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Tomato (Solanum lycopersicumL.) is one of the most important crops in the world for its fruit production. Advances in cutting-edge techniques have enabled the development of numerous critical traits related to the quality and quantity of tomatoes. Genetic engineering techniques, such as gene transformation and gene editing, have emerged as powerful tools for generating new plant varieties with superior traits. In this study, we induced parthenocarpic traits in a population of elite tomato (ET) lines. At first, the adaptability of ET lines to genetic transformation was evaluated to identify the best-performing lines by transforming theSlANT1gene overexpression cassette and then later used to produce theSlIAA9knockout lines using the CRISPR/Cas9 system. ET5 and ET8 emerged as excellent materials for these techniques and showed higher efficiency. Typical phenotypes of knockoutsliaa9were clearly visible in G0 and G1 plants, in which simple leaves and parthenocarpic fruits were observed. The high efficiency of the CRISPR/Cas9 system in developing new tomato varieties with desired traits in a short period was demonstrated by generating T-DNA-free homozygoussliaa9knockout plants in the G1 generation. Additionally, a simple artificial fertilization method was successfully applied to recover seed production from parthenocarpic plants, securing the use of these varieties as breeding materials.

Tomato (Solanum lycopersicumL.) is the second most important vegetable crop after potatoes, and global demands have been steadily increasing in recent years. Conventional breeding has been applied to breed and domesticate tomato varieties to meet the need for higher yield or superior agronomical traits that allow to sustain under different climatic conditions. In the current study, we applied bulk population breeding by crossing eight tomato accessions procured from the Asian Vegetable Research and Development Center (AVRDC) with three heat-resistant tomato inbred lines from Vietnam and generated ten elite tomato (ET) lines in the F8 generation. The individual F8 lines exhibited robust vigor and adaptability to Vietnamese climate conditions. Among the ten lines, ET1 and ET3 displayed indeterminate growth. ET2 showed semi-determinate, while all the other lines had determinate growth. The different ET lines showed distinctive superior agronomical traits, including early maturing (ET4, ET7, and ET10), highly efficient fruit set (ET1), higher yield (ET1, ET8, ET10), jointless pedicels (ET2), and partial parthenocarpy (ET9). Molecular analysis revealed that the ET3 line consisted ofTy-1andTy-3loci that positively contribute toTomato yellow leaf curl virus(TYCLV) resistance in tomato plants. The elite tomato lines developed in this study would contribute significantly to the Vietnamese and Asian gene pool for improved tomato production and may be a valuable resource for various breeding goals.

Highly efficient precision gene editing is ultimately a desirable technology, but implementation remains challenging, especially in plants. Here, we tested a novel CRISPR-Cas-based tool for precision gene replacement in plants employing a microhomology-mediated end joining (MMEJ) mechanism. The MMEJ-mediated precise gene replacement employing 20-bp microhomology MMEJ donors (~100 bp lengths) and a cNHEJ inhibitor, NU7441, produced high targeted editing efficiencies, up to 1.60 ± 0.14, 4.47 ± 1.98, and 8.98 ± 4.73 % in tomato, lettuce, and cabbage, respectively. Our data also revealed the critical impacts of the microhomology length and NU7441 concentration on MMEJ-based precision gene editing in plants.

Main conclusion Genome editing offers revolutionized solutions for plant breeding to sustain food production to feed the world by 2050. Therefore, genome-edited products are increasingly recognized via more relaxed legislation and community adoption. Abstract The world population and food production are disproportionally growing in a manner that would have never matched each other under the current agricultural practices. The emerging crisis is more evident with the subtle changes in climate and the running-off of natural genetic resources that could be easily used in breeding in conventional ways. Under these circumstances, affordable CRISPR-Cas-based gene-editing technologies have brought hope and charged the old plant breeding machine with the most energetic and powerful fuel to address the challenges involved in feeding the world. What makes CRISPR-Cas the most powerful gene-editing technology? What are the differences between it and the other genetic engineering/breeding techniques? Would its products be labeled as "conventional" or "GMO"? There are so many questions to be answered, or that cannot be answered within the limitations of our current understanding. Therefore, we would like to discuss and answer some of the mentioned questions regarding recent progress in technology development. We hope this review will offer another view on the role of CRISPR-Cas technology in future of plant breeding for food production and beyond.

