QTL detection and candidate gene analysis of grape white rot resistance by interspecific grape (Vitis vinifera L. × Vitis davidii Foex.) crossing

Grape white rot,a devastating disease of grapevines caused by Coniella diplodiella(Speg.)Sacc.,leads to significant yield losses in grape.Breeding grape cultivars resistant to white rot is essential to reduce the regular use of chemical treatments.In recent years,Chinese grape species have gained more attention for grape breeding due to their high tolerance to various biotic and abiotic factors along with changing climatic conditions.In this study,we employed whole-genome resequencing(WGR)to genotype the parents of‘Manicure Finger’(Vitis vinifera,female)and‘0940’(Vitis davidii,male),along with 101 F1 mapping population individuals,thereby constructing a linkage genetic map.The linkage map contained 9337 single-nucleotide polymorphism(SNP)markers with an average marker distance of 0.3 cM.After 3 years of phenotypic evaluation of the progeny for white rot resistance,we confirmed one stable quantitative trait locus(QTL)for white rot resistance on chromosome 3,explaining up to 17.9%of the phenotypic variation.For this locus,we used RNA-seq to detect candidate gene expression and identified PR1 as a candidate gene involved in white rot resistance.Finally,we demonstrated that recombinant PR1 protein could inhibit the growth of C.diplodiella and that overexpression of PR1 in susceptible V.vinifera increased grape resistance to the pathogen.

Comparative transcriptome analysis reveals synergistic and disparate defense pathways in the leaves and roots of trifoliate orange(Poncirus trifoliata)autotetraploids with enhanced salt tolerance

Polyploid plants often exhibit enhanced stress tolerance relative to their diploid counterparts,but the physiological and molecular mechanisms of this enhanced stress tolerance remain largely unknown.In this study,we showed that autotetraploid trifoliate orange(Poncirus trifoliata(L.)Raf.)exhibited enhanced salt tolerance in comparison with diploid progenitors.Global transcriptome profiling of diploid and tetraploid plants with or without salt stress by RNAseq revealed that the autotetraploids displayed specific enrichment of differentially expressed genes.Interestingly,the leaves and roots of tetraploids exhibited different expression patterns of a variety of upregulated genes.Genes related to plant hormone signal transduction were enriched in tetraploid leaves,whereas those associated with starch and sucrose metabolism and proline biosynthesis were enriched in roots.In addition,genes encoding different antioxidant enzymes were upregulated in the leaves(POD)and roots(APX)of tetraploids under salt stress.Consistently,the tetraploids accumulated higher levels of soluble sugars and proline but less ROS under salt stress compared to the diploids.Moreover,several genes encoding transcription factors were induced specifically or to higher levels in the tetraploids under salt stress.Collectively,this study demonstrates that the activation of various multifaceted defense systems in leaves and roots contributes to the enhanced salt tolerance of autotetraploids.

Integrated multi-omics analysis of developing‘Newhall’orange and its glossy mutant provide insights into citrus fragrance formation

Sesquiterpene valencene is dominant in flavedo tissues of sweet oranges and imparts a unique woody aroma.However,the interaction between the biosynthetic pathways of valencene and other nutritional compounds is less studied.Sesquiterpenoids were significantly accumulated in a previously reported glossy mutant of orange(MT)than the wild type(WT),especially valencene and caryophyllene.In addition,we identified several other pathways with variations at both the transcriptional and metabolic levels in MT.It’s interesting to found those upregulated metabolites in MT,such as eukaryotic lipids,kaempferol and proline also showed strong positive correlation with valencene along with fruit maturation while those down-regulated metabolites,such as phenylpropanoid coumarins and most of the modified flavonoids exhibited negative correlation.We then categorized these shifted pathways into the‘sesquitepenoid-identical shunt’and the sesquitepenoid-opposite shunt’and confirmed the classification result at transcriptional level.Our results provide important insights into the connections between various fruit quality-related properties.

Lipidomic and transcriptomic analysis reveals reallocation of carbon flux from cuticular wax into plastid membrane lipids in a glossy“Newhall”navel orange mutant

Both cuticle and membrane lipids play essential roles in quality maintenance and disease resistance in fresh fruits.Many reports have indicated the modification of alternative branch pathways in epicuticular wax mutants;however,the specific alterations concerning lipids have not been clarified thus far.Here,we conducted a comprehensive,timeresolved lipidomic,and transcriptomic analysis on the“Newhall”navel orange(WT)and its glossy mutant(MT)“Gannan No.1”.The results revealed severely suppressed wax formation accompanied by significantly elevated production of 36-carbon plastid lipids with increasing fruit maturation in MT.Transcriptomics analysis further identified a series of key functional enzymes and transcription factors putatively involved in the biosynthesis pathways of wax and membrane lipids.Moreover,the high accumulation of jasmonic acid(JA)in MT was possibly due to the need to maintain plastid lipid homeostasis,as the expression levels of two significantly upregulated lipases(CsDAD1 and CsDALL2)were positively correlated with plastid lipids and characterized to hydrolyze plastid lipids to increase the JA content.Our results will provide new insights into the molecular mechanisms underlying the natural variation of plant lipids to lay a foundation for the quality improvement of citrus fruit.

