Recoloring tomato fruit by CRISPR/Cas9-mediated multiplex gene editing

Fruit color is an important horticultural trait,which greatly affects consumer preferences.In tomato,fruit color is determined by the accumulation of different pigments,such as carotenoids in the pericarp and f lavonoids in the peel,along with the degradation of chlorophyll during fruit ripening.Since fruit color is a multigenic trait,it takes years to introgress all color-related genes in a single genetic background via traditional crossbreeding,and the avoidance of linkage drag during this process is difficult.Here,we proposed a rapid breeding strategy to generate tomato lines with different colored fruits from red-fruited materials by CRISPR/Cas9-mediated multiplex gene editing of three fruit color-related genes(PSY1,MYB12,and SGR1).Using this strategy,the red-fruited cultivar‘Ailsa Craig’has been engineered to a series of tomato genotypes with different fruit colors,including yellow,brown,pink,light-yellow,pink-brown,yellow-green,and light green.Compared with traditional crossbreeding,this strategy requires less time and can obtain transgene-free plants with different colored fruits in less than 1 year.Most importantly,it does not alter other important agronomic traits,like yield and fruit quality.Our strategy has great practical potential for tomato breeding and serves as a reference for improving multigene-controlled traits of horticultural crops.

Role of the Arabidopsis thafiana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses

Jasmonic acid （JA） is an important phytohormone that regulates plant defense responses against herbivore attack, pathogen infection and mechanical wounding. In this report, we provided biochemical and genetic evidence to show that the Arabidopsis thaliana NAC family proteins ANAC019 and ANAC055 might function as transcription activators to regulate JA-induced expression of defense genes. The role of the two NAC genes in JA signaling was examined with the anacO19 anac055 double mutant and with transgenic plants overexpressing ANACO19 or ANAC055. The anacO19 anac055 double mutant plants showed attenuated JA-induced VEGETATIVE STORAGE PROTEIN1 （VSP1） and LIPOXYGENASE2 （LOX2） expression, whereas transgenic plants overexpressing the two NAC genes showed enhanced JA-induced VSP1 and LOX2 expression. That the JA-induced expression of the two NAC genes depends on the function of COIl and AtMYC2, together with the finding that overexpression of ANACO19 partially rescued the JA-related phenotype of the atmyc2-2 mutant, has led us to a hypothesis that the two NAC proteins act downstream of AtMYC2 to regulate JA-signaled defense responses. Further evidence to substantiate this idea comes from the observation that the response of the anacO19 anac055 double mutant to a necrotrophic fungus showed high similarity to that of the atmyc2-2 mutant.

The Arabidopsis P450 protein CYP82C2 modulates jasmonateinduced root growth inhibition, defense gene expression and indole glucosinolate biosynthesis

Jasmonic acid （JA） is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies have established that JA also performs a critical role in several aspects of plant development. Here, we describe the characterization of the Arabidopsis mutantjasmonic acid-hypersensitivel-1 （jah1-1）, which is defective in several aspects of JA responses. Although the mutant exhibits increased sensitivity to JA in root growth inhibition, it shows decreased expression of JA-inducible defense genes and reduced resistance to the necrotrophic fungus Botrytis cinerea. Gene cloning studies indicate that these defects are caused by a mutation in the cytochrome P450 protein CYP82C2. We provide evidence showing that the compromised resistance of thejah1-1 mutant to B. cinerea is accompanied by decreased expression of JA-induced defense genes and reduced accumulation of JA-induced indole glucosinolates （IGs）. Conversely, the enhanced resistance to B. cinerea in CYP82C2-overexpressing plants is accompanied by increased expression of JA-induced defense genes and elevated levels of JA-induced IGs. We demonstrate that CYP82C2 affects JA-induced accumulation of the IG biosynthetic precursor tryptophan （Trp）, but not the JA-induced IAA or pathogen-induced camalexin. Together, our results support a hypothesis that CYP82C2 may act in the metabolism of Trp-derived secondary metabolites under conditions in which JA levels are elevated. Thejah1-1 mutant should thus be important in future studies toward understanding the mechanisms underlying the complexity of JA-mediated differential responses, which are important for plants to adapt their growth to the ever-changing environments.

