A Zinc Finger Motif in the P1 N Terminus, Highly Conserved in a Subset of Potyviruses, Is Associated with the Host Range and Fitness of Telosma Mosaic Virus.

P1 is the first protein translated from the genomes of most viruses in the family Potyviridae, and it contains a C-terminal serine-protease domain that cis-cleaves the junction between P1 and HCPro in most cases. Intriguingly, P1 is the most divergent among all mature viral factors, and its roles during viral infection are still far from understood. In this study, we found that telosma mosaic virus (TelMV, genus Potyvirus) in passion fruit, unlike TelMV isolates present in other hosts, has two stretches at the P1 N terminus, named N1 and N2, with N1 harboring a Zn finger motif. Further analysis revealed that at least 14 different potyviruses, mostly belonging to the bean common mosaic virus subgroup, encode a domain equivalent to N1. Using the newly developed TelMV infectious cDNA clones from passion fruit, we demonstrated that N1, but not N2, is crucial for viral infection in both Nicotiana benthamiana and passion fruit. The regulatory effects of N1 domain on P1 cis cleavage, as well as the accumulation and RNA silencing suppression (RSS) activity of its cognate HCPro, were comprehensively investigated. We found that N1 deletion decreases HCPro abundance at the posttranslational level, likely by impairing P1 cis cleavage, thus reducing HCPro-mediated RSS activity. Remarkably, disruption of the Zn finger motif in N1 did not impair P1 cis cleavage and HCPro accumulation but severely debilitated TelMV fitness. Therefore, our results suggest that the Zn finger motif in P1s plays a critical role in viral infection that is independent of P1 protease activity and self-release, as well as HCPro accumulation and silencing suppression. IMPORTANCE Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy. P1, the first protein translated from most potyvirid genomes, is the most variable viral factor and has attracted great scientific interest. Here, we defined a Zn finger motif-encompassing domain (N1) at the N terminus of P1 among diverse potyviruses phylogenetically related to bean common mosaic virus. Using TelMV as a model virus, we demonstrated that the N1 domain is key for viral infection, as it is involved both in regulating the abundance of its cognate HCPro and in an as-yet-undefined key function unrelated to protease processing and RNA silencing suppression. These results advance our knowledge of the hypervariable potyvirid P1s and highlight the importance for infection of a previously unstudied Zn finger domain at the P1 N terminus.

Benefiting others and self: Production of vitamins in plants.

Vitamins maintain growth and development in humans, animals, and plants. Because plants serve as essential producers of vitamins, increasing the vitamin contents in plants has become a goal of crop breeding worldwide. Here, we begin with a summary of the functions of vitamins. We then review the achievements to date in elucidating the molecular mechanisms underlying how vitamins are synthesized, transported, and regulated in plants. We also stress the exploration of variation in vitamins by the use of forward genetic approaches, such as quantitative trait locus mapping and genome-wide association studies. Overall, we conclude that exploring the diversity of vitamins could provide new insights into plant metabolism and crop breeding.

Metabolic GWAS-based dissection of genetic bases underlying the diversity of plant metabolism.

Plants have served as sources providing humans with metabolites for food and nutrition, biomaterials for living, and treatment for pain and disease. Plants produce a huge array of metabolites, with an immense diversity at both the population and individual levels. Dissection of the genetic bases for metabolic diversity has attracted increasing research attention. The concept of genome-wide association study (GWAS) was extended to studies on the diversity of plant metabolome that benefitted from the development of mass-spectrometry-based analytical systems and genome sequencing technologies. Metabolic genome-wide association study (mGWAS) is one of the most powerful tools for global identification of genetic determinants for diversity of plant metabolism. Recently, mGWAS has been performed for various species with continuous improvements, providing deeper insights into the genetic bases of metabolic diversity. In this review, we discuss fully the achievements to date and remaining challenges that are associated with both mGWAS and mGWAS-based multi-dimensional analysis. We begin with a summary of GWAS and its development based on statistical methods and populations. As variation in targeted traits is essential for GWAS, we review metabolic diversity and its rise at both the population and individual levels. Subsequently, the application of mGWAS for plants and its corresponding achievements are fully discussed. We address the current knowledge on mGWAS-based multi-dimensional analysis and emerging insights into the diversity of metabolism.

