DkWRKY transcription factors enhance persimmon resistance to Colletotrichum horii by promoting lignin accumulation through DkCAD1 promotor interaction.

Persimmon anthracnose, a severe disease caused by the hemibiotrophic fungus Colletotrichum horii, poses a substantial threat to China's persimmon industry. Previous research showed that 'Kangbing Jianshi' cultivar exhibits strong resistance to anthracnose. Notably, 'Kangbing Jianshi' branches exhibit greater lignification compared with the susceptible 'Fuping Jianshi' cultivar. In this study, higher lignin content was observed in 'Kangbing Jianshi' compared with 'Fuping Jianshi', and this difference was associated with disease resistance. Transcriptome and metabolome analyses revealed that the majority of differentially expressed genes and differentially accumulated metabolites were primarily enriched in the phenylpropanoid biosynthesis and lignin synthesis pathways. Furthermore, significant upregulation of DkCAD1, a pivotal gene involved in lignin metabolism, was observed in the resistant cultivar when inoculated with C. horii. Transient overexpression of DkCAD1 substantially increased lignin content and improved resistance to C. horii in a susceptible cultivar. Furthermore, through yeast one-hybrid (Y1H) assays, we identified two WRKY transcription factors, DkWRKY8 and DkWRKY10, which interacts with the DkCAD1 promoter and induces its activity. Overexpression of DkWRKY8 and DkWRKY10 not only increased leaf lignin content but also enhanced persimmon tolerance to C. horii. Moreover, the expression levels of DkCAD1, DkWRKY8, and DkWRKY10 were significantly increased in response to salicylic acid and jasmonic acid in the resistant cultivar. These findings enhance our understanding of the molecular functions of DkWRKY8, DkWRKY10, and DkCAD1 in persimmons, as well as their involvement in molecular breeding processes in persimmons.

First Report of Colletotrichum fioriniae Causing Anthracnose on Persimmon in China.

Persimmons (Diospyros kaki Thunb.) have a longstanding history of cultivation in China. Both aesthetically pleasing and edible, they often symbolize a sweet and fulfilling life. During the summer of 2022, a severe outbreak of anthracnose was observed on the lower leaves of persimmon trees in the National Field Genebank for Persimmon (NFGP), located in Yangling, Shaanxi, China (34°17'42.80″ N, 108°04'08.21″ E). The estimated incidence rate of this disease within the NFGP was approximately 30%. The typical symptoms of the disease included the presence of irregular lesions on leaves, and oval sunken lesions on infected fruit. Under high humidity conditions, pink sticky substances appeared in the affected areas. The presence of numerous lesions led to softening and detachment of persimmon fruit. To identify the causal pathogen, 5 × 5mm samples of the diseased leaves were collected from the interface between the infected and healthy leaves. The leaves were disinfected with 70% alcohol for 20 s, followed by rinsing with sterile water. Subsequently, the leaves were immersed in 1% NaClO for 2 to 3 minutes, rinsed with sterile water three times, dried using sterile absorbent paper, and the leaf samples were then transferred onto potato dextrose agar (PDA) medium, and cultured in 25°C incubators. Once the colony reached a certain size, small pieces of hyphae were extracted from edge and transferred for purification and repeated three times. After being cultured on PDA for 7 days, the colony showed a white spongy surface with a pink-orange center. The conidia displayed a fusiform shape and were transparent, measuring 4.58 to 6.53 µm × 9.27 to 13.11 µm (n=50). The isolates share morphological similarities with Colletotrichum fioriniae. The representative isolate HY-7 was selected for molecular identification. The internal transcribed spacers (ITS) region, chitin synthase (CHS-1), actin (ACT), beta-tubulin 2 (TUB2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were amplified using ITS1/4 (White et al. 1990), CHS-79F/CHS-345R (Carbone & Kohn, 1999), ACT512F/ACT (Carbone & Kohn, 1999), T1/BT2B (Glass & Donaldson 1995, O'Donnell et al., 1997), and GDF/GDR (Templeton et al. 1992), respectively. The generated sequences were deposited at GenBank under accession numbers OR878056 (ITS), OR766019 (CHS-1), OR766021(TUB2), OR766018 (ACT) and OR766020 (GAPDH). BLAST analysis revealed the sequences were 100% identical to C. fioriniae (MH865005 for ITS, JQ948953 for CHS-1, JQ949613 for ACT, JQ949943 for TUB2 and JQ948622 for GAPDH). The morphological characteristics and molecular analyses of the isolate matched the description of C. fioriniae. To fulfill Koch's postulates, the twigs and leaves of 'Fupingjianshi' in four different directions were inoculated without wounding in the field, and 10 healthy fruits were selected for wound inoculation. The concentration of conidia used for inoculation was about 1 × 106 conidia/ml, and sterilized water was used as control. The experiment was replicated three times under the same conditions. One week after inoculation, characteristic symptoms resembling those observed on the leaves of primary diseased persimmon trees appeared on the leaves and fruits. No symptoms were observed on the leaves, twigs and fruits in the control treatment. The pathogen from the artificially infected leaves and fruits were reisolated and identified as C. fiorinae based on morphological and molecular characteristics. Persimmon anthracnose is a common disease in regions where the fruit is grown, to the best of our knowledge, this is the first documented occurrence of C. fioriniae-induced anthracnose on persimmons in China, which should be paid more attentions. This report will help identify disease symptoms in the field and provides a basis for determining the occurrence, distribution, and control of C. fioriniae on persimmon leaves and fruits.

