An efficient transformation method for genome editing of elite bread wheat cultivars.

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.

Maize Lethal Necrosis disease: review of molecular and genetic resistance mechanisms, socio-economic impacts, and mitigation strategies in sub-Saharan Africa.

BACKGROUND: Maize lethal necrosis (MLN) disease is a significant constraint for maize producers in sub-Saharan Africa (SSA). The disease decimates the maize crop, in some cases, causing total crop failure with far-reaching impacts on regional food security. RESULTS: In this review, we analyze the impacts of MLN in Africa, finding that resource-poor farmers and consumers are the most vulnerable populations. We examine the molecular mechanism of MLN virus transmission, role of vectors and host plant resistance identifying a range of potential opportunities for genetic and phytosanitary interventions to control MLN. We discuss the likely exacerbating effects of climate change on the MLN menace and describe a sobering example of negative genetic association between tolerance to heat/drought and susceptibility to viral infection. We also review role of microRNAs in host plant response to MLN causing viruses as well as heat/drought stress that can be carefully engineered to develop resistant varieties using novel molecular techniques. CONCLUSIONS: With the dual drivers of increased crop loss due to MLN and increased demand of maize for food, the development and deployment of simple and safe technologies, like resistant cultivars developed through accelerated breeding or emerging gene editing technologies, will have substantial positive impact on livelihoods in the region. We have summarized the available genetic resources and identified a few large-effect QTLs that can be further exploited to accelerate conversion of existing farmer-preferred varieties into resistant cultivars.

Stomatal Development and Gene Expression in Rice Florets.

Stomata play a fundamental role in modulating the exchange of gases between plants and the atmosphere. These microscopic structures form in high numbers on the leaf epidermis and are also present on flowers. Although leaf stomata are well studied, little attention has been paid to the development or function of floral stomata. Here, we characterize in detail the spatial distribution and development of the floral stomata of the indica rice variety IR64. We show that stomatal complexes are present at low density on specific areas of the lemma, palea and anthers and are morphologically different compared to stomata found on leaves. We reveal that in the bract-like organs, stomatal development follows the same cell lineage transitions as in rice leaves and demonstrate that the overexpression of the stomatal development regulators OsEPFL9-1 and OsEPF1 leads to dramatic changes in stomatal density in rice floral organs, producing lemma with approximately twice as many stomata (OsEPFL9-1_oe) or lemma where stomata are practically absent (OsEPF1_oe). Transcriptomic analysis of developing florets also indicates that the cellular transitions during the development of floral stomata are regulated by the same genetic network used in rice leaves. Finally, although we were unable to detect an impact on plant reproduction linked to changes in the density of floral stomata, we report alterations in global gene expression in lines overexpressing OsEPF1 and discuss how our results reflect on the possible role(s) of floral stomata.

Closer vein spacing by ectopic expression of nucleotide-binding and leucine-rich repeat proteins in rice leaves.

KEY MESSAGE: Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves. To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a "Kranz-like" anatomy in leaves that includes decreased interveinal spacing close to that in C4 plant species. As C4 photosynthesis has evolved from C3 photosynthesis independently in multiple lineages, the genes required to facilitate C4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 (CVS1), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty.

Novel Mutant Alleles Reveal a Role of the Extra-Large G Protein in Rice Grain Filling, Panicle Architecture, Plant Growth, and Disease Resistance.

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLGs has the potential to improve agronomic properties in rice.

CRISPR mediated genome engineering to develop climate smart rice: Challenges and opportunities.

Rice is a staple food crop, which ensures the calorie requirement of half of the world's population. With the continued increase in population, rice will play a key role in achieving the food security. However, in the constantly shrinking scenario of rice fields, the necessity of these extra grains of rice must be met by reducing the yield loss due to various abiotic and biotic stresses. The adverse effects of climate impact both quality and quantity of rice production. One of the most desirable applications of CRISPR/Cas technology would be to develop climate smart rice crop to sustain and enhance its productivity in the changing environment. In this review, we analyze the desirable phenotypes and responsible genetic factors, which can be utilized to develop tolerance against major abiotic stresses imposed by climate change through genome engineering. The possibility of utilizing the information from wild resources to engineer the corresponding alleles of cultivated rice has been presented. We have also shed light on available resources for generating genome edited rice lines. The CRISPR/Cas mediated genome editing strategies for engineering of novel genes were proposed to create a plant phenotype, which can face the adversities of climate change. Further, challenges of off-targets and undesirable phenotype were discussed.

