Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome.

Pseudomonas syringae pv. actinidiae (Psa), the bacterium that causes kiwifruit bacterial canker, is a common field occurrence that is difficult to control globally. Currently, exploring the resources for efficient biocontrol bacteria is a hot spot in the field. The common strategy for isolating biocontrol bacteria is to directly isolate biocontrol bacteria that can secrete diffusible antibacterial substances, most of which are members of Bacillus, Pseudomonas and Streptomycetaceae, from disease samples or soil. Here, we report a new approach by adapting the typical isolation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from the branch microbiome. Using this unique approach, we isolated a group of kiwifruit biocontrol agents (KBAs) from the branch microbiome of Psa-resistant varieties. Thirteen of these showed no antagonistic activity in vitro, which depends on the secretion of antibacterial compounds. However, they exhibited antibacterial activity via cell-to-cell contacts mimicked by co-culture on agar plates. Through biocontrol tests on plants, two isolates, KBA13 and KBA19, demonstrated their effectiveness by protecting kiwifruit branches from Psa infection. Using KBA19, identified as Pantoea endophytica, as a representative, we found that this bacterium uses the type VI secretion system (T6SS) as the main contact-dependent antibacterial weapon that acts via translocating toxic effector proteins into Psa cells to induce cell death, and that this capacity expressed by KBA19 is common to various Psa strains from different countries. Our findings highlight a new strategy to identify efficient biocontrol agents that use the T6SS to function in an antibacterial metabolite-independent manner to control wood diseases.

Increased ascorbic acid synthesis by overexpression of AcGGP3 ameliorates copper toxicity in kiwifruit.

The widespread application of copper (Cu) -based fertilizers and pesticides could increase the accumulation of Cu in kiwifruit. According to a global survey, red- and yellow-fleshed kiwifruit contained more elevated amounts of Cu than green-fleshed kiwifruit due to weaker disease resistance and higher use of Cu pesticides. Intriguingly, our research revealed that external and endogenous ascorbic acid (AsA) reduced the phenotypic and physiological injury of Cu toxicity in kiwifruit. Cu stress assays and transcriptional analysis have shown that Cu treatment for 12 h significantly increased the AsA content in kiwifruit leaves and up-regulated key genes involved in AsA biosynthesis, such as GDP-L-galactose phosphorylase3 (GGP3) and GDP-mannose-3',5'-epimerase (GME). Overexpressing GGP3 in transgenic kiwifruit significantly increased the endogenous AsA content of kiwifruit, which was beneficial in mitigating Cu toxicity by decreasing levels of reactive oxygen species, malondialdehyde, and electrolyte leakage, as well as reducing damage to the chloroplast structure and photosystem II. This study presented a novel strategy to ameliorate plant Cu stress by increasing the endogenous antioxidant (AsA) content through transgenesis.

Assembly of an active microbial consortium by engineering compatible combinations containing foreign and native biocontrol bacteria of kiwifruit.

Assembling functional bacterial biocontrol consortia is expected to expand the scope and efficiency of biocontrol agents. Generally, bacterial interspecies interactions lead to incompatibility events, as bacteria can produce antibacterial compounds and/or assemble contact-dependent killing (CDK) devices. Here, we aimed to assemble a bacterial consortium comprising Lysobacter enzymogenes OH11 and Bacillus safensis ZK-1 for the synergistic control of bacterial and fungal diseases of kiwifruit. ZK-1, a native kiwifruit biocontrol bacterium, is effective against Pseudomonas syringae pv. actinidiae (Psa) that causes bacterial kiwifruit canker, but has weak antifungal activity. OH11 is a foreign kiwifruit biocontrol agent with strong antifungal activity. While OH11 was unable to produce anti-Gram-negative metabolites, this strain could utilize type IV secretion system as an antibacterial CDK weapon. We first observed that OH11 could inhibit growth of ZK-1 by generating diffusible anti-Gram-positive antibiotic WAP-8294A2, whereas ZK-1 failed to generate diffusible antibacterial compound to inhibit growth of OH11. To disrupt this interspecies incompatibility, we generated a transgenic OH11-derived strain, OH11W, by deleting the WAP-8294A2 biosynthetic gene and found that OH11W did not kill ZK-1. We further observed that when OH11W and ZK-1 were co-inoculated on agar plates, no CDK effect was observed between them, whereas co-culture of OH11W or ZK-1 with Psa on agar plates resulted in Psa killing, suggesting L. enzymogenes and B. safensis assemble antibacterial CDK weapons against bacterial pathogens, and these CDK weapons did not affect the compatibility between OH11W and ZK-1. Based on these findings, we assembled an OH11W/ZK-1 dependent consortium that was shown to be functional in controlling bacterial canker and several representative fungal diseases of kiwifruit.

