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.

Large-scale interplant exchange of macromolecules between soybean and dodder under nutrient stresses

Parasitic plants and their hosts communicate through haustorial connections.Nutrient deficiency is a common stress for plants,yet little is known about whether and how host plants and parasites communicate during adaptation to such nutrient stresses.In this study,we used transcriptomics and proteomics to analyze how soybean(Glycine max)and its parasitizing dodder(Cuscuta australis)respond to nitrate and phosphate deficiency(-N and-P).After-N and-P treatment,the soybean and dodder plants exhibited substantial changes of transcriptome and proteome,although soybean plants showed very few transcriptional responses to-P and dodder did not show any transcriptional changes to either-N or-P.Importantly,large-scale interplant transport of mRNAs and proteins was detected.Although the mobile mRNAs only comprised at most 0.2%of the transcriptomes,the foreign mobile proteins could reach 6.8%of the total proteins,suggesting that proteins may be the major forms of interplant communications.Furthermore,the interplant mobility of macromolecules was specifically affected by the nutrient regimes and the transport of these macromolecules was very likely independently regulated.This study provides new insight into the communication between host plants and parasites under stress conditions.

Salicylic acid positively regulates maize defenses against lepidopteran insects

In response to insect attack,plants use intricate signaling pathways,including phytohormones,such as jasmonate(JA),ethylene(ET),and salicylic acid(SA),to activate defenses.Maize(Zea mays)is one of the most important staple food crops around the world.Previous studies have shown that the JA and ET signaling play important roles in maize defense against insects,but little is known about whether and how SA regulates maize resistance to insect herbivores.In this study,we ectopically expressed the NahG(salicylate hydroxylase)gene in maize plants(NahG maize)to block the accumulation of SA.It was found that compared with the wild-type(WT)maize,the NahG-maize exhibited decreased resistance to the generalist insects Spodoptera litura and Spodoptera frugiperda and the specialist Mythimna separata,and the compromised resistance in the NahG maize was associated with decreased levels of defensive metabolites benzoxazinoids(Bxs)and chlorogenic acid(CA).Quantification of simulated S.litura feedinginduced JA,JA-isoleucine conjugate(JA-Ile),and ET in the WT and NahG maize indicated that SA does not regulate JA or JA-Ile,but positively controls ET.We provide evidence suggesting that the SA pathway does not crosstalk with the JA or the ET signaling in regulating the accumulation of Bxs and CA.Transcriptome analysis revealed that the bHLH,ERF,and WRKY transcription factors might be involved in SAregulated defenses.This study uncovers a novel and important phytohormone pathway in maize defense against lepidopterous larvae.

HvWRKY2 acts as an immunity suppressor and targets HvCEBiP to regulate powdery mildew resistance in barley

Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of Hv WRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of Hv WRKY2 requires an intact WRKY domain and an upstream sequence of~75 amino acids,and the Hv WRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of Hv WRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of Hv WRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.Hv CEBiP positively regulates resistance against Bgh in barley.Our findings suggest that Hv WRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that Hv CEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.

imple Bioassay for PAMP-Triggered Immunity in Rice Seedlings Based on Lateral Root Growth Inhibition

Pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI)is an essential layer of plant disease resistance.Robust bioassays for PTI are pre-required to dissect its molecular mechanism.In this study,we established that lateral root growth inhibition as a simple and robust measurement of PTI in rice seedlings.Specifically,flg22,a well-characterized PAMP from bacterial flagellin,was used to induce PTI in rice seedlings.While flg22 treatment induced PR gene expression and mitogen-activated protein kinase activation in the roots of rice seedlings to support the PTI triggered,this treatment substantially repressed lateral root growth,but it did not alter primary root growth.Moreover,treatments with chitin(i.e.,a fungal PAMP)and oligogalacturonides(i.e.,classical damage-associated molecular pattern)clearly inhibited the lateral root growth,although a priming step involving ulvan was required for the chitin treatment.The bioassay developed was applicable to various rice cultivars and wild species.Thus,lateral root growth inhibition represents a simple and reliable assay for studying PTI in rice plants.