Comparative analysis of metabolome of rice seeds at three developmental stages using a recombinant inbred line population.

Plants are considered an important food and nutrition source for humans. Despite advances in plant seed metabolomics, knowledge about the genetic and molecular bases of rice seed metabolomes at different developmental stages is still limited. Here, using Zhenshan 97 (ZS97) and Minghui 63 (MH63), we performed a widely targeted metabolic profiling in seeds during grain filling, mature seeds and germinating seeds. The diversity between MH63 and ZS97 was characterized in terms of the content of metabolites and the metabolic shifting across developmental stages. Taking advantage of the ultra-high-density genetic map of a population of 210 recombinant inbred lines (RILs) derived from a cross between ZS97 and MH63, we identified 4681 putative metabolic quantitative trait loci (mQTLs) in seeds across the three stages. Further analysis of the mQTLs for the codetected metabolites across the three stages revealed that the genetic regulation of metabolite accumulation was closely related to developmental stage. Using in silico analyses, we characterized 35 candidate genes responsible for 30 structurally identified or annotated compounds, among which LOC_Os07g04970 and LOC_Os06g03990 were identified to be responsible for feruloylserotonin and l-asparagine content variation across populations, respectively. Metabolite-agronomic trait association and colocation between mQTLs and phenotypic quantitative trait loci (pQTLs) revealed the complexity of the metabolite-agronomic trait relationship and the corresponding genetic basis.

OsTSD2-mediated cell wall modification affects ion homeostasis and salt tolerance.

Salt stress is a major environmental threat to meeting the food demands of an increasing global population. The identification and exploitation of salt adaption mechanisms in plants are therefore vital for crop breeding. We here define the rice mutant (sstm1) whose salt sensitivity was unambiguously assigned to a single T-DNA insertion through segregational analysis following backcrossing to the wild type line. Insertion was within OsTSD2, which encoded a pectin methyltransferase. The sstm1 and allelic mutants, collectively known as tsd2, displayed higher content of Na+ and lower level of K+ in the shoot, which is likely to lead to reduced salt tolerance. Molecular analysis revealed reduced expression of the genes maintaining K+ /Na+ homeostasis in tsd2, including OsHKT1;5, OsSOS1, and OsKAT1. Furthermore, OsTSD2 influenced ion distribution between the hull and the rice seed, which could improve food safety with heavy metal pollution. Amino acid levels tended to be increased in tsd2 mutants, implicating a role of pectin in the regulation of metabolism. Taken together, we have demonstrated an important facet of salt tolerance, which implicated OsTSD2-mediated cell wall pectin modification as a key component that could be widely applied in crop science.

Rice putative methyltransferase gene OsTSD2 is required for root development involving pectin modification.

Pectin synthesis and modification are vital for plant development, although the underlying mechanisms are still not well understood. Here, we report the functional characterization of the OsTSD2 gene, which encodes a putative methyltransferase in rice. All three independent T-DNA insertion lines of OsTSD2 displayed dwarf phenotypes and serial alterations in different zones of the root. These alterations included abnormal cellular adhesion and schizogenous aerenchyma formation in the meristematic zone, inhibited root elongation in the elongation zone, and higher lateral root density in the mature zone. Immunofluorescence (with LM19) and Ruthenium Red staining of the roots showed that unesterified homogalacturonan (HG) was increased in Ostsd2 mutants. Biochemical analysis of cell wall pectin polysaccharides revealed that both the monosaccharide composition and the uronic acid content were decreased in Ostsd2 mutants. Increased endogenous ABA content and opposite roles performed by ABA and IAA in regulating cellular adhesion in the Ostsd2 mutants suggested that OsTSD2 is required for root development in rice through a pathway involving pectin synthesis/modification. A hypothesis to explain the relationship among OsTSD2, pectin methylesterification, and root development is proposed, based on pectin's function in regional cell extension/division in a zone-dependent manner.

Interaction between carbon metabolism and phosphate accumulation is revealed by a mutation of a cellulose synthase-like protein, CSLF6.

