Restricted responses of AcMYB68 and AcERF74/75 enhanced waterlogging tolerance in kiwifruit.

Most of kiwifruit cultivars (e.g. Actinidia chinensis cv. Donghong, "DH") were sensitive to waterlogging, thus, waterlogging resistant rootstocks (e.g. Actinidia valvata Dunn, "Dunn") were widely used for kiwifruit industry. Those different species provided ideal materials to understand the waterlogging responses in kiwifruit. Compared to the weaken growth and root activities in "DH", "Dunn" maintained the relative high root activities under the prolonged waterlogging. Based on comparative analysis, transcript levels of pyruvate decarboxylase (PDCs) and alcohol dehydrogenase (ADHs) showed significantly difference between these two species. Both PDCs and ADHs had been significantly increased by waterlogging in "DH", while they were only limitedly triggered by 2 days stress and subsided during the prolonged waterlogging in "Dunn". Thus, 19 differentially expressed transcript factors (DETFs) had been isolated using weighted gene co-expression network analysis combined with transcriptomics and transcript levels of PDCs and ADHs in waterlogged "DH". Among these DETFs, dual luciferase and electrophoretic mobility shift assays indicated AcMYB68 could bind to and trigger the activity of AcPDC2 promoter. The stable over-expression of AcMYB68 significantly up-regulated the transcript levels of PDCs but inhibited the plant growth, especially the roots. Moreover, the enzyme activities of PDC in 35S::AcMYB68 were significantly enhanced during the waterlogging response than that in wild type plants. Most interestingly, comparative analysis indicated that the expression patterns of AcMYB68 and the previously characterized AcERF74/75 (the direct regulator on ADHs) either showed no responses (AcMYB68 and AcERF74) or very limited response (AcERF75) in "Dunn". Taken together, the restricted responses of AcMYB68 and AcERF74/75 in "Dunn" endow its waterlogging tolerance.

A structural variation in the promoter of the leucoanthocyanidin reductase gene AaLAR1 enhances freezing tolerance by modulating proanthocyanidin accumulation in kiwifruit (Actinidia arguta).

Proanthocyanidins (PAs) are important metabolites that enhance freezing tolerance of plants. Actinidia arguta, especially freezing-tolerant germplasms, accumulate abundant PAs in dormant shoots and thereby enhance freezing tolerance, but the underlying mechanism is unknown. In this study, we used two A. arguta with contrasting cold-resistant phenotypes, KL and RB, to explore the mechanisms in response to cold tolerance. We determined that a leucoanthocyanidin reductase gene (AaLAR1) was more highly expressed in freezing-tolerant KL than in freezing-sensitive RB. Moreover, overexpressing AaLAR1 in kiwifruit promoted PAs biosynthesis and enhanced cold tolerance. The AaLAR1 promoters of various A. arguta germplasms differ due to the presence of a 60-bp deletion in cold-tolerant genotypes that forms a functional binding site for MYC-type transcription factor. Yeast one-hybrid and two-hybrid, dual-luciferase reporter, bimolecular fluorescence complementation and coimmunoprecipitation assays indicated that the AaMYC2a binds to the MYC-core cis-element in the AaLAR1 promoter with the assistance of AaMYB5a, thereby promoting PAs accumulation in the shoots of cold-tolerant kiwifruit. We conclude that the variation in the AaLAR1 promoter and the AaMYC2a-AaMYB5a-AaLAR1 module shape freezing tolerance in A. arguta. The identification of a key structural variation in the AaLAR1 promoter offers a new target for resistance breeding of kiwifruit.

NbPTR1 confers resistance against Pseudomonas syringae pv. actinidiae in kiwifruit.