The Nucleotide-Dependent Interactome of Rice Heterotrimeric G-Protein α -Subunit.

The rice heterotrimeric G-protein complex, a guanine-nucleotide-dependent on-off switch, mediates vital cellular processes and responses to biotic and abiotic stress. Exchange of bound GDP (resting state) for GTP (active state) is spontaneous in plants including rice and thus there is no need for promoting guanine nucleotide exchange in vivo as a mechanism for regulating the active state of signaling as it is well known for animal G signaling. As such, a master regulator controlling the G-protein activation state is unknown in plants. Therefore, an ab initio approach is taken to discover candidate regulators. The rice Gα subunit (RGA1) is used as bait to screen for nucleotide-dependent protein partners. A total of 264 proteins are identified by tandem mass spectrometry of which 32 were specific to the GDP-bound inactive state and 22 specific to the transition state. Approximately, 10% are validated as previously identified G-protein interactors.

CRISPR-Cas9-Mediated Genome Editing of Rice Towards Better Grain Quality.

With continued economic development in Asia the demand for high yielding varieties with premium grain quality traits is set to increase. This presents a significant challenge to plant breeders because varieties must be tailored to meet regional preferences. It is already apparent that traditional breeding techniques cannot meet this challenge and so emerging genomics technologies will have to be utilized. Genome editing tools afford the ability to efficiently and precisely manipulate the genome. Among these, the bacterial clustered, regularly interspaced, short palindromic repeat (CRISPR) associated protein 9 (Cas9) or CRISPR-Cas9 has emerged as the easiest, most economic, and efficient technology to undertake genome editing in rice. This technique allows precise site-specific gene modification or integration. In this chapter we present a method for utilizing CRISPR-Cas9 for improving grain quality traits in rice; this should enable molecular breeders to quickly and efficiently produce high yielding rice varieties tailored to meet specific cultural and regional requirements for grain quality.

Rice with reduced stomatal density conserves water and has improved drought tolerance under future climate conditions.

Much of humanity relies on rice (Oryza sativa) as a food source, but cultivation is water intensive and the crop is vulnerable to drought and high temperatures. Under climate change, periods of reduced water availability and high temperature are expected to become more frequent, leading to detrimental effects on rice yields. We engineered the high-yielding rice cultivar 'IR64' to produce fewer stomata by manipulating the level of a developmental signal. We overexpressed the rice epidermal patterning factor OsEPF1, creating plants with substantially reduced stomatal density and correspondingly low stomatal conductance. Low stomatal density rice lines were more able to conserve water, using c. 60% of the normal amount between weeks 4 and 5 post germination. When grown at elevated atmospheric CO2 , rice plants with low stomatal density were able to maintain their stomatal conductance and survive drought and high temperature (40°C) for longer than control plants. Low stomatal density rice gave equivalent or even improved yields, despite a reduced rate of photosynthesis in some conditions. Rice plants with fewer stomata are drought tolerant and more conservative in their water use, and they should perform better in the future when climate change is expected to threaten food security.

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice.

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C4-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C4 plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C3 plants.

CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice.

KEY MESSAGE: CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.

Action of multiple intra-QTL genes concerted around a co-localized transcription factor underpins a large effect QTL.