Reconstructing early transmission networks of SARS-CoV-2 using a genomic mutation model

The coronavirus disease 2019(COVID-19)pandemic has greatly damaged human society,but the origins and early transmission patterns of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)pathogen remain unclear.Here,we reconstructed the transmission networks of SARS-CoV-2 during the first three and six months since its first report based on ancestor-offspring relationships using BANAL-52-referenced mutations.We explored the position(i.e.,root,middle,or tip)of early detected samples in the evolutionary tree of SARS-CoV-2.In total,6799 transmission chains and 1766 transmission networks were reconstructed,with chain lengths ranging from 1-9 nodes.The root node samples of the 1766 transmission networks were from 58 countries or regions and showed no common ancestor,indicating the occurrence of many independent or parallel transmissions of SARS-CoV-2 when first detected(i.e.,all samples were located at the tip position of the evolutionary tree).No root node sample was found in any sample(n=31,all from the Chinese mainland)collected in the first 15 days from 24 December 2019.Results using six-month data or RaTG13-referenced mutation data were similar.The reconstruction method was verified using a simulation approach.Our results suggest that SARS-CoV-2 may have already been spreading independently worldwide before the outbreak of COVID-19 in Wuhan,China.Thus,a comprehensive global survey of human and animal samples is essential to explore the origins of SARS-CoV-2 and its natural reservoirs and hosts.

Publisher Correction: The RNA-binding domain of DCL3 is required for long-distance RNAi signaling

Correction to:aBIOTECH https://doi.org/10.1007/s42994-023-00124-6 The copyright holder for this article was incorrectly given as‘Agricultural Information Institute,Chinese Academy of Agricultural Sciences’but should have been‘The Authors’This article was originally published with the incorrect licence;it should have been:Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use,sharing,adaptation.

The RNA-binding domain of DCL3 is required for long-distance RNAi signaling

Small RNA(sRNA)-mediated RNA silencing(also known as RNA interference,or RNAi)is a conserved mechanism in eukaryotes that includes RNA degradation,DNA methylation,heterochromatin formation and protein translation repression.In plants,sRNAs can move either cell-to-cell or systemically,thereby acting as mobile silencing signals to trigger noncell autonomous silencing.However,whether and what proteins are also involved in noncell autonomous silencing have not been elucidated.In this study,we utilized a previously reported inducible RNAi plant,PDSi,which can induce systemic silencing of the endogenous PDS gene,and we demonstrated that DCL3 is involved in systemic PDS silencing through its RNA binding activity.We confirmed that the C-terminus of DCL3,including the predicted RNA-binding domain,is capable of binding short RNAs.Mutations affecting RNA binding,but not processing activity,reduced systemic PDS silencing,indicating that DCL3 binding to RNAs is required for the induction of systemic silencing.Cucumber mosaic virus infection assays showed that the RNA-binding activity of DCL3 is required for antiviral RNAi in systemically noninoculated leaves.Our findings demonstrate that DCL3 acts as a signaling agent involved in noncell autonomous silencing and an antiviral effect in addition to its previously known function in the generation of 24-nucleotide sRNAs.

A complete MAP kinase cascade controls hyphopodium formation and virulence of Verticillium dahliae

Phytopathogens develop specialized infection-related structures to penetrate plant cells during infection.Different from phytopathogens that form appressoria or haustoria,the soil-borne root-infecting fungal pathogen Verticillium dahliae forms hyphopodia during infection,which further differentiate into penetration pegs to promote infection.The molecular mechanisms underlying the regulation of hyphopodium formation in V.dahliae remain poorly characterized.Mitogen-activated protein kinases(MAPKs)are highly conserved cytoplasmic kinases that regulate diverse biological processes in eukaryotes.Here we found that deletion of VdKss1,out of the five MAPKs encoded by V.dahliae,significantly impaired V.dahliae hyphopodium formation,in vitro penetration,and pathogenicity in cotton plants.Constitutive activation of MAPK kinase(MAPKK)VdSte7 and MAPK kinase kinase(MAPKKK)VdSte11 specifically activate VdKss1.Deletion of VdSte7 or VdSte11 resulted in a phenotype similar to that of the mutant with VdKss1 deletion.Thus,this study demonstrates that VdSte11-VdSte7-VdKss1 is a core MAPK cascade that regulates hyphopodium formation and pathogenicity in V.dahliae.

