Efficient CRISPR/Cas9-based gene knockout in watermelon.

KEY MESSAGE: CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

Inter-species protein trafficking endows dodder (Cuscuta pentagona) with a host-specific herbicide-tolerant trait.

· Besides photosynthates, dodder (Cuscuta spp.) acquires phloem-mobile proteins from host; however, whether this could mediate inter-species phenotype transfer was not demonstrated. Specifically, we test whether phosphinothricin acetyl transferase (PAT) that confers host plant glufosinate herbicide tolerance traffics and functions inter-specifically. · Dodder tendrils excised from hosts can grow in vitro for weeks or resume in vivo by parasitizing new hosts. The level of PAT in in vivo and in vitro dodder tendrils was quantified by enzyme-linked immunosorbent assay. The glufosinate sensitivity was examined by dipping the distal end of in vivo and in vitro tendrils, growing on or excised from LibertyLink (LL; PAT-transgenic and glufosinate tolerant) and conventional (CN; glufosinate sensitive) soybean hosts, into glufosinate solutions for 5 s. After in vitro tendrils excised from LL hosts reparasitized new CN and LL hosts, the PAT level and the glufosinate sensitivity were also examined. · When growing on LL host, dodder tolerated glufosinate and contained PAT at a level of 0.3% of that encountered in LL soybean leaf. After PAT was largely degraded in dodders, they became glufosinate sensitive. PAT mRNA was not detected by reverse transcription PCR in dodders. · In conclusion, the results indicated that PAT inter-species trafficking confers dodder glufosinate tolerance.

Generation of Herbicide-Resistant Soybean by Base Editing.

Weeds cause the largest yield loss in soybean production. The development of herbicide-resistant soybean germplasm is of great significance for weed control and yield improvement. In this study, we used the cytosine base editor (BE3) to develop novel herbicide-resistant soybean. We have successfully introduced base substitutions in GmAHAS3 and GmAHAS4 and obtained a heritable transgene-free soybean with homozygous P180S mutation in GmAHAS4. The GmAHAS4 P180S mutants have apparent resistance to chlorsulfuron, flucarbazone-sodium, and flumetsulam. In particular, the resistance to chlorsulfuron was more than 100 times that of with wild type TL-1. The agronomic performance of the GmAHAS4 P180S mutants showed no significant differences to TL-1 under natural growth conditions. In addition, we developed allele-specific PCR markers for the GmAHAS4 P180S mutants, which can easily discriminate homozygous, heterozygous mutants, and wild-type plants. This study demonstrates a feasible and effective way to generate herbicide-resistant soybean by using CRISPR/Cas9-mediated base editing.

Metabolomics Analysis Provides New Insights Into the Molecular Mechanisms of Parasitic Plant Dodder Elongation in vitro.

Dodder (Cuscuta spp.) species are obligate parasitic flowering plants that totally depend on host plants for growth and reproduction and severely suppress hosts' growth. As a rootless and leafless plant, excised dodder shoots exhibit rapid growth and elongation for several days to hunt for new host stems, and parasitization could be reestablished. This is one unique ability of the dodder to facilitate its success in nature. Clearly, excised dodder stems have to recycle stored nutrients to elongate as much as possible. However, the mechanism of stored nutrient recycling in the in vitro dodder shoots is still poorly understood. Here, we found that dodder is a carbohydrate-rich holoparasitic plant. During the in vitro dodder shoot development, starch was dramatically and thoroughly degraded in the dodder shoots. Sucrose derived from starch degradation in the basal stems was transported to the shoot tips, in which EMP and TCA pathways were activated to compensate for carbon demand for the following elongation according to the variations of sugar content related to the crucial gene expression, and the metabolomics analysis. Additionally, antioxidants were significantly accumulated in the shoot tips in contrast to those in the basal stems. The variations of phytohormones (jasmonic acid, indole-3-acetic acid, and abscisic acid) indicated that they played essential roles in this process. All these data suggested that starch and sucrose degradation, EMP and TCA activation, antioxidants, and phytohormones were crucial and associated with the in vitro dodder shoot elongation.

Efficient carbon recycling and modulation of antioxidants involved in elongation of the parasitic plant dodder (Cuscuta spp.) in vitro.

Dodder is a holoparasitic flowering plant that re-establishes parasitism with the host when broken off from the host. However, how in vitro dodder shoots recycle stored nutrients to maintain growth for reparasitizing hosts is not well characterized. Here, the spatial and temporal distribution characteristics of carbohydrates and reactive oxygen species (ROS) were analysed to explore the mechanism of recycling stored nutrients in dodder shoots in vitro. Our results showed that in vitro dodder shoots grew actively for more than 10 d, while dry mass decreased continuously. During this process, the transcript levels and activities of amylases gradually increased until 2 d and then declined in basal stems, which induced starch degradation at the tissue, cellular and subcellular levels. Additionally, the distribution characteristics of H2O2 and the activities and transcript levels of antioxidant enzymes indicated that shoot tips exhibited more robust ROS-scavenging capacity, and basal stems maintained higher ROS accumulation. Comparative proteomics analysis revealed that starch in basal stems acted as an energy source, and the glycolysis, TCA cycle and pentose phosphate pathway represented the energy supply for shoot tip elongation with time. These results indicated that efficient nutrient recycling and ROS modulation facilitated the parasitism of dodder grown in vitro by promoting shoot elongation growth to reach the host.

Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing.

Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control. We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase (ALS) gene to confer herbicide tolerance in rice. The four different missense mutations in the P171 codon, P171S, P171A, P171Y and P171F, exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors. For example, P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides. Interestingly, a novel triple mutant (P171F/G628E/G629S) had the highest tolerance to all five tested herbicides. Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron. Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.

A donor-DNA-free CRISPR/Cas-based approach to gene knock-up in rice.

Structural variations (SVs), such as inversion and duplication, contribute to important agronomic traits in crops1. Pan-genome studies revealed that SVs were a crucial and ubiquitous force driving genetic diversification2-4. Although genome editing can effectively create SVs in plants and animals5-8, the potential of designed SVs in breeding has been overlooked. Here, we show that new genes and traits can be created in rice by designed large-scale genomic inversion or duplication using CRISPR/Cas9. A 911 kb inversion on chromosome 1 resulted in a designed promoter swap between CP12 and PPO1, and a 338 kb duplication between HPPD and Ubiquitin2 on chromosome 2 created a novel gene cassette at the joint, promoterUbiquitin2::HPPD. Since the original CP12 and Ubiquitin2 genes were highly expressed in leaves, the expression of PPO1 and HPPD in edited plants with homozygous SV alleles was increased by tens of folds and conferred sufficient herbicide resistance in field trials without adverse effects on other important agronomic traits. CRISPR/Cas-based genome editing for gene knock-ups has been generally considered very difficult without inserting donor DNA as regulatory elements. Our study challenges this notion by providing a donor-DNA-free strategy, thus greatly expanding the utility of CRISPR/Cas in plant and animal improvements.

Generation of imidazolinone herbicide resistant trait in Arabidopsis.

Recently-emerged base editing technologies could create single base mutations at precise genomic positions without generation DNA double strand breaks. Herbicide resistant mutations have been successfully introduced to different plant species, including Arabidopsis, watermelon, wheat, potato and tomato via C to T (or G to A on the complementary strand) base editors (CBE) at the P197 position of endogenous acetolactate synthase (ALS) genes. Additionally, G to A conversion to another conserved amino acid S653 on ALS gene could confer tolerance to imidazolinone herbicides. However, no such mutation was successfully generated via CBE, likely due to the target C base is outside of the classic base editing window. Since CBE driven by egg cell (EC) specific promoter would re-edit the wild type alleles in egg cells and early embryos, we hypothesized the diversity of base editing outcomes could be largely increased at later generations to allow selection of desired herbicide resistant mutants. To test this hypothesis, we aimed to introduce C to T conversion to the complement strand of S653 codon at ALS gene, hosting a C at the 10th position within the 20-nt spacer sequence outside of the classic base editing window. While we did not detect base-edited T1 plants, efficient and diverse base edits emerged at later generations. Herbicide resistant mutants with different editing outcomes were recovered when T3 and T4 seeds were subject to herbicide selection. As expected, most herbicide resistant plants contained S653N mutation as a result of G10 to A10. Our results showed that CBE could create imidazolinone herbicide resistant trait in Arabidopsis and be potentially applied to crops to facilitate weed control.

Generation of herbicide tolerance traits and a new selectable marker in wheat using base editing.

Developing herbicide-tolerant varieties by genome editing holds great promise for addressing the worsening weed problems in wheat cultivation1. Here, we generated transgene-free wheat germplasms harbouring herbicide tolerance mutations that confer tolerance to sulfonylurea-, imidazolinone- and aryloxyphenoxy propionate-type herbicides by base editing the acetolactate synthase (ALS) and acetyl-coenzyme A carboxylase genes. These stackable herbicide tolerance traits provide a potentially powerful tool for weed management. In addition, we found that base editing at the wheat ALS Pro-174 codon (TaALS-P174) endowed wheat with sufficient resistance to nicosulfuron herbicide in MS growth medium to allow selection. When the TaALS-P174 editor was coupled with editors for other targets of interest, co-editing occurred in the nicosulfuron-resistant plants, and selection for resistance in growth medium enriched the frequency of coupled targets by several-fold. This selectable co-editing system has the potential to greatly bolster adoption of base editing for crop improvement applications.

Profiling mRNAs of two Cuscuta species reveals possible candidate transcripts shared by parasitic plants.

Dodders are among the most important parasitic plants that cause serious yield losses in crop plants. In this report, we sought to unveil the genetic basis of dodder parasitism by profiling the trancriptomes of Cuscuta pentagona and C. suaveolens, two of the most common dodder species using a next-generation RNA sequencing platform. De novo assembly of the sequence reads resulted in more than 46,000 isotigs and contigs (collectively referred to as expressed sequence tags or ESTs) for each species, with more than half of them predicted to encode proteins that share significant sequence similarities with known proteins of non-parasitic plants. Comparing our datasets with transcriptomes of 12 other fully sequenced plant species confirmed a close evolutionary relationship between dodder and tomato. Using a rigorous set of filtering parameters, we were able to identify seven pairs of ESTs that appear to be shared exclusively by parasitic plants, thus providing targets for tailored management approaches. In addition, we also discovered ESTs with sequences similarities to known plant viruses, including cryptic viruses, in the dodder sequence assemblies. Together this study represents the first comprehensive transcriptome profiling of parasitic plants in the Cuscuta genus, and is expected to contribute to our understanding of the molecular mechanisms of parasitic plant-host plant interactions.