First Report of Leaf Black Spot Caused by Alternaria alternata on American Persimmons in China.

American persimmons (Diospyros virginiana L.) are native to the United States. After being introduced into China, they were used as a rootstock for expanding persimmon varieties and planted in local areas due to their strong cold resistance and diverse leaf colors. In 2022, 12 plants had similar symptoms to black spot disease on the leaves of 18 American persimmons introduced in the National Field Genebank for Persimmon, Yangling, Shaanxi, China (34°17'42.80″ N, 108°04'08.21″ E). Among them, severity was highest in the 'VM10' variety (almost 100%), 'VM10' is the main cultivar in Shaanxi and Henan regions of China, and the incidence of disease in the two regions ranged between 30 and 60% in 2022. Early symptoms were irregular black-brown spots, which gradually combined into large irregular lesions with a dark brown border. The leaves began to curl, crack, scorch, and abscissed. When relative humidity was high, leaves also had signs of black sporulation and became chlorotic. To isolate the causal agent, 10 symptomatic leaves were collected from 5 diseased plants in the National Field Genebank for Persimmon. The infected leaves were cut into 20 small pieces of 5 × 5 mm from the junction of the diseased and healthy tissues and surface disinfected in 75% alcohol for 15 sec, washed with sterile water and 2% NaClO for 90 sec, rinsed three times with sterile water, dried with sterile absorbent paper, and plated on potato dextrose agar (PDA) medium. After 3 days, 12 strains of fungi were isolated from the tissue by transferring the hyphal tips of the mycelium. Among them, 10 strains had similar morphological characteristics. Fungal colonies developed on the PDA medium were initially white, then gradually changed to gray-brown with neat edges and flocculent hyphae. Conidia (n=50) light brown or medium brown, obovate or pear-shaped, and 8.27 to 15.31×17.51 to 24.31 µm, with 1 to 4 transverse septa and 0 to 2 longitudinal septa. The isolates were morphologically similar to Alternaria alternata (Simmons et al. 2007). For molecular identification, the E.Z.N.A.® Fungal DNA kit (Omega Bio-Tek) was used to extract genomic DNA from 7-day-old mycelium grown on PDA medium. The internal transcribed spacers (ITS) region, translation elongation factor 1-alpha (TEF1-α), Alternaria major allergen (Alt a1) gene, and partial RNA polymerase second largest subunit (RPB2) were amplified using ITS1/4 (Glass et al. 1995), EF1-728F/EF1-968R (Carbone and Kohn 1999), and Alt-4for /Alt-4rev (Hong et al., 2005) and RPB2-5F/RPB2-7CR (Liu et al. 1999) respectively. The sequences of a representative isolate MZS1 were deposited in GeneBank with accession numbers OP198643 for ITS, OP286949 for Tef1-α, OP286948 for Alt a1, and OP951084 for RPB2, and were 100% identical to strains of A. alternata (MN615420 for ITS, MN615423 for EF1-α, MW848792 for Alt a1 and MN615422 for RPB2). A maximum-likelihood (ML) phylogenetic tree was constructed based on the concatenated sequences of ITS, TEF1-α, Alt a1, and RPB2gene, which clustered with the A. alternata strains YZU191238 with high bootstrap support (99%). To fulfill Koch's postulates, three-month-old American persimmon 'VM10' seedlings were tested for pathogenicity. Three seedlings were sprayed with 1 × 106 spores/ml suspension in a spray pot, and the three seedlings were treated with sterilized water as a noninoculated control. All seedlings were cultured in a 25°C incubator. The experiment was performed three times under the same conditions. One week after inoculation, typical symptoms appeared on the leaves, which were similar to those observed on the leaves of the original infected persimmon trees. In the control treatment, the leaves did not show symptoms. To the best of our knowledge, this is the first report of A. alternata causing American persimmon black spots disease in China. This report will contribute to the identification of disease symptoms in the field and provide a basis for the occurrence, distribution, and control of A. alternata on American persimmon leaves.

