Functional genomic screening in Komagataella phaffii enabled by high-activity CRISPR-Cas9 library.

CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guide's targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to metabolic engineering targets, including protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens.

Biochemical characterization of Fsa16295Glu from "Fervidibacter sacchari," the first hyperthermophilic GH50 with ß-1,3-endoglucanase activity and founding member of the subfamily GH50_3.

The aerobic hyperthermophile "Fervidibacter sacchari" catabolizes diverse polysaccharides and is the only cultivated member of the class "Fervidibacteria" within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a ß-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as ß-agarases. The purified enzyme has a wide temperature range of 4-95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa16295Glu possesses a relatively high kcat/KM of 1.82 × 107 s-1 M-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing ß-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from "Fervidibacteria." Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.

eCALIBRATOR: A Comparative Tool to Identify Key Genes and Pathways for Eucalyptus Defense Against Biotic Stressors.

Many pests and pathogens threaten Eucalyptus plantations. The study of defense responses in this economically important wood and fiber crop enables the discovery of novel pathways and genes, which may be adopted to improve resistance. Various functional genomics experiments have been conducted in Eucalyptus-biotic stress interactions following the availability of the Eucalyptus grandis genome, however, comparisons between these studies were limited largely due to a lack of comparative tools. To this end, we developed eCALIBRATOR http://ecalibrator.bi.up.ac.za, a tool for the comparison of Eucalyptus biotic stress interaction. The tool, which is not limited to Eucalyptus, allows the comparison of various datasets, provides a visual output in the form of Venn diagrams and clustering and extraction of lists for gene ontology enrichment analyses. We also demonstrate the usefulness of the tool in revealing pathways and key gene targets to further functionally characterize. We identified 708 differentially expressed E. grandis genes in common among responses to the insect pest Leptocybe invasa, oomycete pathogen Phytophthora cinnamomi and fungus Chrysoporthe austroafricana. Within this set of genes, one of the Gene Ontology terms enriched was "response to organonitrogen compound," with NITRATE TRANSPORTER 2.5 (NRT2.5) being a key gene, up-regulated under susceptible interactions and down-regulated under resistant interactions. Although previous functional genetics studies in Arabidopsis thaliana support a role in nitrate acquisition and remobilization under long-term nitrate starvation, the importance of NRT2.5 in plant defense is unclear. The T-DNA mutants of AtNRT2.5 were more resistant to Pseudomonas syringae pv. tomato pv tomato DC3000 inoculation than the wild-type counterpart, supporting a direct role for NRT2.5 in plant defense. Future studies will focus on characterizing the Eucalyptus ortholog of NRT2.5.

Mediation of plant-mycorrhizal interaction by a lectin receptor-like kinase.

The molecular mechanisms underlying mycorrhizal symbioses, the most ubiquitous and impactful mutualistic plant-microbial interaction in nature, are largely unknown. Through genetic mapping, resequencing and molecular validation, we demonstrate that a G-type lectin receptor-like kinase (lecRLK) mediates the symbiotic interaction between Populus and the ectomycorrhizal fungus Laccaria bicolor. This finding uncovers an important molecular step in the establishment of symbiotic plant-fungal associations and provides a molecular target for engineering beneficial mycorrhizal relationships.

Identification of Populus Small RNAs Responsive to Mutualistic Interactions With Mycorrhizal Fungi, Laccaria bicolor and Rhizophagus irregularis.