Malathion is an organophosphate chemical (OPC) and a toxic contaminant that adversely impacts food quality, human health, biodiversity, and the environment. Due to its small size and unavailability of sensitive sensors, detection of malathion remains a challenging task. Often chromatographic methods employed to analyze OPCs suffer from several shortcomings, including cost, immobility, laboriousness, and unsuitability for point-of-care settings. Hence, developing a specific and sensitive diagnostic sensor for quick and inexpensive food testing is essential. We discovered four unique malathion-specific ssDNA aptamers; designed two independent sensing strategies using fluorescence labeling and Thioflavin T (ThT) displacement. Selected aptamers formed the G4-quadruplex-like (G4Q) structure, which helped develop a label-free detection approach with a 2.01 ppb limit of detection. Additionally, 3D structures of aptamers were generated and validated using a series of computational modeling programs. Furthermore, we explored structural features using CD spectroscopy and molecular docking, probing ligands' binding mode, and revealed vital intermolecular interactions with aptamers. Subsequently, the novel sensors were optimized to detect malathion from food samples. The novel sensors could be further developed to meet the demands of sensing and quantifying toxic contaminants from real food samples in field conditions.

Summary Gene editing and/or allele introgression with absolute precision and control appear to be the ultimate goals of genetic engineering. Precision genome editing in plants has been developed through various approaches, including oligonucleotide‐directed mutagenesis (ODM), base editing, prime editing and especially homologous recombination (HR)‐based gene targeting. With the advent of CRISPR/Cas for the targeted generation of DNA breaks (single‐stranded breaks (SSBs) or double‐stranded breaks (DSBs)), a substantial advancement in HR‐mediated precise editing frequencies has been achieved. Nonetheless, further research needs to be performed for commercially viable applications of precise genome editing; hence, an alternative innovative method for genome editing may be required. Within this scope, we summarize recent progress regarding precision genome editing mediated by microhomology‐mediated end joining (MMEJ) and discuss their potential applications in crop improvement.

CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.

Plasma membranes encapsulated in the symplasmic nanochannels of plasmodesmata (PD) contain abundant lipid rafts, which are enriched with sphingolipids (SLs) and sterols. Reduction of sterols has highlighted the role played by lipid raft integrity in the intercellular trafficking of glycosylphosphatidylinositol (GPI)-anchored PD proteins, particularly in affecting callose enhancement. The presence of callose at PD is strongly attributed to the regulation of callose accumulation and callose degradation by callose synthases and β-1,3-glucanases (BGs), respectively. SLs are implicated in signaling and membrane protein trafficking; however, the underlying processes linking SL composition to the control of symplasmic apertures remain unknown. The wide variety of SLs in plants prompted us to investigate which SL molecules are important for regulating symplasmic apertures in Arabidopsis (Arabidopsis thaliana). We introduced several potential SL pathway inhibitors and genetically modified SL contents using two independent SL pathway mutants. We were able to modulate callose deposition to control symplasmic connectivity through perturbations of SL metabolism. Alteration in glucosylhydroxyceramides or related SL composition particularly disturbed the secretory machinery for the GPI-anchored PdBG2 protein, resulting in an overaccumulation of callose. Moreover, our results revealed that SL-enriched lipid rafts link symplasmic channeling to PD callose homeostasis by controlling the targeting of GPI-anchored PdBG2. This study elevates our understanding of the molecular linkage underlying intracellular trafficking and precise targeting of GPI-anchored PD proteins incorporating glucosyl SLs.

Prolonged periods of drought triggered by climate change hamper plant growth and cause substantial agricultural yield losses every year. In addition to drought, salinity is one of the major abiotic stresses that severely affect crop health and agricultural production. Plant responses to drought and salinity involve multiple processes that operate in a spatiotemporal manner, such as stress sensing, perception, epigenetic modifications, transcription, post-transcriptional processing, translation, and post-translational changes. Consequently, drought and salinity stress tolerance are polygenic traits influenced by genome-environment interactions. One of the ideal solutions to these challenges is the development of high-yielding crop varieties with enhanced stress tolerance, together with improved agricultural practices. Recently, genome-editing technologies, especially clustered regularly interspaced short palindromic repeats (CRISPR) tools, have been effectively applied to elucidate how plants deal with drought and saline environments. In this work, we aim to portray that the combined use of CRISPR-based genome engineering tools and modern genomic-assisted breeding approaches are gaining momentum in identifying genetic determinants of complex traits for crop improvement. This review provides a synopsis of plant responses to drought and salinity stresses at the morphological, physiological, and molecular levels. We also highlight recent advances in CRISPR-based tools and their use in understanding the multi-level nature of plant adaptations to drought and salinity stress. Integrating CRISPR tools with modern breeding approaches is ideal for identifying genetic factors that regulate plant stress-response pathways and for the introgression of beneficial traits to develop stress-resilient crops.