Phenotypic Characterization of Postharvest Fruit Qualities in Astringent and Non-astringent Persimmon (Diospyros kaki) Cultivars.

Phenotypic characterization of postharvest traits is essential for the breeding of high-quality fruits. To compare postharvest traits of different genetic lines, it is essential to use a reference point during fruit development that will be common to all the lines. In this study, we employed a non-destructive parameter of chlorophyll levels to establish a similar physiological age and compared several postharvest traits of ten astringent and seven non-astringent persimmon cultivars. The fruit's traits examined were astringency, weight, total soluble solids (TSS), titratable acidity (TA), chlorophyll levels (I AD ), color (hue), firmness, color development and firmness loss during storage, crack development, and susceptibility to Alternaria infection. Although the chlorophyll (I AD ) index and color (hue) showed a high correlation among mature fruits of all cultivars, the chlorophyll parameter could detect higher variability in each cultivar, suggesting that I AD is a more rigorous parameter for detecting the developmental stage. The average weight, TSS, and TA were similar between astringent and non-astringent cultivars. Cracks appeared only on a few cultivars at harvest. Resistance to Alternaria infection and firmness were lower in astringent than in non-astringent cultivars. Only the astringent cultivar "32" was resistant to infection possibly due to the existence of an efficient peel barrier. It was concluded that a high correlation existed between astringency, susceptibility to Alternaria infection, and firmness. Cracks did not correlate with astringency or firmness. The phenotypic traits evaluated in this work can be used in future breeding programs for elite persimmon fruits.

Harnessing Current Knowledge of DNA N6-Methyladenosine From Model Plants for Non-model Crops.

Epigenetic modifications alter the gene activity and function by causing change in the chromosomal architecture through DNA methylation/demethylation, or histone modifications without causing any change in DNA sequence. In plants, DNA cytosine methylation (5mC) is vital for various pathways such as, gene regulation, transposon suppression, DNA repair, replication, transcription, and recombination. Thanks to recent advances in high throughput sequencing (HTS) technologies for epigenomic "Big Data" generation, accumulated studies have revealed the occurrence of another novel DNA methylation mark, N6-methyladenosine (6mA), which is highly present on gene bodies mainly activates gene expression in model plants such as eudicot Arabidopsis (Arabidopsis thaliana) and monocot rice (Oryza sativa). However, in non-model crops, the occurrence and importance of 6mA remains largely less known, with only limited reports in few species, such as Rosaceae (wild strawberry), and soybean (Glycine max). Given the aforementioned vital roles of 6mA in plants, hereinafter, we summarize the latest advances of DNA 6mA modification, and investigate the historical, known and vital functions of 6mA in plants. We also consider advanced artificial-intelligence biotechnologies that improve extraction and prediction of 6mA concepts. In this Review, we discuss the potential challenges that may hinder exploitation of 6mA, and give future goals of 6mA from model plants to non-model crops.