Phosphorus is an essential macronutrient for plant growth and development. However, the network that affects phosphate (Pi) accumulation in crops is not well established. It is reported here that OsCSLF6, a member of the cellulose synthase-like family (CSLF), which is found only in grasses, is involved in Pi accumulation. The oscslf6 mutants (oscslf6-1 and oscslf6-2) display Pi toxic symptoms and increased Pi accumulation in both roots and shoots under the Pi-sufficient condition, which correlate with the induced expression of Pi transporters in the knockout mutants. Consistent with the over-accumulation of Pi, a significant decrease in primary root length, adventitious root length, and adventitious root number were observed in the oscslf6 mutants when compared with the wild type (WT) under Pi-sufficient conditions. In addition, the sucrose (Suc) level was increased in the oscslf6 mutants and the expression of sucrose synthases (OsSUS4/5) and sucrose transporters (OsSUT1/2/4/OsSweet14) genes were also induced in the shoots of oscslf6 mutants, suggesting that OsCSLF6 may play a role in affecting Pi accumulation by affecting the level of carbon metabolism.

Photoperiod-Dependent Nutrient Accumulation in Rice Cultivated in Plant Factories: A Comparative Metabolomic Analysis.

Plant factories offer a promising solution to some of the challenges facing traditional agriculture, allowing for year-round rapid production of plant-derived foods. However, the effects of conditions in plant factories on metabolic nutrients remain to be explored. In this study, we used three rice accessions (KongYu131, HuangHuaZhan, and Kam Sweet Rice) as objectives, which were planted in a plant factory with strict photoperiods that are long-day (12 h light/12 h dark) or short-day (8 h light/16 h dark). A total of 438 metabolites were detected in the harvested rice grains. The difference in photoperiod leads to a different accumulation of metabolites in rice grains. Most metabolites accumulated significantly higher levels under the short-day condition than the long-day condition. Differentially accumulated metabolites were enriched in the amino acids and vitamin B6 pathway. Asparagine, pyridoxamine, and pyridoxine are key metabolites that accumulate at higher levels in rice grains harvested from the short-day photoperiod. This study reveals the photoperiod-dependent metabolomic differences in rice cultivated in plant factories, especially the metabolic profiling of taste- and nutrition-related compounds.

WTV2.0: A high-coverage plant volatilomics method with a comprehensive selective ion monitoring acquisition mode.

Volatilomics is essential for understanding the biological functions and fragrance contributions of plant volatiles. However, the annotation coverage achieved using current untargeted and widely targeted volatomics (WTV) methods has been limited by low sensitivity and/or low acquisition coverage. Here, we introduce WTV 2.0, which enabled the construction of a high-coverage library containing 2111 plant volatiles, and report the development of a comprehensive selective ion monitoring (cSIM) acquisition method, including the selection of characteristic qualitative ions with the minimal ion number for each compound and an optimized segmentation method, that can acquire the smallest but sufficient number of ions for most plant volatiles, as well as the automatic qualitative and semi-quantitative analysis of cSIM data. Importantly, the library and acquisition method we developed can be self-expanded by incorporating compounds not present in the library, utilizing the obtained cSIM data. We showed that WTV 2.0 increases the median signal-to-noise ratio by 7.6-fold compared with the untargeted method, doubled the annotation coverage compared with the untargeted and WTV 1.0 methods in tomato fruit, and led to the discovery of menthofuran as a novel flavor compound in passion fruit. WTV 2.0 is a Python library with a user-friendly interface and is applicable to profiling of volatiles and primary metabolites in any species.

OsDWARF10, transcriptionally repressed by OsSPL3, regulates the nutritional metabolism of polished rice.

Strigolactone (SL) plays essential roles in plant development and the metabolism of rice leaves. However, the impact of SL on the accumulation of nutritional metabolites in polished rice, as well as the transcription factors directly involved in SL synthesis, remains elusive. In this study, we performed a metabolome analysis on polished rice samples from mutants of an SL biosynthetic gene, OsDWARF10 (OsD10). Compared with those in the wild type plants, primary and secondary metabolites exhibited a series of alterations in the d10 mutants. Notably, the d10 mutants showed a substantial increase in the amino acids and vitamins content. Through a yeast one-hybridization screening assay, we identified OsSPL3 as a transcription factor that binds to the OsD10 promoter, thereby inhibiting OsD10 transcription in vivo and in vitro. Furthermore, we conducted a metabolic profiling analysis in polished rice from plants that overexpressed OsSPL3 and observed enhanced levels of amino acids and vitamins. This study identified a novel transcriptional repressor of the SL biosynthetic gene and elucidated the regulatory roles of OsSPL3 and OsD10 on the accumulation of nutritional metabolites in polished rice.