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) causes a devastating canker disease in yellow-fleshed kiwifruit (Actinidia chinensis). The effector HopZ5, which is present in all isolates of Psa3 causing global outbreaks of pandemic kiwifruit canker disease, triggers immunity in Nicotiana benthamiana and is not recognised in susceptible A. chinensis cultivars. In a search for N. benthamiana nonhost resistance genes against HopZ5, we found that the nucleotide-binding leucine-rich repeat receptor NbPTR1 recognised HopZ5. RPM1-interacting protein 4 orthologues from N. benthamiana and A. chinensis formed a complex with NbPTR1 and HopZ5 activity was able to disrupt this interaction. No functional orthologues of NbPTR1 were found in A. chinensis. NbPTR1 transformed into Psa3-susceptible A. chinensis var. chinensis 'Hort16A' plants introduced HopZ5-specific resistance against Psa3. Altogether, this study suggested that expressing NbPTR1 in Psa3-susceptible kiwifruit is a viable approach to acquiring resistance to Psa3 and it provides valuable information for engineering resistance in otherwise susceptible kiwifruit genotypes.

First Report of Fruit Scab Caused by Alternaria alternata on Actinidia chinensis in Korea.

Kiwi (Actinidia chinesis) is an economically important fruit in Korea, with 1,300 ha cultivated and a production of approximately 25,000 tons per year (Kim and Koh, 2018; Kim and Choi, 2023). In late June 2020, fruit scab symptoms were observed on A. chinensis var. rufopulpa in an orchard in Suncheon, Korea. The incidence of scab symptoms among 20-year-old trees was over 75%, primarily superficial, but rendered the fruit less marketable. In the initial stages of the disease, small, light-brown, circular, and oval spots were formed. As the superficial spots expanded, they became cracked scabs measuring 1 to 7 cm with light edges at the later stages. To isolate the causal pathogen, two lesions were cut from two sections of symptomatic tissue, from each of seven fruits from seven trees. Lesions were surface-sterilized with 70% ethanol for 1 min and washed three times with sterilized distilled water (SDW). The sterilized pieces were placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for one week. After subculturing on PDA, single-spore isolation produced 14 isolates: SYP-410 to 423). All 14 colonies appeared greyish-green and cottony on PDA after 7 d. Conidia were pale brown, ellipsoid to obclavate, with ornamented walls, 1 to 6 transverse and 0 to 3 vertical septa, and length × width of 21.5 to 53.4 × 7.3 to 19.2 µm (avg. 33.0 × 12.0 µm, n = 100). Their morphological characteristics were consistent with Alternaria spp. (van der Waals et al. 2011; Woudenberg et al. 2015). We randomly selected three isolates from the morphologically similar cultures and named them SYP-412 to 414 for further investigation. The ITS (GenBank accession nos.: OR901850 to 52), gapdh (OR924309 to 11), tef1 (OR924312 to 14), rpb2 (OR924315 to 17), Alt a1 (OR924318 to 20), endoPG (OR924321 to 23), and OPA10-2 (OR924324 to 26) sequences from SYP-412 to 414 had a 100% (515 bp/515 bp), 100% (578/578), 100% (240/240), 100% (724/724), 95.55% (451/472), 99.33% (445/448), and 100% (634/634) identity with that of type strain A. alternata CBS 918.96 (AF347032, AY278809, KC584693, KC584435, AY563302, KP124026, and KP124633), respectively. Results from the maximum likelihood phylogenetic analysis, based on the seven concatenated gene sequences, placed the representative isolates in a clade with A. alternata. Pathogenicity of SYP-412 was tested using 12 surface-sterilized two-month-old kiwifruits on a 20-year-old trees. Six kiwifruits were spray-inoculated with 5 mL of a conidial suspension (1 × 106 conidia/ml) generated after culturing in PDA medium for 7 d, with or without wounding. Another six control fruits were inoculated with SDW with and without wounding. The inoculated kiwifruits were enclosed in plastic bags to maintain high humidity for one day. Scab symptoms were observed in both wounded and unwounded fruits six weeks after inoculation, but not in the control. The pathogenicity test was performed on a total of three separate trees twice. To satisfy Koch's postulates, A. alternata was re-isolated from all the symptomatic tissues and confirmed by analyzing the ITS and rpb2 genes. Although scab disease caused by A. tenuissima (now A. alternata) has been previously reported in kiwifruit of A. chinensis var. rufopulpa in China (Woudenberg et al. 2015; Ma et al., 2019), this is the first report of its occurrence on kiwifruit in Korea and will help in future detection and control.