Transcriptome Analysis Revealed the Roles of Carbohydrate Metabolism on Differential Acetaldehyde Production Capacity in Persimmon Fruit in Response to High-CO2 Treatment.

Persimmon (Diospyros kaki Thunb.) fruit is unique due to the continuous accumulation of soluble tannins during fruit development in most cultivars, which causes undesired astringency. High-CO2 treatment was the most effective widely used method for astringency removal. However, differential effects of high-CO2 treatment between cultivars were observed and the molecular basis remained inclusive. Previously, one cultivar ("Luoyangfangtianshengshi," LYFTSS) showed rapid deastringency, while two cultivars ("Shijiazhuanglianhuashi," SJZLHS; "Laopige," LPG) showed slow deastringency in response to high-CO2 (95% CO2) treatment. In this study, the metabolites (acetaldehyde and ethanol) related to deastringency were further analyzed and both acetaldehyde and ethanol were higher in SJZLHS and LYFTSS than that in LPG, where acetaldehyde was undetectable. Based on the RNA-seq data, the weighted gene coexpression network analysis (WGCNA) revealed that one module, comprised of 1773 unigenes, significantly correlated with the contents of acetaldehyde and ethanol (P < 0.001). Further analysis based on the acetaldehyde metabolism pathway indicated that the differentially expressed structural genes, including previously characterized DkADH and DkPDC and also their upstream members (e.g., PFK, phosphofructokinase), showed positive correlations with acetaldehyde production. Quantitative analysis of the precursor substances indicated that sucrose, glucose, and fructose exhibited limited differences between cultivar except for malic acid. However, the content of malic acid is much less than the total soluble sugar content. To verify the correlations between these genes and acetaldehyde production, the fruit from 14 more cultivars were collected and treated with high CO2. After the treatment, acetaldehyde contents in different cultivars ranked in 30.4-255.5 µg/g FW. Real-time polymerase chain reaction (PCR) and correlation analysis indicated that the EVM0002315 (PFK) gene, belonging to carbohydrate metabolism, was significantly correlated with acetaldehyde content in fruit. Thus, it could be proposed that the differentially expressed carbohydrate metabolism related genes (especially PFK) are the basis for the variance of acetaldehyde production among different persimmon cultivars.

DkWRKY interacts with pyruvate kinase gene DkPK1 and promotes natural deastringency in C-PCNA persimmon.

PAs, also known as condensed tannins, cause the astringency sensation in the persimmon fruit. The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree, but the regulatory mechanisms of deastringency remain to be elucidated. In our previous research, DkPK1 was shown to be involved in the natural loss of astringency of C-PCNA persimmon fruit. In the present study, yeast one-hybrid (Y1H) library screening using the DkPK1 promoter as baits identified two DkWRKY transcription factor genes (DkWRKY3 and -15). The transcript levels of both DkWRKY3 and -15 exhibited a positive correlation with the decrease in soluble proanthocyanidin (PA) content during the last developmental stage in C-PCNA persimmon. Multiple sequence analysis and subcellular localization confirmed that DkWRKY3 and -15 belonging to the group II and I families, respectively, were both located in the nucleus. Dual-luciferase and Y1H assays demonstrated that DkWRKY3 and -15 can transactivate the DkPK1 promoters. The combination of DkWRKY3 and -15 most likely produced an additive activation effect compared to a single activator on DkPK1, although the two transcriptional activators were not capable of interacting. Notably, DkWRKY3 and -15 showed ubiquitous expression in various organs and abundant upregulation in seeds. Furthermore, transient overexpression of both DkWRKY3 and -15 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the acetaldehyde metabolism-related DkPK, DkPDC and DkADH genes. Thus, we suggest that DkWRKY3 and -15 are the upstream regulators of DkPK1 and positively regulate the natural deastringency in C-PCNA persimmon.