Interactions between ShPP2-1, an F-box family gene, and ACR11A regulate cold toleranee of tomato

There is a critical need to identify germplasm resources and genes that promote cold tolerance of tomato because global tomato production is threatened by cold stress.We found that the expression of an F-box gene family member named ShPP2-1 from Solanum habrochaites is cold inducible and studied its contribution to cold tolerance.Overexpression of ShPP2-1 in cultivated tomato(AC)reduced cold tolerance by intensifying damage to cell membranes.To explore the underlying molecular mechanism,we conducted a yeast two-hybrid library screen and found that a protein containing ACT domain repeats named ACR11A interacts with PP2-1.Overexpression of SIACR11A in AC enhanced the cold tolerance of seedlings and germinating seeds.Cold tolerance decreased in tomato plants that overexpressed both of these genes.Additionally,we performed seed germination experiments in the cold with 177 tomato accessions and identified two alleles of SlACR11A that differ in one single-nucleotide polymorphism.We found that one of these alleles,SlACR11A G,is significantly enriched in cold-tolerant tomato plants.Taken together,our fi ndings indicate that the combination of low expression levels of PP2-1 and high expression levels of ACR11A can promote cold tolerance.These genes may therefore serve as direct targets for both genetic engineering and improvement projects that aim to enhance the cold tolerance of tomato.

Integrated analysis of miRNA and mRNA expression profiles in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis

Insect gut microbiota has been reported to participate in regulating host multiple biological processes including metabolism and reproduction.However,the corresponding molecular mechanisms remain largely unknown.Recent studies suggest that microRNAs(miRNAs)are involved in complex interactions between the gut microbiota and the host.Here,we used next-generation sequencing technology to characterize miRNA and mRNA expression profiles and construct the miRNA–gene regulatory network in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis.A total of 3016 differentially expressed genes(DEGs)and 18 differentially expressed miRNAs(DEMs)were identified.Based on the integrated analysis of miRNA and mRNA sequencing data,229 negatively correlated miRNA-gene pairs were identified from the miRNA-mRNA network.Gene ontology enrichment analysis indicated that DEMs could target several genes involved in the metabolic process,oxidation-reduction process,oogenesis,and insulin signaling pathway.Finally,real-time quantitative polymerase chain reaction further verified the accuracy of RNA sequencing results.In conclusion,our study provides the profiles of miRNA and mRNA expressions under antibiotics treatment and provides an insight into the roles of miRNAs and their target genes in the interaction between the gut microbiota and its host.

Regulatory mechanisms of microbial homeostasis in insect gut

Insects live in incredibly complex environments.The intestinal epithelium of insects is in constant contact with microorganisms,some of which are beneficial and some harmful to the host.Insect gut health and function are maintained through multidimensional mechanisms that can proficiently remove foreign pathogenic microorganisms while effectively maintaining local symbiotic microbial homeostasis.The basic immune mechanisms of the insect gut,such as the dual oxidase-reactive oxygen species(Duox-ROS)system and the immune deficiency(Imd)-signaling pathway,are involved in the maintenance of microbial homeostasis.This paper reviews the role of physical defenses,the Duox-ROS and Imd signaling pathways,the Janus kinase/signal transducers and activators of transcription signaling pathway,and intestinal symbiotic flora in the homeostatic maintenance of the insect gut microbiome.

