Genome-wide identification of the heat shock transcription factor gene family in two kiwifruit species.

High temperatures have a significant impact on plant growth and metabolism. In recent years, the fruit industry has faced a serious threat due to high-temperature stress on fruit plants caused by global warming. In the present study, we explored the molecular regulatory mechanisms that contribute to high-temperature tolerance in kiwifruit. A total of 36 Hsf genes were identified in the A. chinensis (Ac) genome, while 41 Hsf genes were found in the A. eriantha (Ae) genome. Phylogenetic analysis revealed the clustering of kiwifruit Hsfs into three distinct groups (groups A, B, and C). Synteny analysis indicated that the expansion of the Hsf gene family in the Ac and Ae genomes was primarily driven by whole genome duplication (WGD). Analysis of the gene expression profiles revealed a close relationship between the expression levels of Hsf genes and various plant tissues and stress treatments throughout fruit ripening. Subcellular localization analysis demonstrated that GFP-AcHsfA2a/AcHsfA7b and AcHsfA2a/AcHsfA7b -GFP were localized in the nucleus, while GFP-AcHsfA2a was also observed in the cytoplasm of Arabidopsis protoplasts. The results of real-time quantitative polymerase chain reaction (RT-qPCR) and dual-luciferase reporter assay revealed that the majority of Hsf genes, especially AcHsfA2a, were expressed under high-temperature conditions. In conclusion, our findings establish a theoretical foundation for analyzing the potential role of Hsfs in high-temperature stress tolerance in kiwifruit. This study also offers valuable information to aid plant breeders in the development of heat-stress-resistant plant materials.

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.

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.

Genome-Wide Identification and Structural Characterization of Growth-Regulating Factors (GRFs) in Actinida eriantha and Actinidia chinensis.

Growth-regulating factors (GRFs) encode plant-specific transcription factors that play a vital role in regulation of plant growth, development, and stress response. Although GRFs have been identified in various plants, there is no reported work available in Actinidia (commonly known as kiwifruit) so far. In the present study, we identified 22 GRF genes on A. chinensis (hereafter A. chinensis is referred to as Ac, and GRF genes in A. chinensis are referred to as AcGRF) distributed on 17 chromosomes and one contig, and 26 GRF genes in A. eriantha (hereafter A. eriantha is referred to as Ae, and GRF genes in A. eriantha are referred to as AeGRF) distributed on 21 chromosomes. Phylogenetic analysis showed that kiwifruit GRF proteins were clustered into five distinct groups. Additionally, kiwifruit GRFs showed motif composition and gene structure similarities within the same group. Synteny analysis showed that whole-genome duplication played a key role in the expansion of the GRF family in kiwifruit. The higher expression levels of kiwifruit GRFs in young tissues and under stress conditions indicated their regulatory role in kiwifruit growth and development. We observed two genes in Ae (AeGRF6.1, AeGRF 6.2) and two genes in Ac (AcGRF 6.1, AeGRF 6.2) significantly upregulated in different RNA-seq datasets. The presence of conserved protein structures and cis-regulatory elements caused functional divergence in duplicated gene pairs. The subcellular localization indicated the presence of kiwifruit GRFs in the nucleus of the plant cell. Protein-protein interaction analysis predicted AtGIF protein orthologs for AcGRFs and AeGRFs. Taken together, we systematically analyzed the characterization of kiwifruit GRF family members for their potential role in kiwifruit development and Pseudomonas syringae pv. actinidiae (Psa.) invasion response. Further functional studies of kiwifruit GRFs in plant growth, development, and stress response will provide valuable insights for kiwifruit breeders.

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.

Phylogenetic Analysis of Elaeagnus L. in China: A Basis for Genetic Improvement of a Berry Crop.