The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses

Messenger RNA （mRNA） turnover in eukaryotic cells begins with shortening of the poly （A） tail at the 3＇ end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4- associated factor 1 （CAF1）, two components of the well-characterised protein complex named CCR4-NOT. We report here that AtCAF1a and AtCAF1b, putative Arabidopsis homologs of the yeast CAF1 gene, partially complement the growth defect of the yeast call mutant in the presence of caffeine or at high temperatures. The expression of At-CAF1a and AtCAFlb is induced by multiple stress-related hormones and stimuli. Both AtCAF1a and AtCAFlb show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA insertion mutants disrupting the expression of AtCAF1a and/or AtCAF1b are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAF1 proteins are involved in regulated mRNA deadenylation in vivo. Interestingly, the single and double mutants of AtCAF1a and AtCAFlb show reduced expression of pathogenesis-related （PR） genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 （Pst DC3000） infection, whereas transgenic plants over-expressing AtCAFla show elevated expression of PR1 and PR2 and increased resis-tance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.

Smear-Negative Multidrug-Resistant Tuberculosis a Significance Hidden Problem for MDR-TB Control: An Analysis of Real World Data

Purpose: The drug resistance pattern in tuberculosis (TB) is still under investigated. We analyzed the clinical data from the patients with smear positive TB and applied the model to predict the patients with smear-positive TB. Materials and Methods: Medical records information of 6977 cases was included from 11,950 inpatients from January 2009 to November 2013. The cases data were divided into a training set, test set and prediction set. Logistic regression analysis was applied to the training set data to establish a prediction classification model, the effect of which was then evaluated using the test set by receiver operating characteristic (ROC) analysis. The model was then applied to the prediction set to identify incidence of snMDR-TB. Results: Sixteen factors which correlate with MDR-TB-including frequency of hospitalization, province of origin, anti-TB drugs, and complications, were identified from the comparison between SP-TB and spMDR-TB. The area under the ROC curve (AUC) of the prediction model was 0.752 (sensitivity = 61.3%, specificity = 83.3%). The percentage of all inpatients with snMDR-TB (snMDR-TB/Total) was 28.7% ± 0.02%, while that of all SN-PTB with snMDR-TB (snMDR-TB/SN-PTB) was 26.5% ± 0.03%. The ratio of snMDR-TB to MDR-TB (snMDR-TB/MDR-TB) was 2.09 ± 0.33. Conclusion: snMDR-TB as an important source of MDR-TB is a significant hidden problem for MDR-TB control and can be identified by the prediction model. A kind of vicious circle with a certain delay effect exists between snMDR-TB and MDR-TB. To better control MDR-TB, it is necessary to pay greater attention to snMDR-TB, conduct further research and develop targeted therapeutic strategies.

Tuberculosis-related miRNAs have potential as disease biomarkers

Background: More effective biomarkers for use intuberculosis prevention,diagnosis, and treatmentare urgently needed. The?potential of miRNAsfor use as biomarkers of human disease has received much attention;however, suitable miRNA biomarkers for use in tuberculosis (TB) diagnosis and treatment have not yet been identified. Methods: We used human miRNA arrays to identify miRNAs in Peripheral Blood Mononuclear Cells (PBMCs) that are differentially expressed in subjects with active disease, those with latent TB infections (LTBI) and healthy individuals. The relationship between differentially-expressed miRNAs and mRNAs was examined using Tar- getScanS, Pic-Tar and miRanda. The expression profiles of selected miRNAs in subjects with active disease, those with LTBI and healthy individuals were validated by qRT-PCR. Results: miRNA array analysis of PBMCs from subjects with active disease, those with LTBI and healthy individuals identified 26 differentially-expressed miRNAs. Analysis of gene expression levels in THP-1 cells using mRNA arrays identified 87 differentially-expressed genes, 80 of which were up-regulated (ratio >2) and 7 of which were down-regulated (ratio In silico miRNA target prediction identified target mRNAs for 15of the 26 differentially-expressed miRNAs. Differentially-expressed miRNAs were identified for 90 of the 178 differentially-expressed genes. has-miR-21* and has-miR-26b had the highestnumbers of differentially-expressed target mRNAs.PCR validation of has-miR-21* and has-miR- 15b* demonstrated the fidelity of our microarray results. Conclusion: Whole-genome transcriptional profiling identified differentially-expressed mRNAs and miRNAs. Differentially-expressed miRNAs?combined with predicted differentially-expressed mRNAs from the same whole-genome transcriptional profiling may be used as the new ways to better understand?TB disease.This discovery of differentially-expressed?miRNAsand mRNAs provides a resource for further studies on the role?of miRNAs in tuberculosis.

A snapshot of the Chinese SOL Project

In 2003, the International Solanaceae Project （SOL） was initiated by an international consortium of ten countries including Korea, China, the United Kingdom, India, the Netherlands, France, Japan, Spain, Italy and the United States. The first major effort of the SOL aimed to produce a DNA sequence map for euchromatin regions of 12 chromosomes of tomato （Solanum lycopersicum） before 2010. Here we present an update on Chinese effort for sequencing the euchromatin region of chromosome 3.