Genome-Wide Identification of the DOF Gene Family in Kiwifruit (Actinidia chinensis) and Functional Validation of AcDOF22 in Response to Drought Stress.

DNA-binding one zinc finger (DOF) transcription factors are crucial plant-specific regulators involved in growth, development, signal transduction, and abiotic stress response generation. However, the genome-wide identification and characterization of AcDOF genes and their regulatory elements in kiwifruit (Actinidia chinensis) has not been thoroughly investigated. In this study, we screened the kiwifruit genome database and identified 42 AcDOF genes (AcDOF1 to AcDOF42). Phylogenetic analysis facilitated the categorization of these genes into five subfamilies (DOF-a, DOF-b, DOF-c, DOF-d, and DOF-e). We further analyzed the motifs, conserved domains, gene structures, and collinearity of the AcDOFgene family. Gene ontology (GO) enrichment analysis indicated significant enrichment in the "flower development" term and the "response to abiotic stress" category. Promoter prediction analysis revealed numerous cis-regulatory elements related to responses to light, hormones, and low-temperature and drought stress in AcDOF promoters. RNA-seq expression profiles demonstrated the tissue-specific expression of AcDOF genes. Quantitative real-time PCR results showed that six selected genes (AcDOF04, AcDOF09, AcDOF11, AcDOF13, AcDOF21, and AcDOF22) were differentially induced by abscisic acid (ABA), methyl jasmonate (MeJA), and cold, salt, and drought stresses, with AcDOF22 specifically expressed at high levels in drought-tolerant cultivars. Further experiments indicated that transient AcDOF22 overexpression in kiwifruit leaf disks reduced water loss and chlorophyll degradation. Additionally, AcDOF22 was localized to the nucleus and exhibited transcriptional activation, enhancing drought resistance by activating the downstream drought marker gene AcDREB2A. These findings lay the foundation for elucidating the molecular mechanisms of drought resistance in kiwifruit and offer new insights into drought-resistant breeding.

Genomic architecture of resistance to latania scale (H. lataniae) in kiwifruit (A. chinensis var. chinensis).

BACKGROUND: Latania scale (Hemiberlesia lataniae Signoret) is an armoured scale insect known to cause damage to kiwifruit plants and fruit, which ultimately reduces crop values and creates post-harvest export and quarantine issues. Resistance to H. lataniae does exist in some commercial cultivars of kiwifruit. However, some of the commercial cultivars bred in New Zealand have not inherited alleles for resistance to H. lataniae carried by their parents. To elucidate the architecture of resistance in the parents and develop molecular markers to assist breeding, these experiments analysed the inheritance of resistance to H. lataniae from families related to commercial cultivars. RESULTS: The first experiment identified a 15.97 Mb genomic region of interest for resistance to H. lataniae in rtGBS data of 3.23 to 19.20 Mb on chromosome 10. A larger population was then QTL mapped, which confirmed the region of interest as the sole locus contributing to H. lataniae resistance. inDel markers mapping the region of low recombination under the QTL peak further narrowed the region associated with H. lataniae resistance to a 5.73 Mb region. CONCLUSIONS: The kiwifruit populations and genomic methods used in this study identify the same non-recombinant region of chromosome 10 which confers resistance of A. chinensis var. chinensis to H. lataniae. The markers developed to target the H. lataniae resistance loci will reduce the amount of costly and time-consuming phenotyping required for breeding H. lataniae scale resistance into new kiwifruit cultivars.

Mechanisms of Actinidia chinensis Planch in treating colon cancer based on the integration of network pharmacology, molecular docking, and experimental verification.