Ecto- and endo-mycorrhizal colonization of Populus roots have a positive impact on the overall tree health and growth. A complete molecular understanding of these interactions will have important implications for increasing agricultural or forestry sustainability using plant:microbe-based strategies. These beneficial associations entail extensive morphological changes orchestrated by the genetic reprogramming in both organisms. In this study, we performed a comparative analysis of two Populus species (Populus deltoides and P. trichocarpa) that were colonized by either an arbuscular mycorrhizal fungus (AmF), Rhizophagus irregularis or an ectomycorrhizal fungus (EmF), Laccaria bicolor, to describe the small RNA (sRNA) landscape including small open reading frames (sORFs) and micro RNAs (miRNAs) involved in these mutualistic interactions. We identified differential expression of sRNAs that were, to a large extent, (1) within the genomic regions lacking annotated genes in the Populus genome and (2) distinct for each fungal interaction. These sRNAs may be a source of novel sORFs within a genome, and in this regard, we identified potential sORFs encoded by the sRNAs. We predicted a higher number of differentially-expressed miRNAs in P. trichocarpa (4 times more) than in P. deltoides (conserved and novel). In addition, 44 miRNAs were common in P. trichocarpa between the EmF and AmF treatments, and only 4 miRNAs were common in P. deltoides between the treatments. Root colonization by either fungus was more effective in P. trichocarpa than in P. deltoides, thus the relatively few differentially-expressed miRNAs predicted in P. deltoides might reflect the extent of the symbiosis. Finally, we predicted several genes targets for the plant miRNAs identified here, including potential fungal gene targets. Our findings shed light on additional molecular tiers with a role in Populus-fungal mutualistic associations and provides a set of potential molecular targets for future enhancement.

Conservation and Diversification of Circadian Rhythmicity Between a Model Crassulacean Acid Metabolism Plant Kalanchoë fedtschenkoi and a Model C3 Photosynthesis Plant Arabidopsis thaliana.

Crassulacean acid metabolism (CAM) improves photosynthetic efficiency under limited water availability relative to C3 photosynthesis. It is widely accepted that CAM plants have evolved from C3 plants and it is hypothesized that CAM is under the control of the internal circadian clock. However, the role that the circadian clock plays in the evolution of CAM is not well understood. To identify the molecular basis of circadian control over CAM evolution, rhythmic gene sets were identified in a CAM model plant species (Kalanchoë fedtschenkoi) and a C3 model plant species (Arabidopsis thaliana) through analysis of diel time-course gene expression data using multiple periodicity detection algorithms. Based on protein sequences, ortholog groups were constructed containing genes from each of these two species. The ortholog groups were categorized into five gene sets based on conservation and diversification of rhythmic gene expression. Interestingly, minimal functional overlap was observed when comparing the rhythmic gene sets of each species. Specifcally, metabolic processes were enriched in the gene set under circadian control in K. fedtschenkoi and numerous genes were found to have retained or gained rhythmic expression in K. fedtsechenkoi. Additonally, several rhythmic orthologs, including CAM-related orthologs, displayed phase shifts between species. Results of this analysis point to several mechanisms by which the circadian clock plays a role in the evolution of CAM. These genes provide a set of testable hypotheses for future experiments.

Defining the genetic components of callus formation: A GWAS approach.

A characteristic feature of plant cells is the ability to form callus from parenchyma cells in response to biotic and abiotic stimuli. Tissue culture propagation of recalcitrant plant species and genetic engineering for desired phenotypes typically depends on efficient in vitro callus generation. Callus formation is under genetic regulation, and consequently, a molecular understanding of this process underlies successful generation for propagation materials and/or introduction of genetic elements in experimental or industrial applications. Herein, we identified 11 genetic loci significantly associated with callus formation in Populus trichocarpa using a genome-wide association study (GWAS) approach. Eight of the 11 significant gene associations were consistent across biological replications, exceeding a chromosome-wide-log10 (p) = 4.46 [p = 3.47E-05] Bonferroni-adjusted significance threshold. These eight genes were used as hub genes in a high-resolution co-expression network analysis to gain insight into the genome-wide basis of callus formation. A network of positively and negatively co-expressed genes, including several transcription factors, was identified. As proof-of-principle, a transient protoplast assay confirmed the negative regulation of a Chloroplast Nucleoid DNA-binding-related gene (Potri.018G014800) by the LEC2 transcription factor. Many of the candidate genes and co-expressed genes were 1) linked to cell division and cell cycling in plants and 2) showed homology to tumor and cancer-related genes in humans. The GWAS approach based on a high-resolution marker set, and the ability to manipulate targets genes in vitro, provided a catalog of high-confidence genes linked to callus formation that can serve as an important resource for successful manipulation of model and non-model plant species, and likewise, suggests a robust method of discovering common homologous functions across organisms.