Putrescine, spermidine, and spermine play distinct roles in rice salt tolerance

Polyamines(PAs) play diverse roles in plant growth and development,as well as responses to environmental stimuli.In this study,the effects of PAs on rice salt tolerance were investigated.Salt stress resulted in the alteration of endogenous PAs levels in rice roots and leaves,where spermidine(Spd) and spermine(Spm) contents were increased,and putrescine(Put) content was decreased.RT-qPCR analysis revealed that PAs biosynthesis-related genes ADC1,ODC,and Arginase were significantly downregulated by salt treatment;however,SAMDC transcription was significantly upregulated.Exogenous Spm enhanced rice salt tolerance remarkably;however,exogenous Put and Spd undermined rice salt tolerance.Transgenic rice plants overexpressing SAMDC display a higher ratio of Spm/(Put+Spd) and enhanced salt tolerance.Salt stress also increased polyamine oxidase activities in rice,resulting in elevated reactive oxygen species(ROS) production.Our findings revealed that accumulation of Put and Spd substantially reduced salt tolerance in rice,likely by facilitating ROS production;whereas,conversion of Put and Spd to Spm contributes to rice salt tolerance.

Inter-species mRNA transfer among green peach aphids,dodder parasites,and cucumber host plants

mRNAs are transported within a plant through phloem.Aphids are phloem feeders and dodders(Cuscuta spp.)are parasites which establish phloem connections with host plants.When aphids feed on dodders,whether there is trafficking of mRNAs among aphids,dodders,and host plants and if aphid feeding a匚fects the mRNA transfer between dodders and hosts are unclear.We constructed a green peach aphid(GPA,Myzus persicae)-dodder(Cuscuta austra/is)-cucumber(Cucumis sativus)tritrophic system by infesting GPAs on C.australis,which parasitized cucumber hosts.We found that GPA feeding activated defense-related phytohormonal and transcriptomic responses in both C.australis and cucumbers and large numbers of mRNAs were found to be transferred between C.australis and cucumbers and between C.australis and GPAs;importantly,GPA feeding on C.australis greatly altered inter-species mobile mRNA profiles.Furthermore,three cucumber mRNAs and three GPA mRNAs could be respectively detected in GPAs and cucumbers.Moreover,our statistical analysis indicated that mRNAs with high abundances and long transcript lengths are likely to be mobile.This study reveals the existence of inter-species and even inter-kingdom mRNA movement among insects,parasitic plants,and parasite hosts,and suggests complex regulation of mRNA trafficking.

TRP (transient receptor potential) ion channel family:structures, biological functions and therapeutic interventions for diseases

Transient receptor potential(TRP)channels are sensors for a variety of cellular and environmental signals.Mammals express a total of 28 different TRP channel proteins,which can be divided into seven subfamilies based on amino acid sequence homology:TRPA(Ankyrin),TRPC(Canonical),TRPM(Melastatin),TRPML(Mucolipin),TRPN(NO-mechano-potential,NOMP),TRPP(Polycystin),TRPV(Vanilloid).They are a class of ion channels found in numerous tissues and cell types and are permeable to a wide range of cations such as Ca^(2+),Mg^(2+),Na^(+),K^(+),and others.TRP channels are responsible for various sensory responses including heat,cold,pain,stress,vision and taste and can be activated by a number of stimuli.Their predominantly location on the cell surface,their interaction with numerous physiological signaling pathways,and the unique crystal structure of TRP channels make TRPs attractive drug targets and implicate them in the treatment of a wide range of diseases.Here,we review the history of TRP channel discovery,summarize the structures and functions of the TRP ion channel family,and highlight the current understanding of the role of TRP channels in the pathogenesis of human disease.Most importantly,we describe TRP channel-related drug discovery,therapeutic interventions for diseases and the limitations of targeting TRP channels in potential clinical applications.