BACKGROUND: As an anticancer Chinese herbal medicine, the effective components and mechanism of Actinidia chinensis Planch (ACP, Tengligen) in the treatment of colon cancer are still unclear. In the present study, the integration of network pharmacology, molecular docking, and cell experiments was employed to study the effective mechanism of ACP against colon cancer. METHODS: The Venn diagram and STRING database were used to construct the protein-protein interaction network (PPI) of ACP-colon cancer, and further topological analysis was used to obtain the key target genes of ACP in colon cancer. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to visualize the related functions and pathways. Molecular docking between key targets and compounds was determined using software such as AutoDockTools. Finally, the effect of ACP on CT26 cells was observed in vitro. RESULTS: The study identified 40 ACP-colon key targets, including CASP3, CDK2, GSK3B, and PIK3R1. GO and KEGG enrichment analyses found that these genes were involved in 211 biological processes and 92 pathways, among which pathways in cancer, PI3K-Akt, p53, and cell cycle might be the main pathways of ACP against colon cancer. Molecular docking verified that the key components of ACP could stably bind to the corresponding targets. The experimental results showed that ACP could inhibit proliferation, induce apoptosis, and downregulate the phosphorylation of PIK3R1, Akt, and GSK3B in CT26 cells. CONCLUSION: ACP is an anti-colon cancer herb with multiple components, and involvement of multiple target genes and signaling pathways. ACP can significantly inhibit proliferation and induce apoptosis of colon cancer cells, which may be closely related to the regulation of PI3K/AKT/GSK3B signal transduction.

Genome-Wide Identification of Kiwifruit SGR Family Members and Functional Characterization of SGR2 Protein for Chlorophyll Degradation.

The STAY-GREEN (SGR) proteins play an important role in chlorophyll (Chl) degradation and are closely related to plant photosynthesis. However, the availability of inadequate studies on SGR motivated us to conduct a comprehensive study on the identification and functional dissection of SGR superfamily members in kiwifruit. Here, we identified five SGR genes for each of the kiwifruit species [Actinidia chinensis (Ac) and Actinidia eriantha (Ae)]. The phylogenetic analysis showed that the kiwifruit SGR superfamily members were divided into two subfamilies the SGR subfamily and the SGRL subfamily. The results of transcriptome data and RT-qPCR showed that the expression of the kiwifruit SGRs was closely related to light and plant developmental stages (regulated by plant growth regulators), which were further supported by the presence of light and the plant hormone-responsive cis-regulatory element in the promoter region. The subcellular localization analysis of the AcSGR2 protein confirmed its localization in the chloroplast. The Fv/Fm, SPAD value, and Chl contents were decreased in overexpressed AcSGR2, but varied in different cultivars of A. chinensis. The sequence analysis showed significant differences within AcSGR2 proteins. Our findings provide valuable insights into the characteristics and evolutionary patterns of SGR genes in kiwifruit, and shall assist kiwifruit breeders to enhance cultivar development.

Ethylene-induced potassium transporter AcKUP2 gene is involved in kiwifruit postharvest ripening.

BACKGROUND: Potassium (K) is important in the regulation of plant growth and development. It is the most abundant mineral element in kiwifruit, and its content increases during fruit ripening. However, how K+ transporter works in kiwifruit postharvest maturation is not yet clear. RESULTS: Here, 12 K+ transporter KT/HAK/KUP genes, AcKUP1 ~ AcKUP12, were isolated from kiwifruit, and their phylogeny, genomic structure, chromosomal location, protein properties, conserved motifs and cis-acting elements were analysed. Transcription analysis revealed that AcKUP2 expression increased rapidly and was maintained at a high level during postharvest maturation, consistent with the trend of K content; AcKUP2 expression was induced by ethylene, suggesting that AcKUP2 might play a role in ripening. Fluorescence microscopy showed that AcKUP2 is localised in the plasma membrane. Cis-elements, including DER or ethylene response element (ERE) responsive to ethylene, were found in the AcKUP2 promoter sequence, and ethylene significantly enhanced the AcKUP2 promoter activity. Furthermore, we verified that AcERF15, an ethylene response factor, directly binds to the AcKUP2 promoter to promote its expression. Thus, AcKUP2 may be an important potassium transporter gene which involved in ethylene-regulated kiwifruit postharvest ripening. CONCLUSIONS: Therefore, our study establishes the first genome-wide analysis of the kiwifruit KT/HAK/KUP gene family and provides valuable information for understanding the function of the KT/HAK/KUP genes in kiwifruit postharvest ripening.