The Kalanchoë genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism.

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops.

New technologies accelerate the exploration of non-coding RNAs in horticultural plants.

Non-coding RNAs (ncRNAs), that is, RNAs not translated into proteins, are crucial regulators of a variety of biological processes in plants. While protein-encoding genes have been relatively well-annotated in sequenced genomes, accounting for a small portion of the genome space in plants, the universe of plant ncRNAs is rapidly expanding. Recent advances in experimental and computational technologies have generated a great momentum for discovery and functional characterization of ncRNAs. Here we summarize the classification and known biological functions of plant ncRNAs, review the application of next-generation sequencing (NGS) technology and ribosome profiling technology to ncRNA discovery in horticultural plants and discuss the application of new technologies, especially the new genome-editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, to functional characterization of plant ncRNAs.

Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis.

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as 'effectors', i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa and 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. We conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.

Plant-Derived Terpenes: A Feedstock for Specialty Biofuels.

Research toward renewable and sustainable energy has identified specific terpenes capable of supplementing or replacing current petroleum-derived fuels. Despite being naturally produced and stored by many plants, there are few examples of commercial recovery of terpenes from plants because of low yields. Plant terpene biosynthesis is regulated at multiple levels, leading to wide variability in terpene content and chemistry. Advances in the plant molecular toolkit, including annotated genomes, high-throughput omics profiling, and genome editing, have begun to elucidate plant terpene metabolism, and such information is useful for bioengineering metabolic pathways for specific terpenes. We review here the status of terpenes as a specialty biofuel and discuss the potential of plants as a viable agronomic solution for future terpene-derived biofuels.

The Arabidopsis Domain of Unknown Function 1218 (DUF1218) Containing Proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) Function Redundantly to Alter Secondary Cell Wall Lignin Content.

DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 (MWL-2), the most closely related gene to MWL-1 in the protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.

Cell wall-related proteins of unknown function: missing links in plant cell wall development.

Lignocellulosic biomass is an important feedstock for the pulp and paper industry as well as emerging biofuel and biomaterial industries. However, the recalcitrance of the secondary cell wall to chemical or enzymatic degradation remains a major hurdle for efficient extraction of economically important biopolymers such as cellulose. It has been estimated that approximately 10-15% of about 27,000 protein-coding genes in the Arabidopsis genome are dedicated to cell wall development; however, only about 130 Arabidopsis genes thus far have experimental evidence validating cell wall function. While many genes have been implicated through co-expression analysis with known genes, a large number are broadly classified as proteins of unknown function (PUFs). Recently the functionality of some of these unknown proteins in cell wall development has been revealed using reverse genetic approaches. Given the large number of cell wall-related PUFs, how do we approach and subsequently prioritize the investigation of such unknown genes that may be essential to or influence plant cell wall development and structure? Here, we address the aforementioned question in two parts; we first identify the different kinds of PUFs based on known and predicted features such as protein domains. Knowledge of inherent features of PUFs may allow for functional inference and a concomitant link to biological context. Secondly, we discuss omics-based technologies and approaches that are helping identify and prioritize cell wall-related PUFs by functional association. In this way, hypothesis-driven experiments can be designed for functional elucidation of many proteins that remain missing links in our understanding of plant cell wall biosynthesis.

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion.

Plant biomass, as an abundant renewable carbon source, is a promising alternative to fossil fuels. However, the enzymes most commonly used for depolymerization of lignocellulosic biomass are expensive, and the development of cost-effective alternative conversion technologies would be desirable. One possible option is the heterologous expression of genes encoding lignocellulose-digesting enzymes in plant tissues. To overcome simultaneously issues of toxicity and incompatibility with high-temperature steam explosion processes, the use of heterologous genes encoding hyperthermophilic enzymes may be an attractive alternative. This approach could reduce the need for exogenous enzyme additions prior to fermentation, reducing the cost of the complete processing operation. This review highlights recent advances and future prospects for using hyperthermophilic enzymes in the biofuels industry.