Sub-QTLs and multiple intra-QTL genes are hypothesized to underpin large-effect QTLs. Known QTLs over gene families, biosynthetic pathways or certain traits represent functional gene-clusters of genes of the same gene ontology (GO). Gene-clusters containing genes of different GO have not been elaborated, except in silico as coexpressed genes within QTLs. Here we demonstrate the requirement of multiple intra-QTL genes for the full impact of QTL qDTY12.1 on rice yield under drought. Multiple evidences are presented for the need of the transcription factor 'no apical meristem' (OsNAM12.1) and its co-localized target genes of separate GO categories for qDTY12.1 function, raising a regulon-like model of genetic architecture. The molecular underpinnings of qDTY12.1 support its effectiveness in further improving a drought tolerant genotype and for its validity in multiple genotypes/ecosystems/environments. Resolving the combinatorial value of OsNAM12.1 with individual intra-QTL genes notwithstanding, identification and analyses of qDTY12.1has fast-tracked rice improvement towards food security.

An absolute index (Ab-index) to measure a researcher's useful contributions and productivity.

Bibliographic analysis has been a very powerful tool in evaluating the effective contributions of a researcher and determining his/her future research potential. The lack of an absolute quantification of the author's scientific contributions by the existing measurement system hampers the decision-making process. In this paper, a new metric system, Absolute index (Ab-index), has been proposed that allows a more objective comparison of the contributions of a researcher. The Ab-index takes into account the impact of research findings while keeping in mind the physical and intellectual contributions of the author(s) in accomplishing the task. The Ab-index and h-index were calculated for 10 highly cited geneticists and molecular biologist and 10 young researchers of biological sciences and compared for their relationship to the researchers input as a primary author. This is the first report of a measuring method clarifying the contributions of the first author, corresponding author, and other co-authors and the sharing of credit in a logical ratio. A java application has been developed for the easy calculation of the Ab-index. It can be used as a yardstick for comparing the credibility of different scientists competing for the same resources while the Productivity index (Pr-index), which is the rate of change in the Ab-index per year, can be used for comparing scientists of different age groups. The Ab-index has clear advantage over other popular metric systems in comparing scientific credibility of young scientists. The sum of the Ab-indices earned by individual researchers of an institute per year can be referred to as Pr-index of the institute.

In silico analysis of microsatellites in organellar genomes of major cereals for understanding their phylogenetic relationships.

Microsatellites are abundant across prokaryotic and eukaryotic genomes. However, comparative analysis of microsatellites in the organellar genomes of plants and their utility in understanding phylogeny has not been reported. The purpose of this study was to understand the organization of microsatellites in the coding and non-coding regions of organellar genomes of major cereals viz., rice, wheat, maize and sorghum. About 5.8-14.3% of mitochondrial and 30.5-43.2% of chloroplast microsatellites were observed in the coding regions. About 83.8-86.8% of known mitochondrial genes had at least one microsatellite while this value ranged from 78.6-82.9% among the chloroplast genomes. Dinucleotide repeats were the most abundant in the coding and non-coding regions of the mitochondrial genome while mononucleotides were predominant in chloroplast genomes. Maize harbored more repeats in the mitochondrial genome, which could be due to the larger size of genome. A phylogenetic analysis based on mitochondrial and chloroplast genomic microsatellites revealed that rice and sorghum were closer to each other, while wheat was the farthest and this corroborated with the earlier reported phylogenies based on nuclear genome co-linearity and chloroplast gene-based analysis.

Simple sequence repeats in organellar genomes of rice: frequency and distribution in genic and intergenic regions.

MOTIVATION: Simple sequence repeats (SSRs) are abundant across genomes. However, the significance of SSRs in organellar genomes of rice has not been completely understood. The availability of organellar genome sequences allows us to understand the organization of SSRs in their genic and intergenic regions. RESULTS: We have analyzed SSRs in mitochondrial and chloroplast genomes of rice. We identified 2528 SSRs in the mitochondrial genome and average 870 SSRs in the chloroplast genomes. About 8.7% of the mitochondrial and 27.5% of the chloroplast SSRs were observed in the genic region. Dinucleotides were the most abundant repeats in genic and intergenic regions of the mitochondrial genome while mononucleotides were predominant in the chloroplast genomes. The rps and nad gene clusters of mitochondria had the maximum repeats, while the rpo and ndh gene clusters of chloroplast had the maximum repeats. We identified SSRs in both organellar genomes and validated in different cultivars and species.