Comparative Metabolomic Analysis Reveals the Role of OsHPL1 in the Cold-Induced Metabolic Changes in Rice.

Cytochrome P450 (CYP74) family members participate in the generation of oxylipins and play essential roles in plant adaptation. However, the metabolic reprogramming mediated by CYP74s under cold stress remains largely unexplored. Herein, we report how cold-triggered OsHPL1, a member of the CYP74 family, modulates rice metabolism. Cold stress significantly induced the expression of OsHPL1 and the accumulation of OPDA (12-oxo-phytodienoic acid) and jasmonates in the wild-type (WT) plants. The absence of OsHPL1 attenuates OPDA accumulation to a low temperature. Then, we performed a widely targeted metabolomics study covering 597 structurally annotated compounds. In the WT and hpl1 plants, cold stress remodeled the metabolism of lipids and amino acids. Although the WT and hpl1 mutants shared over one hundred cold-affected differentially accumulated metabolites (DAMs), some displayed distinct cold-responding patterns. Furthermore, we identified 114 and 56 cold-responding DAMs, specifically in the WT and hpl1 mutants. In conclusion, our work characterized cold-triggered metabolic rewiring and the metabolic role of OsHPL1 in rice.

Integrative Analysis of Metabolome and Transcriptome Reveals the Role of Strigolactones in Wounding-Induced Rice Metabolic Re-Programming.

Plants have evolved mechanisms to adapt to wounding, a threat occurring separately or concomitantly with other stresses. During the last decades, many efforts have been made to elucidate the wounding signaling transduction. However, we know little about the metabolic re-programming under wounding, let alone whether and how strigolactones (SLs) participate in this progress. Here, we reported a metabolomic and transcriptomic analysis of SLs synthetic and signal mutants in rice before and after wounding. A series of metabolites differentially responded to wounding in the SLs mutants and wild-type rice, among which flavones were enriched. Besides, the SLs mutants accumulated more jasmonic acid (JA) and jasmonyl isoleucine (JA-lle) than the wild-type rice after wounding, suggesting an interplay of SLs and JAs during responding to wounding. Further transcriptome data showed that cell wall, ethylene, and flavones pathways might be affected by wounding and SLs. In addition, we identified candidate genes regulated by SLs and responding to wounding. In conclusion, our work provides new insights into wounding-induced metabolic re-programming and the SLs' function.

Metabolomic Analysis Reveals Nutritional Diversity among Three Staple Crops and Three Fruits.

More than 2 billion people worldwide are under threat of nutritional deficiency. Thus, an in-depth comprehension of the nutritional composition of staple crops and popular fruits is essential for health. Herein, we performed LC-MS-based non-targeted and targeted metabolome analyses with crops (including wheat, rice, and corn) and fruits (including grape, banana, and mango). We detected a total of 2631 compounds by using non-targeted strategy and identified more than 260 nutrients. Our work discovered species-dependent accumulation of common present nutrients in crops and fruits. Although rice and wheat lack vitamins and amino acids, sweet corn was rich in most amino acids and vitamins. Among the three fruits, mango had more vitamins and amino acids than grape and banana. Grape and banana provided sufficient 5-methyltetrahydrofolate and vitamin B6, respectively. Moreover, rice and grape had a high content of flavonoids. In addition, the three crops contained more lipids than fruits. Furthermore, we also identified species-specific metabolites. The crops yielded 11 specific metabolites, including flavonoids, lipids, and others. Meanwhile, most fruit-specific nutrients were flavonoids. Our work discovered the complementary pattern of essential nutrients in crops and fruits, which provides metabolomic evidence for a healthy diet.

Nondestructive 3D phenotyping method of passion fruit based on X-ray micro-computed tomography and deep learning.