CRISPR-Cas9-mediated mutagenesis of kiwifruit BFT genes results in an evergrowing but not early flowering phenotype.

Phosphatidylethanolamine-binding protein (PEBP) genes regulate flowering and architecture in many plant species. Here, we study kiwifruit (Actinidia chinensis, Ac) PEBP genes with homology to BROTHER OF FT AND TFL1 (BFT). CRISPR-Cas9 was used to target AcBFT genes in wild-type and fast-flowering kiwifruit backgrounds. The editing construct was designed to preferentially target AcBFT2, whose expression is elevated in dormant buds. Acbft lines displayed an evergrowing phenotype and increased branching, while control plants established winter dormancy. The evergrowing phenotype, encompassing delayed budset and advanced budbreak after defoliation, was identified in multiple independent lines with edits in both alleles of AcBFT2. RNA-seq analyses conducted using buds from gene-edited and control lines indicated that Acbft evergrowing plants had a transcriptome similar to that of actively growing wild-type plants, rather than dormant controls. Mutations in both alleles of AcBFT2 did not promote flowering in wild-type or affect flowering time, morphology and fertility in fast-flowering transgenic kiwifruit. In summary, editing of AcBFT2 has the potential to reduce plant dormancy with no adverse effect on flowering, giving rise to cultivars better suited for a changing climate.

A Rapid qPCR for the Detection of Verticillium nonalfalfae MLST2 - A Highly Pathogenic Fungus on Kiwifruit.

A highly pathogenic fungus characterized as Verticillium nonalfalfae multilocus sequence type 2 (MLST2) is an emerging fungal pathogen causing Verticillium wilt on kiwifruit. Although V. nonalfalfae MLST2 has not been reported outside Chile, there is a risk that this pathogen could spread through the global movement of germplasms to other countries. Current diagnostic methods for this fungus rely on a laborious and time-consuming plating assay for morphological identification and DNA sequence analysis. In this study, we describe the development and validation of a novel quantitative polymerase chain reaction (qPCR) assay for rapid and specific detection of V. nonalfalfae MLST2 in plant tissues. The assay targets the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and was shown to detect all tested isolates of V. nonalfalfae MLST2 with a detection limit of approximately 2 pg of pathogen genomic DNA. There was no cross-reaction with V. nonalfalfae MLST1, other Verticillium species, or non-target fungal species found on kiwifruit. This assay was duplexed with a plant internal control for simultaneous detection of the pathogen and cytochrome oxidase gene from the host plant. This new specific and sensitive qPCR assay is a valuable molecular diagnostic tool for rapid screening of imported plant material and would also be useful for testing samples collected from field surveillance activities to monitor the presence of V. nonalfalfae MLST2.

Genomic Variation and Host Interaction among Pseudomonas syringae pv. actinidiae Strains in Actinidia chinensis 'Hongyang'.

Kiwifruit bacterial canker is a recent epidemic disease caused by Pseudomonas syringae pv. actinidiae (Psa), which has undergone worldwide expansion in a short time and resulted in significant economic losses. 'Hongyang' (Actinidia chinensis), a widely grown cultivar because of its health-beneficial nutrients and appreciated red-centered inner pericarp, is highly sensitive to Psa. In this work, ten Psa strains were isolated from 'Hongyang' and sequenced for genome analysis. The results indicated divergences in pathogenicity and pathogenic-related genes among the Psa strains. Significantly, the interruption at the 596 bp of HrpR in two low-pathogenicity strains reemphasized this gene, expressing a transcriptional regulator for the effector secretion system, as an important pathogenicity-associated locus of Psa. The transcriptome analysis of 'Hongyang' infected with different Psa strains was performed by RNA-seq of stem tissues locally (at the inoculation site) and systemically. Psa infection re-programmed the host genes expression, and the susceptibility to Psa might be attributed to the down-regulation of several genes involved in plant-pathogen interactions, especially calcium signaling transduction, as well as fatty acid elongation. This suppression was found in both low- and high-pathogenicity Psa inoculated tissues, but the effect was stronger with more virulent strains. Taken together, the divergences of P. syringae pv. actinidiae in pathogenicity, genome, and resulting transcriptomic response of A. chinensis provide insights into unraveling the molecular mechanism of Psa-kiwifruit interactions and resistance improvement in the kiwifruit crop.

