patterned by the positional identities of cells along developmental axes. Recent progress in functional genomics and chromatin biology illustrates how cells use specific gene expression programs to encode location. Dynamic chromatin states of key genes, notably the Hox loci, serve as the internal representation in cells of their positional identity within the animal.

Maximizing the potential of functional genomics.

Geneticists have made tremendous progress in understanding the genetic basis of phenotypes, and genomics promises to bring further insights at a rapid pace. The progress in functional genomics has been driven primarily by the development of new techniques that are used in a few dedicated research centres. Focusing on selected advances in genomic technologies, we assess the results that have been obtained so far, highlight the challenges faced by these new tools and suggest ways in which they can be overcome. We argue that progress in functional genomics will depend on developing high-throughput technologies that can easily be moved away from dedicated centres and into individual laboratories.

Automated in situ hybridisation using Tecan robotics: a kick-start for Functional Genomics

The completion of the Human Genome Project has been hailed as the most important scientific advance of recent times. Some commentators have even predicted that this definitive knowledge of our genetic makeup will eventually transform medicine by leading to the development of revolutionary new therapies and drugs. Genes will be identified that are responsible for particular diseases, and methods will be developed that allow specific genetic defects to be corrected or circumvented. However, biologists have been quick to point out that these kinds of developments will require much more than just the sequence of the human genome. Even identifying the 50,000 or so genes that this sequence contains will not be enough. The challenge now is to understand in detail what the function of all these thousands of genes is and how they interact. This vast new field has been called “Functional Genomics”, and it may prove to be even more of a challenge than posed by the Human Genome Project. The success of that project depended upon the automation of nucleotide sequencing, and it is clear that significant progress in Functional Genomics will also require the development of automated methods for many of the analytical techniques that are currently carried out manually. A group at the Max Planck Institute of Experimental Endocrinology in Hannover has collaborated with the automated solutions company, Tecan to develop GenePaint, an automated system for carrying out in situ hybridisation. This technique is used to reveal the way in which particular genes are expressed in different tissues, information which is an important first step in understanding gene function. The Director of the Experimental Endocrinology Institute, Professor Gregor Eichele, explained the background to their work: “Gene function is critically illuminated by knowing where and when a gene is expressed. We need to know where genes are expressed in pathological samples, but also of course in normal tissue and during development. When you look at any tissue, human or animal, or even plants, genes are often only expressed in certain cell types. “If you have a piece of tumour tissue, a cancer gene may only be expressed in certain cells, and this is very often used as a diagnostic tool. The sequence of a gene itself is not very useful functionally and there are many tens of thousands of genes whose expression pattern has to be determined.”