CHH methylation of genes associated with fatty acid and jasmonate biosynthesis contributes to cold tolerance in autotetraploids of Poncirus trifoliata

Polyploids have elevated stress tolerance,but the underlying mechanisms remain largely elusive.In this study,we showed that naturally occurring tetraploid plants of trifoliate orange(Poncirus trifoliata(L.) Raf.) exhibited enhanced cold tolerance relative to their diploid progenitors.Transcriptome analysis revealed that whole-genome duplication was associated with higher expression levels of a range of well-characterized cold stress-responsive genes.Global DNA methylation profiling demonstrated that the tetraploids underwent more extensive DNA demethylation in comparison with the diploids under cold stress.CHH methylation in the promoters was associated with up-regulation of related genes,whereas CG,CHG,and CHH methylation in the 3’-regions was relevant to gene down-regulation.Of note,genes involved in unsaturated fatty acids(UFAs) and jasmonate(JA)biosynthesis in the tetraploids displayed different CHH methylation in the gene flanking regions and were prominently up-regulated,consistent with greater accumulation of UFAs and JA when exposed to the cold stress.Collectively,our findings explored the difference in cold stress response between diploids and tetraploids at both transcriptional and epigenetic levels,and gained new insight into the molecular mechanisms underlying enhanced cold tolerance of the tetraploid.These results contribute to uncovering a novel regulatory role of DNA methylation in better cold tolerance of polyploids.

OVEREXPRESSION OF PTRLEA7,A LATE EMBRYOGENESIS ABUNDANT FAMILY GENE FROM PONCIRUS TRIFOLIATA,CONFERS ENHANCED DROUGHT TOLERANCE BY ENHANCING ANTIOXIDANT CAPACITY

Late embryogenesis abundant(LEA)genes encode highly hydrophilic proteins that are essential in abiotic stress responses.However,most LEA genes in higher plants have not yet been investigated.This study identified an LEA family gene(PtrLEA7)from Poncirus trifoliata and studied its function in drought tolerance.The full-length coding sequence of PtrLEA7 was 420 bp encoding a protein of 139 amino acids.Phylogenetic analysis shows that PtrLEA7 protein belongs to the LEA_4 subfamily.Expression profiling by qPCR found that PtrLEA7 was strongly induced by dehydration,cold and ABA treatments,and slightly induced by salt stress.Subcellular localization reveals that PtrLEA7 protein was located in both cytoplasm and nucleus.To investigate its function,transgenic plants of both tobacco and Poncirus trifoliata overexpressing PtrLEA7 were obtained.Stress tolerance assays show that overexpression lines had enhanced dehydration and drought tolerance compared with wild type plants,indicating that PtrLEA7 positively regulates drought tolerance.In addition,transgenic plants had much higher expression levels of three antioxidant enzyme genes(CAT,SOD and POD)and significantly increased catalase enzyme activity,accompanied by reduced reactive oxygen species accumulation in comparison with wild type plants.Collectively,this study demonstrates that PtrLEA7 can confer enhanced drought tolerance partially via enhancing antioxidant capacity.

Early embryonic transcriptomes of Zeugodacus tau provide insight into sex determination and differentiation genes

Zeugodacus tau(Walker)is an invasive pest.The sterile insect technique is an environment-friendly method for pest control.Understanding the mechanism of sex determination will contribute to improving efficiency of this technique.In this study,we identified the transformer(tra)gene in Z.tau.One female-specific and two male-specific isoforms of tra were found in Z.tau,and the male-specific splicing pattern of tra was found to occur 5 h after egg laying.We performed transcriptome sequencing at 1 h(E1),5 h(E5),and 9 h(E9)after egg laying and obtained high-quality transcriptome libraries of early embryo development.We identified 13297 and 11713 differentially expressed genes(DEGs)from E5 versus E1 and E9 versus E1 comparisons,respectively.To explore the potential functions of the DEGs during embryonic development,Gene Ontology,Clusters of Orthologous Groups of proteins,and Kyoto Encyclopedia of Genes and Genomes analyses were performed.Twenty-six genes potentially involved in sex determination or differentiation,including Maleness-on-the-Y(MoY),were identified in Z.tau.To verify the transcriptome results,15 genes were selected for quantitative real-time PCR validation.The results were consistent with the transcriptome sequencing results.Moreover,U2 small nuclear riboprotein auxiliary factor(U2AF-50),female lethal d(fl(2)d),and virilizer(vir)were highly expressed at E5,indicating that they may be related to the sex-specific splicing of tra.Further functional analysis is needed to confirm this speculation.Our data provide an insight into the mechanism underlying sex determination and differentiation in tephritid species.