Passion fruit is a tropical liana of the Passiflora family that is commonly planted throughout the world due to its abundance of nutrients and industrial value. Researchers are committed to exploring the relationship between phenotype and genotype to promote the improvement of passion fruit varieties. However, the traditional manual phenotyping methods have shortcomings in accuracy, objectivity, and measurement efficiency when obtaining large quantities of personal data on passion fruit, especially internal organization data. This study selected samples of passion fruit from three widely grown cultivars, which differed significantly in fruit shape, size, and other morphological traits. A Micro-CT system was developed to perform fully automated nondestructive imaging of the samples to obtain 3D models of passion fruit. A designed label generation method and segmentation method based on U-Net model were used to distinguish different tissues in the samples. Finally, fourteen traits, including fruit volume, surface area, length and width, sarcocarp volume, pericarp thickness, and traits of fruit type, were automatically calculated. The experimental results show that the segmentation accuracy of the deep learning model reaches more than 0.95. Compared with the manual measurements, the mean absolute percentage error of the fruit width and length measurements by the Micro-CT system was 1.94% and 2.89%, respectively, and the squares of the correlation coefficients were 0.96 and 0.93. It shows that the measurement accuracy of external traits of passion fruit is comparable to manual operations, and the measurement of internal traits is more reliable because of the nondestructive characteristics of our method. According to the statistical data of the whole samples, the Pearson analysis method was used, and the results indicated specific correlations among fourteen phenotypic traits of passion fruit. At the same time, the results of the principal component analysis illustrated that the comprehensive quality of passion fruit could be scored using this method, which will help to screen for high-quality passion fruit samples with large sizes and high sarcocarp content. The results of this study will firstly provide a nondestructive method for more accurate and efficient automatic acquisition of comprehensive phenotypic traits of passion fruit and have the potential to be extended to more fruit crops. The preliminary study of the correlation between the characteristics of passion fruit can also provide a particular reference value for molecular breeding and comprehensive quality evaluation.

Development of a widely targeted volatilomics method for profiling volatilomes in plants.

Volatile organic compounds play essential roles in plant environment interactions as well as determining the fragrance of plants. Although gas chromatography-mass spectrometry-based untargeted metabolomics is commonly used to assess plant volatiles, it suffers from high spectral convolution, low detection sensitivity, a limited number of annotated metabolites, and relatively poor reproducibility. Here, we report a widely targeted volatilomics (WTV) method that involves using a "targeted spectra extraction" algorithm to address spectral convolution, constructing a high-coverage MS2 spectral tag library to expand volatile annotation, adapting a multiple reaction monitoring mode to improve sensitivity, and using regression models to adjust for signal drift. The newly developed method was used to profile the volatilome of rice grains. Compared with the untargeted method, the newly developed WTV method shows higher sensitivity (for example, the signal-to-noise ratio of guaicol increased from 4.1 to 18.8), high annotation coverage (the number of annotated volatiles increased from 43 to 132), and better reproducibility (the number of volatiles in quality control samples with relative standard deviation value below 30.0% increased from 14 to 92 after normalization). Using the WTV method, we studied the metabolic responses of tomato to environmental stimuli and profiled the volatilomes of different rice accessions. The results identified benzothiazole as a potential airborne signal priming tomato plants for enhanced defense and 2-nonanone and 2-heptanone as novel aromatic compounds contributing to rice fragrance. These case studies suggest that the widely targeted volatilomics method is more efficient than those currently used and may considerably promote plant volatilomics studies.

Integration of rhythmic metabolome and transcriptome provides insights into the transmission of rhythmic fluctuations and temporal diversity of metabolism in rice.