Genome-Wide Identification of the LHC Gene Family in Kiwifruit and Regulatory Role of AcLhcb3.1/3.2 for Chlorophyll a Content.

Light-harvesting chlorophyll a/b-binding (LHC) protein is a superfamily that plays a vital role in photosynthesis. However, the reported knowledge of LHCs in kiwifruit is inadequate and poorly understood. In this study, we identified 42 and 45 LHC genes in Actinidia chinensis (Ac) and A. eriantha (Ae) genomes. Phylogenetic analysis showed that the kiwifruit LHCs of both species were grouped into four subfamilies (Lhc, Lil, PsbS, and FCII). Expression profiles and qRT-PCR results revealed expression levels of LHC genes closely related to the light, temperature fluctuations, color changes during fruit ripening, and kiwifruit responses to Pseudomonas syringae pv. actinidiae (Psa). Subcellular localization analysis showed that AcLhcb1.5/3.1/3.2 were localized in the chloroplast while transient overexpression of AcLhcb3.1/3.2 in tobacco leaves confirmed a significantly increased content of chlorophyll a. Our findings provide evidence of the characters and evolution patterns of kiwifruit LHCs genes in kiwifruit and verify the AcLhcb3.1/3.2 genes controlling the chlorophyll a content.

Cytotype distribution and chloroplast phylogeography of the Actinidia chinensis complex.

BACKGROUND: Plant phylogeographic studies of species in subtropical China have mainly focused on rare and endangered species, whereas few studies have been conducted on taxa with relatively wide distribution, especially polyploid species. We investigated the cytotype and haplotype distribution pattern of the Actinidia chinensis complex, a widespread geographically woody liana with variable ploidy in subtropical China comprising two varieties, with three chloroplast fragments DNA (ndhF-rpl132, rps16-trnQ and trnE-trnT). Macroevolutionary, microevolutionary and niche modeling tools were also combined to disentangle the origin and the demographic history of the species or cytotypes. RESULTS: The ploidy levels of 3338 individuals from 128 populations sampled throughout the species distribution range were estimated with flow cytometry. The widespread cytotypes were diploids followed by tetraploids and hexaploids, whereas triploids and octoploids occurred in a few populations. Thirty-one chloroplast haplotypes were detected. The genetic diversity and genetic structure were found to be high between varieties (or ploidy races) chinensis and deliciosa. Our results revealed that these two varieties inhabit significantly different climatic niche spaces. Ecological niche models (ENMs) indicate that all varieties' ranges contracted during the Last Inter Glacial (LIG), and expanded eastward or northward during the Last Glacial Maximum (LGM). CONCLUSIONS: Pliocene and Plio-Pleistocene climatic fluctuations and vicariance appear to have played key roles in shaping current population structure and historical demography in the A. chinensis complex. The polyploidization process also appears to have played an important role in the historical demography of the complex through improving their adaptability to environmental changes.

Draft Genome Resources Sequences of Six Pseudomonas syringae pv. actinidiae Strains Isolated from Actinidia chinensis var. deliciosa Leaves in Portugal.

Pseudomonas syringae pv. actinidiae is a quarantine bacterium affecting all the Portuguese main areas of kiwifruit production. We report the draft genome of six P. syringae pv. actinidiae strains isolated from symptomatic leaves of Actinidia chinensis var. deliciosa in a study that determined the genetic population structure of the endophytic and epiphytic populations in two consecutive seasons. Average nucleotide identity values were above 99% similarity with reference strains from P. syringae pv. actinidiae biovar 3. The genomic differences found between these strains confirm the genetic diversity described for P. syringae pv. actinidiae population in Portugal. Furthermore, data provide evidence that the initial clonal expansion of P. syringae pv. actinidiae in Europe was followed by a genomic diversification constituting a valuable resource for epidemiological and evolutionary studies, namely when adopting strategies for epidemics management.

