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.

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.

Efficient Breeding and Cultivation of Type 2 Red-fleshed Apple Cultivars Using a Search System for Suitable Apple Cultivar Combination

As apple possesses self-incompatibility, knowledge of the S-genotypes is very important for apple breeding and cultivation. We investigated the S-RNase genotypes of 13 type 2 red-fleshed apple cultivars and 8 lineages. All of the investigated type 2 red-fleshed apples contained the S3-RNase allele closely linked to the red-fleshed trait. We made a search system for ovule parents to efficiently produce new type 2 red-fleshed cultivars based on the S-RNase allele information(http://www.agr.nagoya-u.ac.jp/～hort/apple/red/). More than 90% of progenies from crossing a selected white-fleshed ovule parent with a type 2 red-fleshed pollen parent are expected to show the red-fleshed phenotype. We also compiled a database of the S-RNase genotypes of more than 1 000 apple cultivars, including a survey system of cultivar combinations showing those that were fully incompatible, semicompatible and fully compatible, written in Chinese(http://www.agr.nagoya-u.ac.jp/～hort/apple/ch/) and Japanese(http://www.agr.nagoya-u.ac.jp/～hort/apple/ja/).

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.

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.