Tomato CYP94C1 inactivates bioactive JA-lle to attenuate jasmonate-mediated defense during fruit ripening

Dear Editor,Fruit rot caused by necrotrophic pathogens results in substantial reductions in fruit yield and revenues worldwide(Petrasch et al.,2019).A widespread phenomenon in fleshy fruit species is the susceptibility of ripe fruits to necrotrophs(Silva et al.,2021),which facilitates seed dispersal(Forlani et al.,2019)but causes severe post-harvest losses in production.As most of the nutritional and sensory qualities of fruits are elaborated at the ripening stage(Liu et al.,2015),balancing fruit ripening and pathogen resistance to maintain fruit quality has proven to be challenging.A deeper understanding of the mechanisms underlying the increased susceptibility of fruits to necrotrophs during ripening could lead to new strategies for producing necrotrophy-resistant fruits without compromising ripening-relatedquality.

Regulation of jasmonate signaling by reversible acetylation of TOPLESS in Arabidopsis

The plant hormone jasmonate(JA)regulates plant immunity and adaptive growth by orchestrating a genome-wide transcriptional program.Key regulators of JA-responsive gene expression include the master transcription factor MYc2,which is repressed by the conserved Groucho/Tup1-like corepressor TOPLESS(TPL)in the resting state.However,the mechanisms underlying TPL-mediated transcriptional repression of MYc2 activity and hormone-dependent switching between repression and de-repression remainenigmatic.Here,we report the regulation of TPLactivity and JAsignaling byreversibleacetylation of TPL.We found that the histone acetyltransferase GCN5 could mediate TPL acetylation,which enhances its interaction with the NOVEL-INTERACTOR-OF-JAZ(NINJA)adaptor and promotes its recruitment to MYc2 target promoters,facilitating transcriptional repression.Conversely,TPL deacetylation by the histonedeacetylase HDA6 weakens TPL-NINJA interaction and inhibitsTPL recruitmentto MYC2 target promoters,facilitating transcriptional activation.In the resting state,the opposing activities of GCN5 and HDA6 maintain TPL acetylation homeostasis,promoting transcriptional repression activity of TPL.In response to JA elicitation,HDA6 expression is transiently induced,resulted in decreased TPL acetylation and repressor activity,thereby transcriptional activation of MYC2 target genes.Thus,the GCN5-TPL-HDA6 module main tains the homeostasis of acetylated TPL,thereby determining the transcriptional state of JA-responsive genes.Our findings uncovered a mechanism by which the TPL corepressor activity in JA signaling is activelytuned inarapidandreversiblemanner.

Mediator tail module subunits MED16 and MED25 differentially regulate abscisic acid signaling in Arabidopsis

MED25 has been implicated as a negative regulator of the abscisic acid(ABA)signaling pathway.However,it is unclear whether other Mediator subunits could associate with MED25 to participate in the ABA response.Here,we used affinity purification followed by mass spectrometry to uncover Mediator subunits that associate with MED25 in transgenic plants.We found that at least26 Mediator subunits,belonging to the head,middle,tail,and CDK8 kinase modules,were copurified with MED25 in vivo.Interestingly,the tail module subunit MED16 was identified to associate with MED25 under both mock and ABA treatments.We further showed that the disruption of MED16 led to reduced ABA sensitivity compared to the wild type.Transcriptomic analysis revealedthattheexpressionofseveral ABA-responsive genes was significantly lower in med16 than those in wild type.Furthermore,we discovered that MED16 may possibly compete with MED25 to interact with the key transcription factor ABA INSENSITIVE 5(ABI5)to positively regulate ABA signaling.Consistently,med16 and med25 mutants displayed opposite phenotypes in ABA response,cuticle permeability,and differential ABI5-mediated EM1 and EM6 expression.Together,our data indicate that MED16 and MED25 differentially regulate ABA signaling byantagonisticallyaffectingABI5-mediated transcription in Arabidopsis.