Protuberances are organized distinct regions of long-term callus: histological and transcriptomic analyses in kiwifruit.

KEY MESSAGE: Macroscopic, ultrastructural, and molecular features-like a ball shape, the presence of starch granules, and the up-regulation of genes involved in carbohydrate metabolism and secondary metabolite biosynthesis-distinguish PT regions within a callus. The modification of the mass of pluripotent cells into de novo shoot bud regeneration is highly relevant to developmental biology and for agriculture and biotechnology. This study deals with protuberances (PT), structures that appear during the organogenic long-term culturing of callus (OC) in kiwifruit. These ball-shaped regions of callus might be considered the first morphological sign of the subsequent shoot bud development. Sections of PT show the regular arrangement of some cells, especially on the surface, in contrast to the regions of OC beyond the PT. The cells of OC possess chloroplasts; however, starch granules were observed only in PTs' plastids. Transcriptomic data revealed unique gene expression for each kind of sample: OC, PT, and PT with visible shoot buds (PT-SH). Higher expression of the gene involved in lipid (glycerol-3-phosphate acyltransferase 5 [GPAT5]), carbohydrate (granule-bound starch synthase 1 [GBSS1]), and secondary metabolite (beta-glucosidase 45 [BGL45]) pathways were detected in PT and could be proposed as the markers of these structures. The up-regulation of the regulatory associated protein of TOR (RAPTOR1) was found in PT-SH. The highest expression of the actinidain gene in leaves from two-year-old regenerated plants suggests that the synthesis of this protein takes place in fully developed organs. The findings indicate that PT and PT-SH are specific structures within OC but have more features in common with callus tissue than with organs.

Chemical constituents of radix Actinidia chinensis planch by UPLC-QTOF-MS.

Radix Actinidia decoction and its prescriptions are used to treat tumors and other diseases. Although some chemical components have been isolated from Radix Actinidia, systematic analysis of its chemical components has not been reported, which hinders the basic research on its effective substances and its quality control. In this work, a UPLC-QTOF-MS method was employed to profile and characterize the chemical constituents of water extracts from Radix Actinidia Chinensis Planch (RACP). We unambiguously or tentatively identified 295 chemical components from RACP, including 46 pentacyclic triterpenes, 72 flavonoids, 53 phenolic acids, 24 coumarins, three anthraquinones and other compounds. Most of the chemical components have not been described so far in Actinidia. More than 180 phytochemicals are reported in Actinidia for the first time. 2α,3α,24-trihydroxyurs-12-en-28-oic acid, asiatic acid, syringic acid, fraxin, esculetin, 5,7-dihydroxychromone, esculin, (+)-catechin, (-)-epi-catechin, vanillic acid, ferulic acid, protocatechuic acid and rutin were unambiguously identified by comparison with the reference standards. Catechin derivatives, coumarin derivatives and phenolic acid derivatives were the main water-soluble components in RACP. This study broadened the chemical profiles of RACP, and laid the foundation for subsequent research on the effective components and their mechanism of action. This work also provides an important reference for the quality control and evaluation of RACP.

Real-Time PCR and Droplet Digital PCR Are Accurate and Reliable Methods To Quantify Pseudomonas syringae pv. actinidiae Biovar 3 in Kiwifruit Infected Plantlets.