Heterologous viral suppressor of RNA silencing breaks protein-based viral immunity in mixed viral infection

Dear Editor:RNA silencing confers immunity against most viruses and is initiated by host Dicer processing of viral doublestranded RNA into virus-derived small interfering RNA(siRNA)(Guo et al.,2019).Viruses that cause plant disease usually evolve viral suppressor of RNA silencing(VSR)to inhibit antiviral silencing(Li and Ding,2006).Some plant nucleotide-binding leucine-rich repeat proteins(NLRs)emerge as immune receptors to recognize specific virus and trigger rapid hypersensitive response(HR)at infection site to protect plants from devastating viruses(Cui et al.,2015;Deng et al.,2018).Recent studies showed that NLR immune receptors are subjected to negative regulation by plant miRNAs(Deng et al.,2018).It is well known that in some cases,mixed virus infections lead to synergism in susceptible plants due to cooperative inhibition of antiviral silencing by multiple VSRs(Pruss et al.,1997).In case of mixed virus infection in a plant with NLR targeting a single virus,heterologous VSR might play dual roles for the NLR-targeted virus.On one hand,the VSR may promote virulence by cooperative inhibition of antiviral silencing.

IQ67 DOMAIN protein 21 is critical for indentation formation in pavement cell morphogenesis

In plants, cortical microtubules anchor to the plasma membrane in arrays and play important roles in cell shape. However, the molecular mechanism of microtubule binding proteins, which connect the plasma membrane and cortical microtubules in cell morphology remains largely unknown. Here, we report that a plasma membrane and microtubule duallocalized IQ67 domain protein, IQD21, is critical for cotyledon pavement cell(PC) morphogenesis in Arabidopsis. iqd21 mutation caused increased indentation width, decreased lobe length, and similar lobe number of PCs, whereas IQD21 overexpression had a different effect on cotyledon PC shape. Weak overexpression led to increased lobe number, decreased indentation width, and similar lobe length, while moderate or great overexpression resulted in decreased lobe number, indentation width, and lobe length of PCs. Live-cell observations revealed that IQD21 accumulation at indentation regions correlates with lobe initiation and outgrowth during PC development. Cell biological and genetic approaches revealed that IQD21 promotes transfacial microtubules anchoring to the plasma membrane via its polybasic sites and bundling at the indentation regions in both periclinal and anticlinal walls. IQD21 controls cortical microtubule organization mainly through promoting Katanin 1-mediated microtubule severing during PC interdigitation. These findings provide the genetic evidence that transfacial microtubule arrays play a determinant role in lobe formation, and the insight into the molecular mechanism of IQD21 in transfacial microtubule organization at indentations and puzzle-shaped PC development.

MicroRNA targets the ecdysone signaling pathway E75 gene to control larval-pupal development in Bactrocera dorsalis

MicroRNAs (miRNAs) regulate various biological processes during insect developme nt;however, their role in larval-pupal development in oriental fruit fly, Bactrocera dorsalis (Hendel) remains unknown. In the current study, we address the biological function of a conserved miRNA, Bdo-Let-7 in the regulation of BdE75 gene, which belongs to the ecdysone signaling pathway and participates in the larval-pupal development in B. dorsalis. Using dual luciferase reporter assay in HEK293T cells we show that Bdo-Let-7 miRNA interacts with the 3' untranslated region of BdE75 gene and suppresses its expression. The Bdo-Let?7 and BdE75 are also co-expressed in the larval-pupal stages and in different tissues of B. dorsalis .In in vivo experiments, the injectio n of Bdo-Let-7 agomir and antagomir in third instar larvae down- and up-regulated the expression of BdE75、 respectively. The 20-hydroxyecdysone (20E) injection assay shows that 20E up-regulated the expression of Bdo-Let-7 on the 5th day of the larvae. Moreover, abnormal pupation and eclosion were observed after larval Bdo-Let-7 antagomir injection. Based on these results, we show that Bdo-Let-7 regulates the ecdysone signaling pathway through the exact dose of BdE75 gene, and is indispensable for normal larval-pupal development in B. dorsalis.

microRNA-mediated R gene regulation: molecular scabbards for double-edged swords

Plant resistance(R) proteins are immune receptors that recognize pathogen effectors and trigger rapid defense responses, namely effector-triggered immunity. R protein-mediated pathogen resistance is usually race specific. During plant-pathogen coevolution,plant genomes accumulated large numbers of R genes. Even though plant R genes provide important natural resources for breeding disease-resistant crops, their presence in the plant genome comes at a cost. Misregulation of R genes leads to developmental defects, such as stunted growth and reduced fertility. In the past decade, many microRNAs(miRNAs) have been identified to target various R genes in plant genomes. miRNAs reduce R gene levels under normal conditions and allow induction of R gene expression under various stresses. For these reasons, we consider R genes to be double-edged "swords" and miRNAs as molecular "scabbards". In the present review, we summarize the contributions and potential problems of these "swords" and discuss the features and production of the "scabbards", as well as the mechanisms used to pull the "sword" from the "scabbard"when needed.