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in

Plants perceive an assortment of external cues during their life cycle, including abiotic and biotic stressors. Biotic stress from a variety of pathogens, including viruses, oomycetes, fungi, and bacteria, is considered to be a substantial factor hindering plant growth and development. To hijack the host cell's defence machinery, plant pathogens have evolved sophisticated attack strategies mediated by numerous effector proteins. Several studies have indicated that plasmodesmata (PD), symplasmic pores that facilitate cell-to-cell communication between a cell and neighbouring cells, are one of the targets of pathogen effectors. However, in contrast to plant-pathogenic viruses, reports of fungal- and bacterial-encoded effectors that localize to and exploit PD are limited. Surprisingly, a recent study of PD-associated bacterial effectors has shown that a number of bacterial effectors undergo cell-to-cell movement via PD. Here we summarize and highlight recent advances in the study of PD-associated fungal/oomycete/bacterial effectors. We also discuss how pathogen effectors interfere with host defence mechanisms in the context of PD regulation.

Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs.

Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.

Mutations are the basis of all genetic variation, and classical plant breeding has exploited the power of natural mutations in the development of high-yielding varieties. Since the discovery of chemical and radiation mutagens, various mutagenesis methods have been employed effectively in molecular plant breeding to study gene functions, to identify crucial genetic mutations for conferring novel traits to plants. Here, we review the historical development and use of plant mutagenesis tools, mainly physical and chemical mutagens, PCR-based methods, T-DNA insertions, transposon insertions, RNA interference, and meganucleases. The unique feature of meganucleases such as the CRISPR/Cas system is the control of site-directed DNA changes. We highlight the recent advancements in CRISPR/Cas-based mutagenesis tools that enable various applications of genetic manipulations, including gene knock-out, gene replacement, targeted base substitutions, and nucleotide diversification of user-defined sites. In addition, we review the use of CRISPR/Cas-based mutagenesis tools in agriculture.

Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we optimize and advance our GT system by using a temperature-tolerant LbCas12a (ttLbCas12a) in combination with various crRNA forms and chemical treatments to suppress the canonical non-homologous end-joining pathway in tomato. Our work demonstrates the significance of the selection of gene scissors, the appropriate design and number of LbCas12a crRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.

Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we customize and advance our GT system by using a temperature-tolerant LbCas12a (ttLbCas12a) in combination with various crRNA forms and chemical treatments to suppress the canonical non-homologous end-joining pathway in tomato. Our work demonstrates the significance of the selection of gene scissors, the appropriate design of LbCas12a gRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a “priming” extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement.

Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.

Endophytes are symptomless microbes found in different plant tissues. Endophytes are beneficial for the growth of the host plant and essential part of biogeochemical cycling in the environment. The host range of endophytes is comprehensive, that is, cereals, pulses, oilseeds, medicinal plants, and trees. The habitat of endophytes is host-dependent and varies from tissue to tissue. They produce growth hormones, antibiotics, secondary metabolites, siderophores, and volatile compounds, which help in plant health management (PHM). The role of plant–endophyte interactions thoroughly investigated for PHM, especially in some cereal and vegetable (CV) crops. Recently, microbial formulations, including endophytes, are one of the most innovative and attractive approaches for organic farming and alternative biocontrol agents. Before commercial exploitation of beneficial properties of CV endophytes in agriculture, further research is needed about their mode of action, host interaction, and suitable delivery methods before application into the open fields. In this chapter, the biology of CV endophytes, mechanisms of plant–endophyte interactions, mode of action for plant growth promotion activity, biotic and abiotic stress management are discussed.

