A novel miRNA negatively regulates resistance to Glomerella leaf spot by suppressing expression of an NBS gene in apple

Glomerella leaf spot(GLS)of apple(Malus×domestica Borkh.),caused by Glomerella cingulata,is an emerging fungal epidemic threatening the apple industry.Little is known about the molecular mechanism underlying resistance to this devastating fungus.In this study,high-throughput sequencing technology was used to identify microRNAs(miRNAs)involved in GLS resistance in apple.We focused on miRNAs that target genes related to disease and found that expression of a novel miRNA,Md-miRln20,was higher in susceptible apple varieties than in resistant ones.Furthermore,its target gene Md-TN1-GLS exhibited the opposite expression pattern,which suggested that the expression levels of Md-miRln20 and its target gene are closely related to apple resistance to GLS.Furthermore,downregulation of MdmiRln20 in susceptible apple leaves resulted in upregulation of Md-TN1-GLS and reduced the disease incidence.Conversely,overexpression of Md-miRln20 in resistant apple leaves suppressed Md-TN1-GLS expression,with increased disease incidence.We demonstrated that Md-miRln20 negatively regulates resistance to GLS by suppressing Md-TN1-GLS expression and showed,for the first time,a crucial role for miRNA in response to GLS in apple.

Two pathogenesis-related proteins interact with leucine-rich repeat proteins to promote Alternaria leaf spot resistance in apple

Alternaria leaf spot in apple(Malus x domestica),caused by the fungal pathogen Alternaria alternata f.sp.mali(also called A.mali),is a devastating disease resulting in substantial economic losses.We previously established that the resistance(R)protein MdRNL2,containing a coiled-coil,nucleotide-binding,and leucine-rich repeat(CC R-NB-LRR)domain,interacts with another CC R-NB-LRR protein,MdRNL6,to form a MdRNL2–MdRNL6 complex that confers resistance to A.mali.Here,to investigate the function of the MdRNL2–MdRNL6 complex,we identified two novel pathogenesis-related(PR)proteins,MdPR10-1 and MdPR10-2,that interact with MdRNL2.Yeast two-hybrid(Y2H)assays and bimolecular fluorescence complementation(BiFC)assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain.Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A.mali that we tested:ALT1,GBYB2,BXSB5,and BXSB7.In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf spot resistance by inhibiting fungal growth.Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth,thereby contributing to Alternaria leaf spot resistance in apple.The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.

Functional identification of lncRNAs in sweet cherry(Prunus avium)pollen tubes via transcriptome analysis using single-molecule long-read sequencing

Sweet cherry(Prunus avium)is a popular fruit with high nutritional value and excellent flavor.Although pollen plays an important role in the double fertilization and subsequent fruit production of this species,little is known about its pollen tube transcriptome.In this study,we identified 16,409 transcripts using single-molecule sequencing.After filtering 292 transposable elements,we conducted further analyses including mRNA classification,gene function prediction,alternative splicing(AS)analysis,and long noncoding RNA(lncRNA)identification to gain insight into the pollen transcriptome.The filtered transcripts could be matched with 3,438 coding region sequences from the sweet cherry genome.GO and KEGG analyses revealed complex biological processes during pollen tube elongation.A total of 2043 AS events were predicted,7 of which were identified in different organs,such as the leaf,pistil and pollen tube.Using BLASTnt and the Coding-Potential Assessment Tool(CPAT),we distinguished a total of 284 lncRNAs,among which 154 qualified as natural antisense transcripts(NATs).As the NATs could be the reverse complements of coding mRNA sequences,they might bind to coding sequences.Antisense transfection assays showed that the NATs could regulate the expression levels of their complementary sequences and even affect the growth conditions of pollen tubes.In summary,this research characterizes the transcripts of P.avium pollen and lays the foundation for elucidating the physiological and biochemical mechanisms underlying sexual reproduction in the male gametes of this species.

Cloning and Characterization of miRNAs and Their Targets, Including a Novel miRNA-Targeted NBS-LRR Protein Class Gene in Apple （Golden Delicious）

MicroRNA （miRNA） has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple （Malus domestica cv. Golden Delicious） by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 （Malus domestica microRNA Ln11）, targeted an apple nucleotide-binding site （NBS）-Ieucine-rich repeat （LRR） class protein coding gene （Md-NBS）. 5/ RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11-12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety ＇JiGuan＇ were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety ＇FuJi＇ infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS-LRR protein class genes in woody plants.

Nanomaterials for neurodegenerative diseases:Molecular mechanisms guided design and applications

Successful treatment of neurodegenerative diseases(NDDs),including Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD),remains a significant challenge for neurologists due to the undesirable curative outcomes.Apart from surgeries,most drugs are only used to relieve the patients’symptoms without a permanent cure of the disease.The development of novel biomaterials targeting NDDs is greatly hindered by the limited understanding of underlying molecular mechanisms.Considering the difficulties in NDD drug development and clinical trials,a comprehensive and up-to-date review of disease pathogenesis and related novel therapies are needed.In the current article,the basic concepts and pathogenic characteristics of NDDs are firstly illustrated.Following the detailed description of molecular mechanisms underlying three common NDDs,recent advances of drug development based on targeting different pathogenic mechanisms are clarified.Hopefully,this review will be beneficial to address the gap between materials and targeted mechanisms while simultaneously provide suggestions for the future design of precise NDD medicine.

一种乳酸菌-金属有机框架复合物用于强化肿瘤乏氧光动力和免疫治疗

开发长期有效的癌症治疗技术仍然是科研工作者和临床医生面临的重要任务.厌氧型乳酸菌(LAB)具有天然的乏氧倾向性,可自发富集在缺氧的肿瘤微环境中.金属有机框架(MOF)是一类独特的纳米材料,其中的ZIF-67可以自发分解过氧化氢产氧,且析氧能力可被光照增强.然而,LAB或MOF单独应用于肿瘤治疗的效果并不令人满意.在本文中,我们制备了一种MOF修饰的乳酸菌制剂,用以在缺氧性肿瘤中局部产生活性氧(ROS)助力光动力治疗,同时精准诱导宿主肿瘤免疫.这些细菌表面被ZIF-67纳米材料修饰(LAB@ZIF-67),经体内循环后富集在肿瘤微环境中.细菌表面的ZIF-67可以被CT-26细胞吸收,并且在光照后检测到细胞内快速生成ROS.体内实验表明,该细菌复合物可通过光动力疗法消灭肿瘤,同时诱导肿瘤特异的炎性反应,招募巨噬细胞和T细胞实现肿瘤的免疫治疗.此类材料-细菌复合物为今后研发肿瘤主动靶向药物和癌症免疫疗法提供了新思路.