Small GTPase Rab40C is upregulated by 20-hydroxyecdysone and insulin pathways to regulate ovarian development and fecundity

The insulin and 20-hydroxyecdysone(20E)pathways coordinately regulate in-sect vitellogenesis and ovarian development.However,the detailed molecular mechanisms such as the genes mediating the cooperation of the interaction of these 2 pathways in reg-ulating insect reproductive development are not well understood.In the present study,a small GTPase,Rab4oC,was identified from the notorious agricultural pest Bactrocera dorsalis.In addition to the well-known RAB domain,it also has a unique SOCS-box do-main,which is different from other Rab-GTPases.Moreover,we found that Rab40C was enriched in the ovaries of sexually mature females.RNA interference(RNAi)-mediated knockdown of BdRab40C resulted in a decrease in vitellogenin synthesis,underdevel-oped ovaries,and low fertility.Furthermore,depletion of insulin receptor InR or the het-erodimer receptor of 20E(EcRor USP)by RNAi significantlydecreased the transcription of BdRab40C and resulted in lower fecundity.Further studies revealed that the transcrip-tion of BdRab40C could be upregulated by the injection of insulin or 20E.These results indicate that Rab40C participates in the insulin and 20E pathways to coordinately regulate reproduction in B.dorsalis.Our results not only provide new insights into the insulin-and 20E-stimulated regulatory pathways controlling female reproduction in insects but also contribute to the development of potential eco-friendly strategies for pest control.

Gut fungal community and its probiotic effect on Bactrocera dorsalis

The oriental fruit fly,Bactrocera dorsalis(Diptera:Tephritidae)is a destructive horticultural pest which causes considerable economic losses every year.A collection of microorganisms live within the B.dorsalis gut,and they are involved in its development,physiology,and behavior.However,knowledge regarding the composition and function of the gut mycobiota in B.dorsalis are still limited.Here,we comprehensively characterized the gut mycobiota in B.dorsalis across different developmental stages.High-throughput sequencing results showed a significant difference in fungal species abundance and diversity among different developmental stages of B.dorsalis.Quantitative polymerase chain reaction and culture-dependent methods showed that yeast species was the dominant group in the larval stage.We isolated 13 strains of yeast from the larval gut,and found that GF(germ-free)larvae mono-associated with strain Hanseniaspora uvarum developed faster than those mono-associated with other tested fungal strains.Supplementing the larval diet with H.uvarum fully rescued B.dorsalis development,shortened the larval developmental time,and increased adult wing lengths,as well as the body sizes and weights of both pupae and adults.Thus,our study highlights the close interactions between gut fungi,especially H.uvarum,and B.dorsalis.These findings can be applied to the sterile insect technique program to promote host development during mass insect rearing.

Transcription factor CsESE3 positively modulates both jasmonic acid and wax biosynthesis in citrus

PLIP lipases can initiate jasmonic acid(JA)biosynthesis.However,little is known about the transcriptional regulation of this process.In this study,an ERF transcription factor(CsESE3)was found to be co-expressed with all necessary genes for JA biosynthesis and several key genes for wax biosynthesis in transcriptomes of‘Newhall’navel orange.CsESE3 shows partial sequence similarity to the well-known wax regulator SHINEs(SHNs),but lacks a complete MM protein domain.Ectopic overexpression of CsESE3 in tomato(OE)resulted in reduction of fruit surface brightness and dwarf phenotype compared to the wild type.The OE tomato lines also showed significant increases in the content of wax and JA and the expression of key genes related to their biosynthesis.Overexpression of CsESE3 in citrus callus and fruit enhanced the JA content and the expression of JA biosynthetic genes.Furthermore,CsESE3 could bind to and activate the promoters of two phospholipases from the PLIP gene family to initiate JA biosynthesis.Overall,this study indicated that CsESE3 could mediate JA biosynthesis by activating PLIP genes and positively modulate wax biosynthesis.The findings provide important insights into the coordinated control of two defense strategies of plants represented by wax and JA biosynthesis.

Novel polymorphic EST-based microsatellite marker isolation and characterization from Poncirus trifoliata(Rutaceae)

Novel Poncirus trifoliata simple sequence repeat(SSR) markers were developed to evaluate their utility for genetic diversity and breeding studies of P. trifoliata and related species. A total of 108 primer pairs were characterized by PCR amplification experiments. Among these, 61 were polymorphic and transferable to other citrus species. The number of alleles per locus ranged from 2 to 6, with an average of 2.37 alleles per locus. The expected heterozygosity and observed heterozygosity ranged from 0 to 0.83 and 0 to 1.00, respectively.These novel polymorphic SSR markers will be useful for citrus cultivar identification and evaluation as well as breeding studies.