The central dogma (CD) of molecular biology is the transfer of genetic information from DNA to RNA to protein. Major CD processes governing genetic flow include the cell cycle, DNA replication, chromosome packaging, epigenetic changes, transcription, posttranscriptional alterations, translation, and posttranslational modifications. The CD processes are tightly regulated in plants to maintain genetic integrity throughout the life cycle and to pass genetic materials to next generation. Engineering of various CD processes involved in gene regulation will accelerate crop improvement to feed the growing world population. CRISPR technology enables programmable editing of CD processes to alter DNA, RNA, or protein, which would have been impossible in the past. Here, an overview of recent advancements in CRISPR tool development and CRISPR-based CD modulations that expedite basic and applied plant research is provided. Furthermore, CRISPR applications in major thriving areas of research, such as gene discovery (allele mining and cryptic gene activation), introgression (de novo domestication and haploid induction), and application of desired traits beneficial to farmers or consumers (biotic/abiotic stress-resilient crops, plant cell factories, and delayed senescence), are described. Finally, the global regulatory policies, challenges, and prospects for CRISPR-mediated crop improvement are discussed.

Lateral root development is known to be regulated by Aux/IAA-ARF modules in Arabidopsis thaliana. As components, several Aux/IAAs have participated in these Aux/IAA-ARF modules. In this study, to identify the biological function of IAA15 in plant developments, transgenic plant overexpressing the gain-of-function mutant of IAA15 (IAA15P75S OX) under the control of dexamethasone (DEX) inducible promoter, in which IAA15 protein was mutated by changing Pro-75 residue to Ser at the degron motif in conserved domain II, was constructed. As a result, we found that IAA15P75S OX plants show a decreased number of lateral roots. Coincidently, IAA15 promoter-GUS reporter analysis revealed that IAA15 transcripts were highly detected in all stages of developing lateral root tissues. It was also verified that the IAA15P75S protein is strongly stabilized against proteasome-mediated protein degradation by inhibiting its poly-ubiquitination, resulting in the transcriptional repression of auxin-responsive genes. In particular, transcript levels of LBD16 and LBD29, which are positive regulators of lateral root formation, dramatically repressed in IAA15P75S OX plants. Furthermore, it was elucidated that IAA15 interacts with ARF7 and ARF19 and binds to the promoters of LBD16 and LBD29, strongly suggesting that IAA15 represses lateral root formation through the transcriptional suppression of LBD16 and LBD29 by inhibiting ARF7 and ARF19 activity. Taken together, this study suggests that IAA15 also plays a key negative role in lateral root formation as a component of Aux/IAA-ARF modules.

CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.

The plasma membranes encapsulated in the plasmodesmata (PDs) with symplasmic nano-channels contain abundant lipid rafts, which are enriched by sphingolipids and sterols. The attenuation of sterol compositions has demonstrated the role played by lipid raft integrity in the intercellular trafficking of glycosylphosphatidylinositol (GPI)-anchored PD proteins, particularly affecting in the callose enhancement. The presence of callose at PD is tightly attributed to the callose metabolic enzymes, callose synthases (CalSs) and β-1,3-glucanases (BGs) in regulating callose accumulation and callose degradation, respectively. Sphingolipids have been implicated in signaling and membrane protein trafficking, however the underlying processes linking sphingolipid compositions to the control of symplasmic apertures remain unknown. A wide variety of sphingolipids in plants prompts us to investigate which sphingolipid molecules are important in regulating symplasmic apertures. Here, we demonstrate that perturbations of sphingolipid metabolism by introducing several potential sphingolipid (SL) pathway inhibitors and genetically modifying SL contents from two independent SL pathway mutants are able to modulate callose deposition to control symplasmic connectivity. Our data from pharmacological and genetic approaches show that the alteration in glucosylhydroxyceramides (GlcHCers) particularly disturb the secretory machinery for GPI-anchored PdBG2 protein, resulting in an over accumulated callose. Moreover, our results reveal that SL-enriched lipid rafts link symplasmic channeling to PD callose homeostasis by controlling the targeting of GPI-anchored PdBG2. This study elevates our understanding of the molecular linkage underlying intracellular trafficking and precise targeting to specific destination of GPI-anchored PD proteins incorporated with GlcHCers contents.