Spindle-E is essential for gametogenesis in the silkworm, Bombyx mori

As a defense mechanism against transposable elements,the PIWI-interacting RNA(piRNA)pathway maintains genomic integrity and ensures proper gametogenesis in gonads.Numerous factors are orchestrated to ensure normal operation of the piRNA pathway.Spindle-E(Spn-E)gene was one of the first genes shown to participate in the piRNA pathway.In this study,we performed functional analysis of Spn-E in the model lepidopteran insect,Bombyx mori.Unlike the germline-specific expression pattern observed in Drosophila and mouse,BmSpn-E was ubiquitously expressed in all tissues tested,and it was highly expressed in gonads.Immunofluorescent staining showed that BmSpn-E was localized in both germ cells and somatic cells in ovary and was expressed in spermatocytes in testis.We used a binary transgenic CRISPR/Cas9 system to construct BmSpn-E mutants.Loss of BmSpn-E expression caused derepression of transposons in gonads.We also found that mutant gonads were much smaller than wild-type gonads and that the number of germ cells was considerably lower in mutant gonads.Quantitative real-time PCR analysis and TUNEL staining revealed that apoptosis was greatly enhanced in mutant gonads.Further,we found that the BmSpn-E mutation impacted gonadal development and gametogenesis at the early larval stage.In summary,our data provided the first evidence that BmSpn-E plays vital roles in gonadal development and gametogenesis in B.mori.

The combined effect of bromadiolone and ivermectin (iBr) in controlling both rodents and their fleas

Rodent pests not only cause severe agricultural loss but also spread zoonotic pathogens to human beings.Antico-agulant rodenticides are widely used to decrease the population densities of rodents but often lead to the spillover of ectoparasites becausefleas and ticks may gather on surviving rodents.Therefore,it is necessary to killfleas and ticks before culling rodents to minimize the risk of pathogen transmission.In this study,we used a mixture of ivermectin(an antiparasitic drug)and bromadiolone(an anticoagulant rodenticide)to control both rodent andflea/tick abundances.We found that in a laboratory test,0.01%ivermectin bait was not lethal for greater long-tailed hamsters after 7 days of treatment,while 0.1%ivermectin bait was lethal for approximately 33%of treated rodents.In afield test,bait containing 0.001%,0.005%,0.01%,and 0.05%ivermectin decreased the number offleas per vole of Brandt’s voles to 0.42,0.22,0.12,and 0.2,respectively,compared with 0.77 in the control group,indicating that 0.01%ivermectin bait performed best in removingfleas.In another laboratory test,bait containing a 0.01%ivermectin and 0.005%bromadiolone mixture caused the death of all voles within 6–14 days after the intake of the bait.In thefield test,the bait containing 0.01%ivermectin and 0.005%bromadiolone reduced the average number offleas per vole to 0.35,which was significantly lower than the 0.77 of the control group.Our results indicate that a 0.01%ivermectin and 0.005%bromadiolone mixture could be used to control both rodents andfleas to minimize the spillover risk of disease transmission when using traditional rodenticides.

Antifertility effects of EP-1 (quinestrol and levonorgestrel) on Pacific rats (Rattus exulans)

Pest rodents pose a serious threat to island biodiversity.Fertility control could be an alternative approach to control the impact of rodents on these islands.In this study,we examined the antifertility effects of EP-1 baits containing quinestrol(E)and levonorgestrel(P)using a dose of 50 ppm E and P at three different ratios(E:P ratio=1:2,1:1,and 2:1)on Pacific rats(Rattus exulans)in the Xisha Islands,Hainan,China.Compared to the control group,all animals in EP-1 treatment groups showed significantly decreased food intake and body weight.In treated males,there were obvious abnormalities in testis structure and a significant decrease of relative seminal vesicle weight,but no significant effect on relative uterine and ovarian weights(g kg-1 body weight),or ovarian structure in females.Adding 8%sucrose to the original 50-ppm baits(E:P ratio=1:1)significantly increased bait palatability for males and females.This dose induced uterine edema and abnormalities of ovarian structure in females but had no significant negative effect on the relative testis,epididymis,and seminal vesicle weights(g kg-1 body weight)or sperm density in males.In summary,50-ppm EP-1(1:1)baits have the potential to disrupt the fertility of females,and 8%sucrose addition to the EP-1 baits(E:P ratio=1:1)could improve bait palatability.This dose disrupted the testis structure in males.Future studies are needed to improve bait acceptance and assess the antifertility effects of EP-1(1:1)on Pacific rats in captive breeding trials and underfield conditions.