Elaeagnus L. is found in wild or grown as ornamental plants and is increasingly regarded as underutilized berry shrubs by breeders. This genus has cosmopolitan distribution with various species widely distributed in China, Europe, the United States, and Canada. Interspecific hybrids, which have been reported several times, have attracted intense interest from plant breeders attempting to develop a fruit crop of Elaeagnus. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) is a powerful statistical modeling tool that provides insights into separations between experimental groups. In this study, the molecular phylogeny of Elaeagnus species was first discussed using the ITS and matK sequences for guiding the construction of a genetic basis pool. A morphological OPLS-DA clustering model based on the genetic divergence was also constructed for the first time, which effectively realized the morphological grouping of Chinese Elaeagnus species. The results showed that a total of 10 wild species widely distributed in China have the potential to develop fruit crops. Particularly, Elaeagnus conferta has the potential to provide a founder species with a large fruit size, while Elaeagnus Gonyanthes has the potential to provide important genetic resources with long pedicel. Elaeagnus lanceolata and Elaeagnus delavayi could be used to domesticate hybrids without spines, and the other five climbing shrubs could be used to develop high-yield crown-type commercial cultivars for automated field management. The top five contributing morphological traits affecting the current clustering model were V9 (flower color), V1 (flowering), V5 (evergreen or deciduous), V3 (leaf size), and V2 (fruiting). Furthermore, the grouping analysis indicated that the V9 was the most important factor affecting morphological clustering. Thereafter, the temporally calibrated phylogeny inferred from the matK sequence was used to reconstruct the origin and evolution of the genus Elaeagnus, and the results inferred an interesting geographic distribution pattern and potential cross-species interactions of Elaeagnus species at low latitudes in China. Our study also highlighted dispersal pattern investigation and genetic background analysis to improve future practices and policies related to species introduction of genetic basis pool.

Genome-wide identification and characterization of the TIFY gene family in kiwifruit.

BACKGROUND: The TIFY gene family is a group of plant-specific transcription factors involved in regulation of plant growth and development and a variety of stress responses. However, the TIFY family has not yet been well characterized in kiwifruit, a popular fruit with important nutritional and economic value. RESULTS: A total of 27 and 21 TIFY genes were identified in the genomes of Actinidia eriantha and A. chinensis, respectively. Phylogenetic analyses showed that kiwifruit TIFY genes could be classified into four major groups, JAZ, ZML, TIFY and PPD, and the JAZ group could be further clustered into six subgroups (JAZ I to JAZ VI). Members within the same group or subgroup have similar exon-intron structures and conserved motif compositions. The kiwifruit TIFY genes are unevenly distributed on the chromosomes, and the segmental duplication events played a vital role in the expansion of the TIFY genes in kiwifruit. Syntenic analyses of TIFY genes between kiwifruit and other five plant species (including Arabidopsis thaliana, Camellia sinensis, Oryza sativa, Solanum lycopersicum and Vitis vinifera) and between the two kiwifruit species provided valuable clues for understanding the potential evolution of the kiwifruit TIFY family. Molecular evolutionary analysis showed that the evolution of kiwifruit TIFY genes was primarily constrained by intense purifying selection. Promoter cis-element analysis showed that most kiwifruit TIFY genes possess multiple cis-elements related to stress-response, phytohormone signal transduction and plant growth and development. The expression pattern analyses indicated that TIFY genes might play a role in different kiwifruit tissues, including fruit at specific development stages. In addition, several TIFY genes with high expression levels during Psa (Pseudomonas syringae pv. actinidiae) infection were identified, suggesting a role in the process of Pas infection. CONCLUSIONS: In this study, the kiwifruit TIFY genes were identified from two assembled kiwifruit genomes. In addition, their basic physiochemical properties, chromosomal localization, phylogeny, gene structures and conserved motifs, synteny analyses, promoter cis-elements and expression patters were systematically examined. The results laid a foundation for further understanding the function of TIFY genes in kiwifruit, and provided a new potential approach for the prevention and treatment of Psa infection.

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.

In silico identification, characterization expression profile of WUSCHEL-Related Homeobox (WOX) gene family in two species of kiwifruit.