Kiwifruit bZIP transcription factor AcePosF21 elicits ascorbic acid biosynthesis during cold stress.

Cold stress seriously affects plant development, resulting in heavy agricultural losses. L-ascorbic acid (AsA, vitamin C) is an antioxidant implicated in abiotic stress tolerance and metabolism of reactive oxygen species (ROS). Understanding whether and how cold stress elicits AsA biosynthesis to reduce oxidative damage is important for developing cold-resistant plants. Here, we show that the accumulation of AsA in response to cold stress is a common mechanism conserved across the plant kingdom, from single-cell algae to angiosperms. We identified a basic leucine zipper domain (bZIP) transcription factor (TF) of kiwifruit (Actinidia eriantha Benth.), AcePosF21, which was triggered by cold and is involved in the regulation of kiwifruit AsA biosynthesis and defense responses against cold stress. AcePosF21 interacted with the R2R3-MYB TF AceMYB102 and directly bound to the promoter of the gene encoding GDP-L-galactose phosphorylase 3 (AceGGP3), a key conduit for regulating AsA biosynthesis, to up-regulate AceGGP3 expression and produce more AsA, which neutralized the excess ROS induced by cold stress. On the contrary, VIGS or CRISPR-Cas9-mediated editing of AcePosF21 decreased AsA content and increased the generation of ROS in kiwifruit under cold stress. Taken together, we illustrated a model for the regulatory mechanism of AcePosF21-mediated regulation of AceGGP3 expression and AsA biosynthesis to reduce oxidative damage by cold stress, which provides valuable clues for manipulating the cold resistance of kiwifruit.

Comprehensive Analysis of Metabolome and Transcriptome in Fruits and Roots of Kiwifruit.

Kiwifruit (Actinidia chinensis) roots instead of fruits are widely used as Chinese medicine, but the functional metabolites remain unclear. In this study, we conducted comparative metabolome analysis between root and fruit in kiwifruit. A total of 410 metabolites were identified in the fruit and root tissues, and of them, 135 metabolites were annotated according to the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway. Moreover, 54 differentially expressed metabolites (DEMs) were shared in root and fruit, with 17 DEMs involved in the flavonoid pathway. Of the 17 DEMs, three flavonols (kaempferol-3-rhamnoside, L-Epicatechin and trifolin) and one dihydrochalcone (phloretin) showed the highest differences in the content level, suggesting that flavonols and dihydrochalcones may act as functional components in kiwifruit root. Transcriptome analysis revealed that genes related to flavonols and dihydrochalcones were highly expressed in root. Moreover, two AP2 transcription factors (TFs), AcRAP2-4 and AcAP2-4, were highly expressed in root, while one bHLH TF AcbHLH62 showed extremely low expression in root. The expression profiles of these TFs were similar to those of the genes related to flavonols and dihydrochalcones, suggesting they are key candidate genes controlling the flavonoid accumulation in kiwifruit. Our results provided an insight into the functional metabolites and their regulatory mechanism in kiwifruit root.

Two haplotype-resolved, gap-free genome assemblies for Actinidia latifolia and Actinidia chinensis shed light on the regulatory mechanisms of vitamin C and sucrose metabolism in kiwifruit.