Pseudomonas syringae pv. actinidiae is the etiological agent of kiwifruit canker disease, causing severe economic losses in kiwifruit production areas around the world. Rapid diagnosis, understanding of bacterial virulence, and rate of infection in kiwifruit cultivars are important in applying effective measures of disease control. P. syringae pv. actinidiae load in kiwifruit is currently determined by a labor-intense colony counting method with no high-throughput and specific quantification method being validated. In this work, we used three alternative P. syringae pv. actinidiae quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative PCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared with the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of P. syringae pv. actinidiae biovar 3 (Psa3) inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation (r > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 102 CFU/cm2), highly correlated to each other (r = 0.955), and flexible, allowing for sample storage. The inclusion of a kiwifruit biomass marker increased the methods' accuracy. The qPCR method was efficient and allowed for high-throughput processing, and the ddPCR method showed highly accurate results but was more expensive and time consuming. While not ideal for high-throughput processing, ddPCR was useful in developing accurate standard curves for the qPCR method. The combination of the two methods is high-throughput, specific for Psa3 quantification, and useful for research studies (e.g., disease phenotyping and host-pathogen interactions).

ABF2 and MYB transcription factors regulate feruloyl transferase FHT involved in ABA-mediated wound suberization of kiwifruit.

Suberin is a cell-wall biopolymer with aliphatic and aromatic domains that is synthesized in the wound tissues of plants in order to restrict water loss and pathogen infection. ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (FHT) is required for cross-linking of the aliphatic and aromatic domains. ABA is known to play a positive role in suberin biosynthesis but it is not known how it interacts with FHT. In this study, the kiwifruit (Actinidia chinensis) AchnFHT gene was isolated and was found to be localized in the cytosol. Transient overexpression of AchnFHT in leaves of Nicotiana benthamiana induced massive production of ferulate, ω-hydroxyacids, and primary alcohols, consistent with the in vitro ability of AchnFHT to catalyse acyl-transfer from feruloyl-CoA to ω-hydroxypalmitic acid and 1-tetradecanol. A regulatory function of four TFs (AchnABF2, AchnMYB4, AchnMYB41, and AchnMYB107) on AchnFHT was identified. These TFs localized in the nucleus and directly interacted with the AchnFHT promoter in yeast one-hybrid assays. Dual-luciferase analysis indicated that AchnABF2, AchnMYB41, and AchnMYB107 activated the AchnFHT promoter while AchnMYB4 repressed it. These findings were supported by the results of transient overexpression in N. benthamiana, in which AchnABF2, AchnMYB41, and AchnMYB107 induced expression of suberin biosynthesis genes (including FHT) and accumulation of suberin monomers, whilst AchnMYB4 had the opposite effect. Exogenous ABA induced the expression of AchnABF2, AchnMYB41, AchnMYB107, and AchnFHT and induced suberin monomer formation, but it inhibited AchnMYB4 expression. In addition, fluridone (an inhibitor of ABA biosynthesis) was found to counter the inductive effects of ABA. Activation of suberin monomer biosynthesis by AchnFHT was therefore controlled in a coordinated way by both repression of AchnMYB4 and promotion of AchnABF2, AchnMYB41, and AchnMYB107.

Pseudomonas syringae pv. actinidiae: Ecology, Infection Dynamics and Disease Epidemiology.

Since 2008, the kiwifruit industry has been devastated by a pandemic outbreak of Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker. This disease has become the most significant limiting factor in kiwifruit production. Psa colonizes different organs of the host plant, causing a specific symptomatology on each of them. In addition, the systemic invasion of the plant may quickly lead to plant death. Despite the massive risk that this disease poses to the kiwifruit industry, studies focusing on Psa ecology have been sporadic, and a comprehensive description of the disease epidemiology is still missing. Optimal environmental conditions for infection, dispersal and survival in the environment, or the mechanisms of penetration and colonization of host tissues have not been fully elucidated yet. The present work aims to provide a synthesis of the current knowledge, and a deeper understanding of the epidemiology of kiwifruit bacterial canker based on new experimental data. The pathogen may survive in the environment or overwinter in dormant tissues and be dispersed by wind or rain. Psa was observed in association with several plant structures (stomata, trichomes, lenticels) and wounds, which could represent entry points for apoplast infection. Environmental conditions also affect the bacterial colonization, with lower optimum values of temperature and humidity for epiphytic than for endophytic growth, and disease incidence requiring a combination of mild temperature and leaf wetness. By providing information on Psa ecology, these data sets may contribute to plan efficient control strategies for kiwifruit bacterial canker.