Plant virology in the 21st century in China:Recent advances and future directions

Plant viruses are a group of intracellular pathogens that persistently threaten global food security.Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years,including basic research and technologies for preventing and controlling plant viral diseases.Here,we review these milestones and advances,including the identification of new crop-infecting viruses,dissection of pathogenic mechanisms of multiple viruses,examination of multilayered interactions among viruses,their host plants,and virus-transmitting arthropod vectors,and in-depth interrogation of plantencoded resistance and susceptibility determinants.Notably,various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants.We also recommend future plant virology studies in China.

Species recognition and the divergences in the chemical and ultrasonic signals between two coexisting Rattus species

The ability to recognize and differentiate between conspecifics and heterospecifics as well as their signals is critical for the coexistence of closely related species.In the genus Rattus,species are morphologically similar and multiple species often coexist.Here,we investigated the interspecific recognition and signal differentiation of two sympatric rat species,the brown rat(Rattus norvegicus,RN)and the Asian house rat(Rattus tanezumi,RT).In a two-way choice test,both RN and RT females showed a preference for conspecific male rats to heterospecific ones.RT females showed a significant preference for accessible urine of males of same species to those of other species,but not for the inaccessi-ble urine.On the other hand,there were significant differences in the structural characteristics of the ultrasonic vocalization emitted by males of these two rat species.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)and isoelectric focusing electrophoresis unveiled that major urinary proteins(MUPs)in voided urine were more highly expressed in RN males versus RT males.The interspecific dif-ferences of urinary volatile compounds were also discussed.In conclusion,female rats had the ability to distinguish between males of either species.

New insight into Ca^(2+)-permeable channel in plant immunity

Calcium ions(Ca^(2+)) are crucial intracellular second messengers in eukaryotic cells. Upon pathogen perception, plants generate a transient and rapid increase in cytoplasmic Ca^(2+)levels, which is subsequently decoded by Ca^(2+)sensors and effectors to activate downstream immune responses. The elevation of cytosolic Ca^(2+)is commonly attributed to Ca^(2+)influx mediated by plasma membranelocalized Ca^(2+)–permeable channels. However, the contribution of Ca^(2+)release triggered by intracellular Ca^(2+)-permeable channels in shaping Ca^(2+)signaling associated with plant immunity remains poorly understood. This review discusses recent advances in understanding the mechanism underlying the shaping of Ca^(2+)signatures upon the activation of immune receptors, with particular emphasis on the identification of intracellular immune receptors as non-canonical Ca^(2+)-permeable channels. We also discuss the involvement of Ca^(2+)release from the endoplasmic reticulum in generating Ca^(2+)signaling during plant immunity.

Functional characterization of Bmcap in uric acid metabolism in the silkworm

After a millennium of domestication,numerous silkworm mutants have emerged that exhibit transparent epidermis,which is caused by abnormally low levels of uric acid.We identified the Bombyx mori gene Bmcap(BMSK0003832)as the homolog of cappuccino,a subunit of the biogenesis of lysosome-related organelles complex-1(BLOC-1)that has been extensively characterized in human,mouse,and insect species,by analyzing the amino acid sequences of putative purine metabolism genes.Using the clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9 system,we disrupted Bmcap,resulting in decreased uric acid levels in the silkworm epidermis and a translucent skin phenotype.In the Bmcap mutant,the purine metabolism,nitrogen metabolism,pyrimidine metabolism,and membrane system were altered compared to the wild type.Biogenesis of lysosome-related organelle complex genes play a role in the pigmentation and biogenesis of lysosome-related organelles(LROs)in platelets,melanocytes,and megakaryocytes.LROs exhibit unique morphologies and functions in various tissues and cells.Investigation of the Bmcap mutant will enhance our understanding of the uric acid metabolic pathway in silkworms,and this mutant offers a valuable silkworm model for LRO studies.

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.