The persimmon (Diospyros oleifera Cheng) genome provides new insights into the inheritance of astringency and ancestral evolution.

Persimmon (Diospyros kaki) is an oriental perennial woody fruit tree whose popular fruit is produced and consumed worldwide. The persimmon fruit is unique because of the hyperaccumulation of proanthocyanidins during fruit development, causing the mature fruit of most cultivars to have an astringent taste. In this study, we obtained a chromosome-scale genome assembly for 'Youshi' (Diospyros oleifera, 2n = 2x = 30), the diploid species of persimmon, by integrating Illumina sequencing, single-molecule real-time sequencing, and high-throughput chromosome conformation capture techniques. The assembled D. oleifera genome consisted of 849.53 Mb, 94.14% (799.71 Mb) of which was assigned to 15 pseudochromosomes, and is the first assembled genome for any member of the Ebenaceae. Comparative genomic analysis revealed that the D. oleifera genome underwent an ancient γ whole-genome duplication event. We studied the potential genetic basis for astringency development (proanthocyanidin biosynthesis) and removal (proanthocyanidin insolublization). Proanthocyanidin biosynthesis genes were mainly distributed on chromosome 1, and the clustering of these genes is responsible for the genetic stability of astringency heredity. Genome-based RNA-seq identified deastringency genes, and promoter analysis showed that most of their promoters contained large numbers of low oxygen-responsive motifs, which is consistent with the efficient industrial application of high CO2 treatment to remove astringency. Using the D. oleifera genome as the reference, SLAF-seq indicated that 'Youshi' is one of the ancestors of the cultivated persimmon (2n = 6x = 90). Our study provides significant insights into the genetic basis of persimmon evolution and the development and removal astringency, and it will facilitate the improvement of the breeding of persimmon fruit.

Comparative transcriptomic analysis reveals genetic divergence and domestication genes in Diospyros.

BACKGROUND: Persimmon (Diospyros kaki) is the most economically cultivated species belonging to the genus Diospyros. However, little is known about the interspecific diversity and mechanism of domestication, partly due to the lack of genomic information that is available for closely related species of D. kaki (DK). Here, we performed transcriptome sequencing on nine samples, including DK, a variety of DK and seven closely related species, to evaluate the interspecific genetic divergence and to identify candidate genes involved in persimmon domestication. RESULTS: We obtained a total of 483,421 unigenes with N50 at 1490 bp in the nine Diospyros samples and identified 2603 orthogroups that were shared among all the samples using OrthoMCL analysis. A phylogenetic tree was established based on the tandem 2603 one-to-one single copy gene alignments, showing that DK was closely related to D. kaki var. silvestris (DKV) and that it clustered with the clade of D. deyangnsis (DD) and was farthest from the D. cathayensis (DC) species. The nonsynonymous substitutions (Ka), via synonymous substitution (Ks) ratios, was directly proportional to the genetic relationship of the different species. The higher the Ka/Ks ratios, the longer the distance was. Moreover, 31 positively selected genes (PSGs) involved in carbohydrate metabolism and phenolic metabolism were identified and isolated, and nearly all PSGs except the MATE gene had a high expression in the DK or DKV species. It was hypothesized that these genes might contribute to the domestication of the DK species. Finally, we developed the expressed sequence tag-simple sequence repeat (EST-SSR) and identified 2 unique amplicons DKSSR10 and DKSSR39: the former was absent in the DC species but was present in the other species, the latter had a long amplification product in the DJ species. CONCLUSION: This study presents the first transcriptome resources for the closely related species of persimmon and reveals interspecific genetic divergence. It is speculated that DK is derived from the hybridization of DD and DO species. Furthermore, our analysis suggests candidate PSGs that may be crucial for the adaptation, domestication, and speciation of persimmon relatives and suggests that DKSSR10 and DKSSSR39 could potentially serve as species-specific molecular markers.