Kiwifruit is a recently domesticated horticultural fruit crop with substantial economic and nutritional value, especially because of the high content of vitamin C in its fruit. In this study, we de novo assembled two telomere-to-telomere kiwifruit genomes from Actinidia chinensis var. 'Donghong' (DH) and Actinidia latifolia 'Kuoye' (KY), with total lengths of 608 327 852 and 640 561 626 bp for 29 chromosomes, respectively. With a burst of structural variants involving inversion, translocations, and duplications within 8.39 million years, the metabolite content of DH and KY exhibited differences in saccharides, lignans, and vitamins. A regulatory ERF098 transcription factor family has expanded in KY and Actinidia eriantha, both of which have ultra-high vitamin C content. With each assembly phased into two complete haplotypes, we identified allelic variations between two sets of haplotypes, leading to protein sequence variations in 26 494 and 27 773 gene loci and allele-specific expression of 4687 and 12 238 homozygous gene pairs. Synchronized metabolome and transcriptome changes during DH fruit development revealed the same dynamic patterns in expression levels and metabolite contents; free fatty acids and flavonols accumulated in the early stages, but sugar substances and amino acids accumulated in the late stages. The AcSWEET9b gene that exhibits allelic dominance was further identified to positively correlate with high sucrose content in fruit. Compared with wild varieties and other Actinidia species, AcSWEET9b promoters were selected in red-flesh kiwifruits that have increased fruit sucrose content, providing a possible explanation on why red-flesh kiwifruits are sweeter. Collectively, these two gap-free kiwifruit genomes provide a valuable genetic resource for investigating domestication mechanisms and genome-based breeding of kiwifruit.

Effects of Short-Term High Temperature on Gas Exchange in Kiwifruits (Actinidia spp.).

Climate changes occurring today require detailed research of the effects of increasing temperatures on photosynthesis in different species and cultivars. Temperature variability is a crucial determinant of the yield and quality of plants, particularly when high-temperature episodes coincide with their growth and fruit development. The effect of high temperature (HT) on higher plants primarily concerns photosynthetic functions, but the sensitivity of photosynthesis to high temperature is not well-understood in kiwifruits. In this study, we designed a new environmental monitoring system to evaluate the effects of environmental factors on the photosynthetic physiology of different kiwifruit species and cultivars. A significant positive correlation was determined between the main photosynthetic indices of kiwifruits, such as transpiration rate and net photosynthetic rate. The net photosynthetic rate of commercial kiwifruit cultivars was strongly inhibited when the temperature exceeded 44.5 °C, and the leaves of kiwifruits were irreversibly damaged when the temperature increased to 52 °C. Kiwifruit cultivars with different ploidy levels (diploid, tetraploid and hexaploid) were found to be sensitive to high temperature, whereas tetraploids had higher HT resistance and hexaploids had the highest net photosynthetic rate. Further research showed that the HT tolerance of kiwifruits existed not only between species but also among cultivars. A. eriantha had the highest net photosynthetic rate at more than 44.7 °C, but those of A. deliciosa and A. arguta declined sharply as the temperature exceeded 43.5 °C. As a result, it was determined that high temperatures have important effects on the photosynthetic activities of kiwifruit plants with different ploidy levels, and that these effects can significantly change their development according to how they differ among different species/cultivars.

Biocontrol Ability and Action Mechanism of Meyerozyma guilliermondii 37 on Soft Rot Control of Postharvest Kiwifruit.

Postharvest soft rot of kiwifruit has resulted in substantial market losses, yet there were few antagonistic yeasts reported to control the disease. This study screened 1113 yeast strains for potential antagonistic yeast to control soft rot of kiwifruit caused by Botryosphaeria dothidea and Diaporthe actinidiae, and strain 37 was selected to evaluate the control efficacy and mechanisms, which was identified as Meyerozyma guilliermondii via molecular biological identification. Our results showed that M. guilliermondii 37 effectively reduced pathogen spore germination rate to 28.52% and decay incidence of inoculated kiwifruit to 42.11% maximumly, whereas cell-free supernatant lacked antifungal activity, implying that M. guilliermondii 37 didn't produce direct antifungal compounds against the two pathogens. In addition, M. guilliermondii 37 adhered tenaciously to the pathogens' mycelium and colonized rapidly in kiwifruit flesh. Moreover, yeast strain 37 induced kiwifruit resistance by elevating the defense-related enzyme activity, increasing the antioxidant substances content, and suppressing the cell wall-degrading enzyme activity. Gene expression was consistent with the corresponding enzyme activity. Further postharvest yeast immersion treatment significantly reduced natural decay to 35.69% while maintaining soft-ripe quality. These results indicated that M. guilliermondii 37 might serve as a biocontrol agent against postharvest soft rot in kiwifruit.

The antifungal activity and mechanism of silver nanoparticles against four pathogens causing kiwifruit post-harvest rot.

Post-harvest rot causes enormous economic loss to the global kiwifruit industry. Currently, there are no effective fungicides to combat the disease. It is unclear whether silver nanoparticles (AgNPs) are effective in controlling post-harvest rot and, if so, what the underlying antifungal mechanism is. Our results indicated that 75 ppm AgNPs effectively inhibited the mycelial growth and spore germination of four kiwifruit rot pathogens: Alternaria alternata, Pestalotiopsis microspora, Diaporthe actinidiae, and Botryosphaeria dothidea. Additionally, AgNPs increased the permeability of mycelium's cell membrane, indicating the leakage of intracellular substance. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed that AgNPs induced pathogen hypha shrinkage and distortion, as well as vacuolation in hypha cells, implying that AgNPs caused cellular and organelle structural degradation. The transcriptome sequencing of mycelium treated with AgNPs (24 h / 48 h) was performed on the Illumina Hiseq 4000 sequencing (RNA-Seq) platform. For the time points of 24 h and 48 h, AgNPs treatment resulted in 1,178 and 1,461 differentially expressed genes (DEGs) of A. alternata, 517 and 91 DEGs of P. microspora, 1,287 and 65 DEGs of D. actinidiae, 239 and 55 DEGs of B. dothidea, respectively. The DEGs were found to be involved in "catalytic activity," "small molecule binding," "metal ion binding," "transporter activity," "cellular component organization," "protein metabolic process," "carbohydrate metabolic process," and "establishment of localization." Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis also revealed that "carbohydrate metabolism," "amino acid metabolism," "energy metabolism," and "xenobiotics biodegradation and metabolism" of "metabolism processes" were the most highly enriched pathways for these DEGs in four pathogens, with "cellular processes" being particularly enriched for B. dothidea. Furthermore, quantitative polymerase chain reactions (qPCRs) were used to validate the RNA-seq results. It was also confirmed that AgNPs could significantly reduce the symptoms of kiwifruit rot without leaving any Ag+ residue on the peel and flesh of kiwifruit. Our findings contributed to a better understanding of the antifungal effect and molecular mechanisms of AgNPs against pathogens causing kiwifruit post-harvest rot, as well as a new perspective on the application of this novel antifungal alternative to fruit disease control.

Characterizing Tetraploid Populations of Actinidia chinensis for Kiwifruit Genetic Improvement.

Understanding genetic diversity and structure in natural populations and their suitable habitat response to environmental changes is critical for the protection and utilization of germplasm resources. We evaluated the genetic diversity and structure of 24 A. chinensis populations using simple sequence repeat (SSR) molecular markers. The potential suitable distribution of tetraploid A. chinensis estimated under the current climate and predicted for the future climate was generated with ecological niche modeling (ENM). The results indicated that the polyploid populations of A.chinensis have high levels of genetic diversity and that there are distinct eastern and western genetic clusters. The population structure of A. chinensis can be explained by an isolation-by-distance model. The results also revealed that potentially suitable areas of tetraploids will likely be gradually lost and the habitat will likely be increasingly fragmented in the future. This study provides an extensive overview of tetraploid A. chinensis across its distribution range, contributing to a better understanding of its germplasm resources. These results can also provide the scientific basis for the protection and sustainable utilization of kiwifruit wild resources.

Genomic characterization of two nickie-like bacteriophages that infect the kiwifruit canker phytopathogen Pseudomonas syringae pv. actinidiae.

Here, we report the genomic characterization of two siphophages, named hairong and ZY21, that infect the kiwifruit canker phytopathogen Pseudomonas syringae pv. actinidiae. The genome sequences of hairong (112,842 bp) and ZY21 (112,006 bp) were determined. Global sequence comparison showed that hairong, ZY21, and two phages of the genus Nickievirus (nickie and psageB1) are similar but are not closely related to any other known phage, and they comprise a unique phylogenetic cluster. Moreover, hairong represents a new genus related to Nickievirus. Comparative genomic analysis revealed some common features shared by the four nickie-like phages.

High CO2 Reduces Spoilage Caused by Botrytis cinerea in Strawberry Without Impairing Fruit Quality.

High CO2 (> 20 kPa) conditions are beneficial for suppressing spoilage caused by Botrytis cinerea in strawberry fruit; however, these conditions are often accompanied by discoloration, off-flavors, and faster softening. Stepwise increments of CO2 concentrations have been proposed to alleviate injuries in fruits caused by high CO2. In this study, we investigated whether stepwise increments of CO2, up to 30 kPa and under a reduced O2 concentration, are beneficial for reducing fungal spoilage without inducing CO2 injury symptoms in strawberry fruit. Based on recommended settings (5-10 kPa O2 with 15-20 kPa CO2), we first selected optimal O2 and CO2 concentrations that best-reduced spoilage caused by B. cinerea in red ripe "Sonsation" strawberry fruit. We found that higher O2 (10 kPa) and CO2 (20 kPa) concentrations were most beneficial for prolonging strawberry fruit shelf life. Subsequently, we studied the performance of red ripe "Arabella" strawberry fruit stored at 5°C under different controlled atmosphere (CA) conditions (10 kPa O2 with either 0, 20, or 30 kPa CO2). The CO2 concentrations were achieved either within 8 h or in a stepwise manner within the first 4 days of storage. As a control, 21 kPa O2 and 0 kPa CO2 were used. Following storage for up to 11 days, the spoilage incidence was assessed at 12°C for 5 days. The application of high CO2 (20 and 30 kPa) combined with 10 kPa O2 greatly suppressed fruit spoilage during storage and subsequent shelf life. High CO2 suppressed respiration as well as maintained a higher pH and firmness in treated fruit. The level of total sugars did not change, but during storage, a substantial part of sucrose was converted into glucose and fructose, especially under high CO2 conditions. High CO2 did not affect ascorbic acid and anthocyanin levels. The stepwise increments of CO2 did not result in beneficial effects compared to the static application of high CO2. Our results show that "Arabella" strawberry fruit are highly tolerant to elevated CO2 and can be stored under 30 kPa CO2 to prolong the shelf life.

Kiwifruit MYBS1-like and GBF3 transcription factors influence l-ascorbic acid biosynthesis by activating transcription of GDP-L-galactose phosphorylase 3.

Plant-derived Vitamin C (l-ascorbic acid (AsA)) is crucial for human health and wellbeing and thus increasing AsA content is of interest to plant breeders. In plants GDP-l-galactose phosphorylase (GGP) is a key biosynthetic control step and here evidence is presented for two new transcriptional activators of GGP. AsA measurement, transcriptomics, transient expression, hormone application, gene editing, yeast 1/2-hybrid, and electromobility shift assay (EMSA) methods were used to identify two positively regulating transcription factors. AceGGP3 was identified as the most highly expressed GGP in Actinidia eriantha fruit, which has high fruit AsA. A gene encoding a 1R-subtype myeloblastosis (MYB) protein, AceMYBS1, was found to bind the AceGGP3 promoter and activate its expression. Overexpression and gene-editing show AceMYBS1 effectively increases AsA accumulation. The bZIP transcription factor AceGBF3 (a G-box binding factor), also was shown to increase AsA content, and was confirmed to interact with AceMYBS1. Co-expression experiments showed that AceMYBS1 and AceGBF3 additively promoted AceGGP3 expression. Furthermore, AceMYBS1, but not GBF3, was repressed by abscisic acid, resulting in reduced AceGGP3 expression and accumulation of AsA. This study sheds new light on the roles of MYBS1 homologues and ABA in modulating AsA synthesis, and adds to the understanding of mechanisms underlying AsA accumulation.

Comparative Study on Physicochemical and Nutritional Qualities of Kiwifruit Varieties.

In order to study the physicochemical and nutritional characteristics of kiwifruit varieties, 14 kiwifruits from different species with different flesh colors were selected for research. The pectin content was significantly higher in green-fleshed kiwifruits than those in red-fleshed and yellow-fleshed kiwifruits. Red-fleshed kiwifruits had the highest total flavonoid content, and green-fleshed kiwifruits in A. eriantha had the highest chlorophyll a content, chlorophyll b content and total carotenoid content. The energy and carbohydrate contents of yellow-fleshed kiwifruits were significantly lower than those of red-fleshed kiwifruit. Moreover, the protein contents in A. chinensis and A. chinensis var. deliciosa were higher than those in other species. The content of vitamin C in A. eriantha was far higher than in other kiwifruits. Red-fleshed kiwifruits had a significantly higher vitamin E and vitamin B1 content than green-fleshed kiwifruits. In addition, 1-pentanol, trans-2-hexen-1-ol, n-hexane and styrene presented only in red-fleshed kiwifruits. Therefore, these could be used as a characteristic fragrance for red-fleshed kiwifruits. Moreover, the varieties were ranked comprehensively by principal component analysis (PCA), among which the top four highest-ranking kiwifruits among the 14 varieties were 'Huate', 'MHYX', 'Jinkui' and 'Xuxiang', respectively. This study provides a reference for consumers and markets on quality improvement and processing.

The genome sequencing and comparative analysis of a wild kiwifruit Actinidia eriantha.

The current kiwifruit industry is mainly based on the cultivars derived from the species Actinidia chinensis (Ac) which may bring risks such as canker disease. Introgression of desired traits from wild relatives is an important method for improving kiwifruit cultivars. Actinidia eriantha (Ae) is a particularly important taxon used for hybridization or introgressive breeding of new kiwifruit cultivars because of its valued species-specific traits. Here, we assembled a chromosome-scale high-quality genome of a Ae sample which was directly collected from its wild populations. Our analysis revealed that 41.3% of the genome consists of repetitive elements, comparable to the percentage in Ac and Ae cultivar "White" genomes. The genomic structural variation, including the presence/absence-variation (PAV) of genes, is distinct between Ae and Ac, despite both sharing the same two kiwifruit-specific whole genome duplication (WGD) events. This suggests that a post-WGD divergence mechanism occurred during their evolution. We further investigated genes involved in ascorbic acid biosynthesis and disease-resistance of Ae, and we found introgressive genome could contribute to the complex relationship between Ae and other representative kiwifruit taxa. Collectively, the Ae genome offers valuable genetic resource to accelerate kiwifruit breeding applications.

Comparative Transcriptome Analysis Revealed the Key Genes Regulating Ascorbic Acid Synthesis in Actinidia.

Actinidia (kiwifruit) is known as 'the king of vitamin C' due to its rich ascorbic acid (AsA) concentration, which makes it an important model for studying the regulation of AsA metabolism. Herein, transcriptomic analysis was employed to identify candidate genes that regulate AsA synthesis in Actinidia species with 100-fold variations in fruit AsA content (A. latifolia and A. rufa). Approximately 1.16 billion high-quality reads were generated, and an average of 66.68% of the data was uniquely aligned against the reference genome. AsA-associated DEGs that predominately respond to abiotic signals, and secondary metabolic pathways were identified. The key candidate genes, for instance, GDP-L-galactose phosphorylase-3 (GGP3), were explored according to integrated analysis of the weighted gene co-expression network and L-galactose pathway. Transgenic kiwifruit plants were generated, and the leaves of GGP3 (OE-GGP3) overexpressing lines had AsA contents 2.0- to 6.4-fold higher than those of the wild type. Transcriptomic analysis of transgenic kiwifruit lines was further implemented to identify 20 potential downstream target genes and understand GGP3-regulated cellular processes. As a result, two transcription factors (AcESE3 and AcMYBR) were selected to carry out yeast two-hybrid and BiFC assays, which verified that there were obvious AcESE3-AcMYBR and AcESE3-AcGGP3 protein-protein interactions. This study provides insight into the mechanism of AsA synthesis and provides candidate factors and genes involved in AsA accumulation in kiwifruit.

Cas12c-DETECTOR: A specific and sensitive Cas12c-based DNA detection platform.

The robust and precise nucleic acid detection platform enormously influences the clinical diagnosis of human and plant pathogens, drastically affecting disease pandemic control. CRISPR-based nucleic acid detection tools have been successfully applied for rapid and sensitive nucleic acid detection. However, the T-rich protospacer adjacent motif (PAM), specificity, and sensitivity of the CRISPR-based nucleic acid detection tools limited its wide application. We first developed a new Cas12c-based nucleic acid detection platform (Cas12c-DETECTOR), recognizing a 5'-TG PAM and showing high sensitivity and specificity on examined targets. Our results indicate that Cas12c-DETECTOR coupling with the optimized single-guide RNA (sgRNA) can be applied to specifically identity single nucleotide polymorphism (SNP). Moreover, combined with pre-amplification and lateral flow strips or the visual fluorescence detection method, Cas12c-DETECTOR can be used to diagnose human and plant pathogens in practice. Therefore, our findings illustrated that Cas12c-DETECTOR is a robust, sensitive, precise, and practiced nucleic acid detection platform.

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.

ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana.

Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.