Oryza sativa mediator subunit OsMED25 interacts with OsBZR1 to regulate brassinosteroid signaling and plant architecture in rice

Brassinosteroids(BRs)are plant-specific steroid hormones which regulate plant growth,development,and adaptation.Transcriptional regulation plays key roles in plant hormone signaling.A mediator can serve as a bridge between gene-specific transcription factors and the RNA polymerase machinery,functioning as an essential component in regulating the transcriptional process.However,whether a mediator is involved in BR signaling is unknown.Here,we discovered that Oryza sativa mediator subunit 25(Os MED25)is an important regulator of rice BR signaling.Phenotypic analyses showed that the Os MED25-RNAi and osmed25 mutant presented erect leaves,as observed in BR-deficient mutants.In addition,the Os MED25-RNAi and osmed25 mutant exhibited decreased BR sensitivity.Genetic analysis indicated that Os MED25-RNAi could suppress the enhanced BR signaling phenotype of Osbzr1-D.Further biochemical analysis showed that Os MED25 interacts with Os BZR1 in vivo,and Os MED25 is enriched on the promoter of Os BZR1 target genes.RNA sequencing analysis indicated that Os MED25 affects the expression of approximately 45%of Os BZR1-regulated genes and mainly functions as a corepressor of Os BZR1.Together,these findings revealed that Os MED25 regulates rice BR signaling by interacting with Os BZR1 and modulating the expression of Os BZR1 target genes,thus expanding our understanding of the roles of mediators in plant hormone signaling.

Construction and characterization of a bacterial artificial chromosome library of rice 5460F

Thermo-sensitive genie male sterile (TGMS) rice has a number of desirable characteristics for hybrid rice production. Many studies have demonstrated that the sterility of TGMS rice is controlled by a single recessive gene. It has been mapped for the first time on chromosome 8 and named tms1. Several AFLP markers which tightly linked to the tms1 gene have been identified recently. In order to develop a detailed physical map of the tms1 gene-encompassing region and finally clone the tms1 gene, a bacterial artificial chromosome (BAC)

WRKY33-mediated indolic glucosinolate metabolic pathway confers resistance against Alternaria brassicicola in Arabidopsis and Brassica crops

The tryptophan(Trp)-derived plant secondary metabolites,including camalexin,4-hydroxyindole-3-carbonylnitrile,and indolic glucosinolate(IGS),show broad-spectrum antifungal activity.However,the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied.In this study,our results revealed that WRKY33 directly regulates IGS biosynthesis,notably the production of 4-methoxyindole-3-ylmethyl glucosinolate(4MI3G),conferring resistance to Alternaria brassicicola,an important pathogen which causes black spot in Brassica crops.WRKY33 directly activates the expression of CYP81F2,IGMT1,and IGMT2 to drive sidechain modification of indole-3-ylmethyl glucosinolate(I3G)to 4MI3G,in both Arabidopsis and Chinese kale(Brassica oleracea var.alboglabra Bailey).However,Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola.Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola.As a result,the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola.Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.

A Transcriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh

Dietary anthocyanins are important health-promoting antioxidants that make a major contribution to the quality of fruits. It is intriguing that most tomato cultivars do not produce anthocyanins in fruit. However, the purple tomato variety Indigo Rose, which has the dominant Aft locus combined with the recessive atv locus from wild tomato species, exhibits light-dependent anthocyanin accumulation in the fruit skin. Here, we report that Aft encodes a functional anthocyanin activator named SlAN2-like, while atv encodes a nonfunctional version of the anthocyanin repressor SlMYBATV. The expression of SlAN2-like is responsive to light, and the functional SlAN2-like can activate the expression of both anthocyanin biosynthetic genes and their regulatory genes, suggesting that SlAN2-like acts as a master regulator in the activation of anthocyanin biosynthesis. We further showed that cultivated tomatoes contain nonfunctional alleles of SlAN2-like and therefore fail to produce anthocyanins. Consistently, expression of a functional SlAN2-like gene driven by the fruit-specific promoter in a tomato cultivar led to the activation of the entire anthocyanin biosynthesis pathway and high-level accumulation of anthocyanins in both the peel and flesh. Taken together, our study exemplifies that efficient engineering of complex metabolic pathways could be achieved through tissue-specific expression of master transcriptional regulators.

Rapid breeding of pink-fruited tomato hybrids using the CRISPR/Cas9 system

As one of the most important vegetables,tomato (Solanum lycopersicum) is extensively produced and consumed worldwide and substantially contributes to human nutrition and health (The Tomato Genome Consortium,2012).Although red tomatoes are the most common,pink tomatoes are more popular in Asia,particularly in China and Japan,because of their better taste (Ballester et al.,2010;Zhu et al.,2018).Compared with red tomatoes,pink tomatoes fail to accumulate the yellow-colored flavonoid pigment,naringenin chalcone (NarCh),in their peels,resulting in a colorless peel phenotype (Adato et al,2009;Ballester et al.,2010).

Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system

Tomato （Solanum lycopersicum） is the leading vegetable crop worldwide and an essential component of a healthy diet （Lin et al., 2014; Du et al., 2017）. Fruit color is regarded as one of the most important commercial traits in tomato （The Tomato Genome Consortium, 2012）. Consumers in different regions have different color preferences. For example, European and American consumers prefer red tomatoes, while pink tomatoes are more pop- ular in Asia countries, particularly in China and Japan （Ballester et al., 2010; Lin et al., 2014）. However, most of tomato breeding ma- terials are red-fruited, thus the generation of pink-fruited materials is very important for Asian tomato production. Metabolomics and genetics studies demonstrate that the pink trait results from the absence of yellow-colored flavonoid naringenin chalcone （NarCh） in the peels,

Rapid and efficient method for isolating plant high molecular weight(HMW)DNA with high purity

PREPARATION of HMW DNA (Megabase-size) is the basis for construction of genomic library with large DNA inserts such as bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC), and for long-range physical mapping. It can also be used for the macro-study of repeat sequences. Since HMW DNA during preparation is inclined to be sheared physically and digested by internal nucleases, it is very difficult to prepare the HMW DNA. Initially, plant HMW DNA was prepared by embedding protoplasts in the low melting-point (LMP) agarose; however, it had several disadvantages: (ⅰ) Culture of protoplasts was time-consuming, costly and tedious. ( ⅱ ) It was only used successfully for limited

OsRLR4 binds to the OsAUX1 promoter to negatively regulate primary root development in rice

Root architecture is one of the most important agronomic traits that determines rice crop yield. The primary root(PR) absorbs mineral nutrients and provides mechanical support;however, the molecular mechanisms of PR elongation remain unclear in rice. Here, the two loss-of-function T-DNA insertion mutants of root length regulator 4(Os RLR4), osrlr4-1 and osrlr4-2 with longer PR, and three Os RLR4 overexpression lines, OE-Os RLR4-1/-2/-3 with shorter PR compared to the wild type/Hwayoung(WT/HY), were identified. Os RLR4 isone of five members of the PRAF subfamily of the regulator chromosome condensation1(RCC1) family. Phylogenetic analysis of Os RLR4 from wild and cultivated rice indicated that it is under selective sweeps, suggesting its potential role in domestication. Os RLR4 controls PR development by regulating auxin accumulation in the PR tip and thus the root apical meristem activity. A series of biochemical and genetic analyses demonstrated that Os RLR4 functions directly upstream of the auxin transporter Os AUX1. Moreover, Os RLR4 interacts with the TRITHORAX-like protein Os Trx1 to promote H3 K4 me3 deposition at the Os AUX1 promoter, thus altering its transcription level. This work provides insight into the cooperation of auxin and epigenetic modifications in regulating root architecture and provides a genetic resource for plant architecture breeding.

Mediator complex subunit MED25 physically interacts with DST to regulate spikelet number in rice

Grain number is a flexible trait and contributes significantly to grain yield.In rice,the zinc finger transcription factor DROUGHT AND SALT TOLERANCE(DST)controls grain number by directly regulating cytokinin oxidase!dehydrogenase 2(OsCKX2)expression.Although specific upstream regulators of the DST-OsCKX2 module have been identified,the mechanism employed by DST to regulate the expression of OsCKX2 remains unclear.Here,we demonstrate that DST-interacting protein 1(DIP1),known as Mediator subunit OsMED25,acts as an interacting coactivator of DST.Phenotypic analyses revealed that OsMED25-RNAi and the osmed25 mutant plants exhibited enlarged panicles,with enhanced branching and spikelet number,similar to the dst mutant.Genetic analysis indicated that OsMED25 acts in the same pathway as the DST-OsCKX2 module to regulate spikelet number per panicle.Further biochemical analysis showed that OsMED25 physically interacts with DST at the promoter region of OsCKX2,and then recruits RNA polymerase II(Pol II)to activate OsCKX2 transcription.Thus,OsMED25 was involved in the communication between DST and Pol II general transcriptional machinery to regulate spikelet number.In general,our findings reveal a novel function of OsMED25 in DST-OsCKX2 modulated transcriptional regulation,thus enhancing our un derstanding of the regulatory mechanism underlying DST-OsCKX2-mediated spikelet number.

Toward Understanding the Stem-Cell Origin and Molecular Regulation of Rice Tillering

Rice tiller is a specialized grain-bearing branch that contrib- utes greatly to grain production. Therefore, rice tillering is an important agronomic trait and provides a model system for the study of branching in monocots. Owing its importance both to agriculture and to fundamental science, much attention has been given to understand the molecular mechanisms under- lying rice tillering.