DkPK Genes Promote Natural Deastringency in C-PCNA Persimmon by Up-regulating DkPDC and DkADH Expression.

The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree. This process is controlled by a single locus and is dominant against other types of persimmons; therefore, this variant is an important candidate for commercial cultivation and the breeding of PCNA cultivars. In our previous study, six full-length coding sequences (CDS) for pyruvate kinase genes (DkPK1-6) were isolated, and DkPK1 is thought to be involved in the natural deastringency of C-PCNA persimmon fruit. Here, we characterize the eight other DkPK genes (DkPK7-14) from C-PCNA persimmon fruit based on transcriptome data. The transcript changes in DkPK7-14 genes and correlations with the proanthocyanidin (PA) content were investigated during different fruit development stages in C-PCNA, J-PCNA, and non-PCNA persimmon; DkPK7 and DkPK8 exhibited up-regulation patterns during the last developmental stage in C-PCNA persimmon that was negatively correlated with the decrease in soluble PAs. Phylogenetic analysis and subcellular localization analysis revealed that DkPK7 and DkPK8 are cytosolic proteins. Notably, DkPK7 and DkPK8 were ubiquitously expressed in various persimmon organs and abundantly up-regulated in seeds. Furthermore, transient over-expression of DkPK7 and DkPK8 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the pyruvate decarboxylase (DkPDC) and alcohol dehydrogenase genes (DkADH), which are closely related to acetaldehyde metabolism. The accumulated acetaldehyde that results from the up-regulation of the DkPDC and DkADH genes can combine with soluble PAs to form insoluble PAs, resulting in the removal of astringency from persimmon fruit. Thus, we suggest that both DkPK7 and DkPK8 are likely to be involved in natural deastringency via the up-regulation of DkPDC and DkADH expression during the last developmental stage in C-PCNA persimmon.

Isolation and Characterization of DkPK Genes Associated with Natural Deastringency in C-PCNA Persimmon.

Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) is considered to be an important germplasm resource for the breeding of PCNA cultivars, though its molecular mechanisms of astringency removal remain to be elucidated. Previously, we showed that the abundance of pyruvate kinase gene transcripts increased rapidly during astringency removal in C-PCNA persimmon fruit. Here, we report the full-length coding sequences of six novel DkPK genes from C-PCNA persimmon fruit isolated based on a complementary DNA (cDNA) library and transcriptome data. The expression patterns of these six DkPK genes and correlations with the soluble proanthocyanidin (PA) content were analyzed during various fruit development stages in different types of persimmon, with DkPK1 showing an expression pattern during the last stage in C-PCNA persimmon that was positively correlated with a decrease in soluble PAs. Phylogenetic analysis revealed that DkPK1 belongs to cytosolic-1 subgroup, and subcellular localization analysis confirmed that DkPK1 is located in the cytosol. Notably, tissue expression profiling revealed ubiquitous DkPK1 expression in different persimmon organs, with the highest expression in seeds. Furthermore, transient over-expression of DkPK1 in persimmon leaves resulted in a significant decrease in the content of soluble PAs but a significant increase in the transcript levels of pyruvate decarboxylase genes (DkPDC1, -3, -4, -5), which catalyze the conversion of pyruvate to acetaldehyde. Thus, we propose that an acetaldehyde-based coagulation effect reduces the content of soluble PAs. Taken together, our results suggest that DkPK1 might be involved in the natural removal of astringency at the last developmental stage in C-PCNA persimmon. This is the first report to identify several novel full-length DkPK genes as well as their potential roles in the natural loss of astringency in C-PCNA persimmon.