Various aspects of the organisms adapt to cyclically changing environmental conditions via transcriptional regulation. However, the role of rhythmicity in altering the global aspects of metabolism is poorly characterized. Here, we subjected four rice (Oryza sativa) varieties to a range of metabolic profiles and RNA-seq to investigate the temporal relationships of rhythm between transcription and metabolism. More than 40% of the rhythmic genes and a quarter of metabolites conservatively oscillated across four rice accessions. Compared with the metabolome, the transcriptome was more strongly regulated by rhythm; however, the rhythm of metabolites had an obvious opposite trend between day and night. Through association analysis, the time delay of rhythmic transmission from the transcript to the metabolite level was ∼4 h under long-day conditions, although the transmission was nearly synchronous for carbohydrate and nucleotide metabolism. The rhythmic accumulation of metabolites maintained highly coordinated temporal relationships in the metabolic network, whereas the correlation of some rhythmic metabolites, such as branched-chain amino acids (BCAAs), was significantly different intervariety. We further demonstrated that the cumulative diversity of BCAAs was due to the differential expression of branched-chain aminotransferase 2 at dawn. Our research reveals the flexible pattern of rice metabolic rhythm existing with conservation and diversity.

A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification.

CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors ( Liu et al., 2020 ). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing. In this protocol, we describe the detailed step-by-step instructions for using this system.

Cross-Species Comparison of Metabolomics to Decipher the Metabolic Diversity in Ten Fruits.

Fruits provide humans with multiple kinds of nutrients and protect humans against worldwide nutritional deficiency. Therefore, it is essential to understand the nutrient composition of various fruits in depth. In this study, we performed LC-MS-based non-targeted metabolomic analyses with ten kinds of fruit, including passion fruit, mango, starfruit, mangosteen, guava, mandarin orange, grape, apple, blueberry, and strawberry. In total, we detected over 2500 compounds and identified more than 300 nutrients. Although the ten fruits shared 909 common-detected compounds, each species accumulated a variety of species-specific metabolites. Additionally, metabolic profiling analyses revealed a constant variation in each metabolite's content across the ten fruits. Moreover, we constructed a neighbor-joining tree using metabolomic data, which resembles the single-copy protein-based phylogenetic tree. This indicates that metabolome data could reflect the genetic relationship between different species. In conclusion, our work enriches knowledge on the metabolomics of fruits, and provides metabolic evidence for the genetic relationships among these fruits.

Integrative Metabolomic and Transcriptomic Analyses Reveal Metabolic Changes and Its Molecular Basis in Rice Mutants of the Strigolactone Pathway.

Plants have evolved many metabolites to meet the demands of growth and adaptation. Although strigolactones (SLs) play vital roles in controlling plant architecture, their function in regulating plant metabolism remains elusive. Here we report the integrative metabolomic and transcriptomic analyses of two rice SL mutants, d10 (a biosynthesis mutant) and d14 (a perception mutant). Both mutants displayed a series of metabolic and transcriptional alterations, especially in the lipid, flavonoid, and terpenoid pathways. Levels of several diterpenoid phytoalexins were substantially increased in d10 and d14, together with the induction of terpenoid gene cluster and the corresponding upstream transcription factor WRKY45, an established determinant of plant immunity. The fact that WRKY45 is a target of IPA1, which acted as a downstream transcription factor of SL signaling, suggests that SLs contribute to plant defense through WRKY45 and phytoalexins. Moreover, our data indicated that SLs may modulate rice metabolism through a vast number of clustered or tandemly duplicated genes. Our work revealed a central role of SLs in rice metabolism. Meanwhile, integrative analysis of the metabolome and transcriptome also suggested that SLs may contribute to metabolite-associated growth and defense.

A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice.

Rapidly growing genetics and bioinformatics studies provide us with an opportunity to obtain a global view of the genetic basis of traits, but also give a challenge to the function validation of candidate genes. CRISPR/Cas9 is an emerging and efficient tool for genome editing. To construct expression clones for the CRISPR/Cas9, most current methods depend on traditional cloning using Gateway reaction or specific type IIS restriction enzymes and DNA ligation, based on multiple steps of PCR. We developed a system for introducing sgRNA expression cassette(s) directly into plant binary vectors in one step. In this system, one sgRNA expression cassette(s) is generated by an optimized multiplex PCR, in which an overlapping PCR took place. Whilst, two sgRNA expression cassettes were amplified in a single round of PCR. Subsequently, an LR or Golden gate reaction was set up with unpurified PCR product and befitting destination vector. We are able to construct expression clones within 36 h, which greatly improves efficiency and saves cost. Furthermore, the efficiency of this system was verified by an agrobacterium-mediated genetic transformation in rice. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing.