CRISPR for Crop Improvement: An Update Review

The availability of genome sequences for several crops and advances in genome editing approaches has opened up possibilities to breed for almost any given desirable trait. Advancements in genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) has made it possible for molecular biologists to more precisely target any gene of interest. However, these methodologies are expensive and time-consuming as they involve complicated steps that require protein engineering. Unlike first-generation genome editing tools, CRISPR/Cas9 genome editing involves simple designing and cloning methods, with the same Cas9 being potentially available for use with different guide RNAs targeting multiple sites in the genome. After proof-of-concept demonstrations in crop plants involving the primary CRISPR-Cas9 module, several modified Cas9 cassettes have been utilized in crop plants for improving target specificity and reducing off-target cleavage (e.g., Nmcas9, Sacas9, and Stcas9). Further, the availability of Cas9 enzymes from additional bacterial species has made available options to enhance specificity and efficiency of gene editing methodologies. This review summarizes the options available to plant biotechnologists to bring about crop improvement using CRISPR/Cas9 based genome editing tools and also presents studies where CRISPR/Cas9 has been used for enhancing biotic and abiotic stress tolerance. Application of these techniques will result in the development of non-genetically modified (Non-GMO) crops with the desired trait that can contribute to increased yield potential under biotic and abiotic stress conditions

Unravelling the physiological basis of salinity stress tolerance in cultivated and wild rice species

Wild rice species provide a rich source of genetic diversity for possible introgression of salinity stress tolerance in cultivated rice. We investigated the physiological basis of salinity stress tolerance in Oryza species by using six rice genotypes (Oryza sativa L.) and four wild rice species. Three weeks of salinity treatment significantly (P < 0.05) reduced physiological and growth indices of all cultivated and wild rice lines. However, the impact of salinity-induced growth reduction differed substantially among accessions. Salt tolerant accessions showed better control over gas exchange properties, exhibited higher tissue tolerance, and retained higher potassium ion content despite higher sodium ion accumulation in leaves. Wild rice species showed relatively lower and steadier xylem sap sodium ion content over the period of 3 weeks analysed, suggesting better control over ionic sodium xylem loading and its delivery to shoots with efficient vacuolar sodium ion sequestration. Contrary to this, saline sensitive genotypes managed to avoid initial Na+ loading but failed to accomplish this in the long term and showed higher sap sodium ion content. Conclusively, our results suggest that wild rice genotypes have more efficient control over xylem sodium ion loading, rely on tissue tolerance mechanisms and allow for a rapid osmotic adjustment by using sodium ions as cheap osmoticum for osmoregulation

Comparative analysis of Root Na+ relation under salinity between Oryza sativa and Oryza coarctata

Na+ toxicity is one of the major physiological constraints imposed by salinity on plant performance. At the same time, Na+ uptake may be beneficial under some circumstances as an easily accessible inorganic ion that can be used for increasing solute concentrations and maintaining cell turgor. Two rice species, Oryza sativa (cultivated rice, salt-sensitive) and Oryza coarctata (wild rice, salt-tolerant), demonstrated different strategies in controlling Na+ uptake. Glasshouse experiments and gene expression analysis suggested that salt-treated wild rice quickly increased xylem Na+ loading for osmotic adjustment but maintained a non-toxic level of stable shoot Na+ concentration by increased activity of a high affinity K+ transporter HKT1;5 (essential for xylem Na+ unloading) and a Na+ /H+ exchanger NHX (for sequestering Na+ and K+ into root vacuoles). Cultivated rice prevented Na+ uptake and transport to the shoot at the beginning of salt treatment but failed to maintain it in the long term. While electrophysiological assays revealed greater net Na+ uptake upon salt application in cultivated rice, O. sativa plants showed much stronger activation of the root plasma membrane Na+ /H+ Salt Overly Sensitive 1 (SOS1) exchanger. Thus, it appears that wild rice limits passive Na+ entry into root cells while cultivated rice relies heavily on SOS1-mediating Na+ exclusion, with major penalties imposed by the existence of the “futile cycle” at the plasma membrane

Vacuolar Targeting of Cry1Ac and its Effects on Expression and Stability in Tobacco.

Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes.Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes

Proto Kranz-like leaf traits and cellular ionic regulation are associated with salinity tolerance in a halophytic wild rice

Species of wild rice (Oryza spp.) possess a wide range of stress tolerance traits that can be potentially utilized in breeding climate-resilient cultivated rice cultivars (Oryza sativa) thereby aiding global food security. In this study, we conducted a greenhouse trial to evaluate the salinity tolerance of six wild rice species, one cultivated rice cultivar (IR64) and one landrace (Pokkali) using a range of electrophysiological, imaging, and whole-plant physiological techniques. Three wild species (O. latifolia, O. officinalis and O. coarctata) were found to possess superior salinity stress tolerance. The underlying mechanisms, however, were strikingly different. Na+ accumulation in leaves of O. latifolia, O. officinalis and O. coarctata were significantly higher than the tolerant landrace, Pokkali. Na+ accumulation in mesophyll cells was only observed in O. coarctata, suggesting that O. officinalis and O. latifolia avoid Na+ accumulation in mesophyll by allocating Na+ to other parts of the leaf. The finding also suggests that O. coarctata might be able to employ Na+ as osmolyte without affecting its growth. Further study of Na+ allocation in leaves will be helpful to understand the mechanisms of Na+ accumulation in these species. In addition, O. coarctata showed Proto Kranz-like leaf anatomy (enlarged bundle sheath cells and lower numbers of mesophyll cells), and higher expression of C4-related genes (e.g., NADPME, PPDK) and was a clear outlier with respect to salinity tolerance among the studied wild and cultivated Oryza species. The unique phylogenetic relationship of O. coarctata with C4 grasses suggests the potential of this species for breeding rice with high photosynthetic rate under salinity stress in the future

Age matters: Life-stage, tissue, and sex-specific gene expression dynamics in Ips typographus (Coleoptera: Curculionidae: Scolytinae)

The Eurasian spruce bark beetle (ESBB), Ips typographus, has recently caused catastrophic damage to Norway spruce (Picea abies) forests in Europe, resulting in the loss of more than 100 million cubic meters of wood. Traditional forest management strategies have failed to constrain the growing infestation rate; hence, novel measures must be deployed. A better understanding of ESBB physiology and adaptation to host allelochemicals may provide a platform for future management strategies using molecular tools such as RNA interference. To understand ESBB physiology and adaptation, the current study unraveled the gene expression dynamics of ESBB in different life stages and tissues. We obtained ESBB transcriptomes for different life stages [larvae (L1, L2, and L3), pupa, callow, and sclerotized adult] and male/female tissues (gut, fat body, and head) from callow and sclerotized adult beetles. Differential gene expression analysis (DGE) identified multiple gene families related to detoxification, digestion, resistance, and transport in different life stages and tissues of the beetle. Gene Ontology (GO) enrichment revealed 61 critical metabolic pathways enriched across all DGE comparisons. DGE analysis further pinpointed the differential expression of essential genes involved in detoxification, digestion, transport, and defense in various tissues and life stages. RT-qPCR experiments and enzymatic assays corroborated the findings further. The catalogue of differentially expressed genes identified in ESBB could aid better understanding of ESBB physiology and adaptation to hosts and serve as targets for future RNAi-based ESBB management

Microhair on the adaxial leaf surface of salt secreting halophytic Oryza coarctata Roxb. show distinct morphotypes : isolation for molecular and functional analysis

Halophytic Oryza coarctata is a good model system to examine mechanisms of salinity tolerance in rice. O. coarctata leaves show the presence of microhairs in adaxial leaf surface furrows that secrete salt under salinity. However, detailed molecular and physiological studies of O. coarctata microhairs are limited due to their relative inaccessibility. This work presents a detailed characterization of O. coarctata leaf features. O. coarctata has two types of microhairs on the adaxial leaf surface: longer microhairs (three morphotypes) lining epidermal furrow walls and shorter microhairs (reported first time) arising from bulliform cells. Microhair morphotypes include (i) finger-like, tubular structures, (ii) tubular hairs with bilobed and flattened heads and (iii) bi-or trifurcated hairs. The unicellular nature of microhairs was confirmed by propidium iodide (PI) staining. An efficient method for the isolation and enrichment of O. coarctata microhairs is presented (yield averaging ˜2 Ã 10 5 /g leaf tissue). The robustness of the microhair isolation procedure was confirmed by subsequent viability staining (PI), total RNA isolation and RT-PCR amplification of O. coarctata trichome-specific WUSCHEL-related homeobox 3B (OcWox3B) and transporter gene-specific cDNA sequences. The present microhair isolation work from O. coarctata paves the way for examining genes involved in ion secretion in this halophytic wild rice model

Reduced apoplastic barriers in tissues of shoot-proximal rhizomes of Oryza coarctata are associated with Na+ sequestration

Oryza coarctata is the only wild rice species with significant salinity tolerance. The present work examines the role of the substantial rhizomatous tissues of O. coarctata in conferring salinity tolerance. Transition to an erect phenotype (shoot emergence) from prostrate growth of rhizome tissues is characterized by marked lignification and suberization of supporting sclerenchymatous tissue, epidermis, and bundle sheath cells in aerial shoot-proximal nodes and internodes in O. coarctata. With salinity, however, aerial shoot-proximal internodal tissues show reductions in lignification and suberization, most probably related to re-direction of carbon flux towards synthesis of the osmporotectant proline. Concurrent with hypolignification and reduced suberization, the aerial rhizomatous biomass of O. coarctata appears to have evolved mechanisms to store Na+ in these specific tissues under salinity. This was confirmed by histochemical staining, quantitative real-time reverse transcription–PCR expression patterns of genes involved in lignification/ suberization, Na+ and K+ contents of internodal tissues, as well as non-invasive microelectrode ion flux measurements of NaCl-induced net Na+, K+, and H+ flux profiles of aerial nodes were determined. In O. coarctata, aerial proximal internodes appear to act as ‘traffic controllers’, sending required amounts of Na+ and K+ into developing leaves for osmotic adjustment and turgor-driven growth, while more deeply positioned internodes assume a Na+ buffering/storage role

To exclude or to accumulate? : revealing the role of the sodium HKT1;5 transporter in plant adaptive responses to varying soil salinity

Arid/semi-arid and coastal agricultural areas of the world are especially vulnerable to climate change-driven soil salinity. Salinity tolerance in plants is a complex trait, with salinity negatively affecting crop yield. Plants adopt a range of mechanisms to combat salinity, with many transporter genes being implicated in Na+-partitioning processes. Within these, the high-affinity K+ (HKT) family of transporters play a critical role in K+ and Na+ homeostasis in plants. Among HKT transporters, Type I transporters are Na+-specific. While Arabidopsis has only one Na + -specific HKT (AtHKT1;1), cereal crops have a multiplicity of Type I and II HKT transporters. AtHKT1; 1 (Arabidopsis thaliana) and HKT1; 5 (cereal crops) ‘exclude’ Na+ from the xylem into xylem parenchyma in the root, reducing shoot Na+ and hence, confer sodium tolerance. However, more recent data from Arabidopsis and crop species show that AtHKT1;1/HKT1;5 alleles have a strong genetic association with ‘shoot sodium accumulation’ and concomitant salt tolerance. The review tries to resolve these two seemingly contradictory effects of AtHKT1;1/HKT1;5 operation (shoot exclusion vs shoot accumulation), both conferring salinity tolerance and suggests that contrasting phenotypes are attributable to either hyper-functional or weak AtHKT1;1/HKT1;5 alleles/haplotypes and are under strong selection by soil salinity levels. It also suggests that opposite balancing mechanisms involving xylem ion loading in these contrasting phenotypes exist that require transporters such as SOS1 and CCC. While HKT1; 5 is a crucial but not sole determinant of salinity tolerance, investigation of the adaptive benefit(s) conferred by naturally occurring intermediate HKT1;5 alleles will be important under a climate change scenario