The WUSCHEL (WUS)-related homeobox (WOX) gene family is a class of plant-specific transcriptional factors and plays a crucial role in forming the shoot apical meristem and embryonic development, stem cell maintenance, and various other developmental processes. However, systematic identification and characterization of the kiwifruit WOX gene family have not been studied. This study identified 17 and 10 WOX genes in A. chinensis (Ac) and A. eriantha (Ae) genomes, respectively. Phylogenetic analysis classified kiwifruit WOX genes from two species into three clades. Analysis of phylogenetics, synteny patterns, and selection pressure inferred that WOX gene families in Ac and Ae had undergone different evolutionary patterns after whole-genome duplication (WGD) events, causing differences in WOX gene number and distribution. Ten conserved motifs were identified in the kiwifruit WOX genes, and motif architectures of WOXs belonging to different clades highly diverged. The cis-element analysis and expression profiles investigation indicated the functional differentiation of WOX genes and identified the potential WOXs in response to stresses. Our results provided insight into general characters, evolutionary patterns, and functional diversity of kiwifruit WOXs.

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.

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.

Evolution and Diversification of Kiwifruit Mitogenomes through Extensive Whole-Genome Rearrangement and Mosaic Loss of Intergenic Sequences in a Highly Variable Region.

Angiosperm mitochondrial genomes (mitogenomes) are notable for their extreme diversity in both size and structure. However, our current understanding of this diversity is limited, and the underlying mechanism contributing to this diversity remains unclear. Here, we completely assembled and compared the mitogenomes of three kiwifruit (Actinidia) species, which represent an early divergent lineage in asterids. We found conserved gene content and fewer genomic repeats, particularly large repeats (>1 kb), in the three mitogenomes. However, sequence transfers such as intracellular events are variable and dynamic, in which both ancestral shared and recently species-specific events as well as complicated transfers of two plastid-derived sequences into the nucleus through the mitogenomic bridge were detected. We identified extensive whole-genome rearrangements among kiwifruit mitogenomes and found a highly variable V region in which fragmentation and frequent mosaic loss of intergenic sequences occurred, resulting in greatly interspecific variations. One example is the fragmentation of the V region into two regions, V1 and V2, giving rise to the two mitochondrial chromosomes of Actinidia chinensis. Finally, we compared the kiwifruit mitogenomes with those of other asterids to characterize their overall mitogenomic diversity, which identified frequent gain/loss of genes/introns across lineages. In addition to repeat-mediated recombination and import-driven hypothesis of genome size expansion reported in previous studies, our results highlight a pattern of dynamic structural variation in plant mitogenomes through global genomic rearrangements and species-specific fragmentation and mosaic loss of intergenic sequences in highly variable regions on the basis of a relatively large ancestral mitogenome.

Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit.

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired-sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired-sgRNA cloning, our strategy only requires the synthesis of two gRNA-containing primers which largely reduces the cost. We further compared efficiencies of paired-sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA-sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10-fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired-sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418-resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.

Whole transcriptome sequencing of Pseudomonas syringae pv. actinidiae-infected kiwifruit plants reveals species-specific interaction between long non-coding RNA and coding genes.

An outbreak of kiwifruit bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) beginning in 2008 caused disaster to the kiwifruit industry. However the mechanisms of interaction between kiwifruit and Psa are unknown. Long noncoding RNAs (lncRNAs) are known to regulate many biological processes, but comprehensive repertoires of kiwifruit lncRNAs and their effects on the interaction between kiwifruit and Psa are unknown. Here, based on in-depth transcriptomic analysis of four kiwifruit materials at three stages of infection with Psa, we identified 14,845 transcripts from 12,280 loci as putative lncRNAs. Hierarchical clustering analysis of differentially-expressed transcripts reveals that both protein-coding and lncRNA transcripts are expressed species-specifically. Comparing differentially-expressed transcripts from different species, variations in pattern-triggered immunity (PTI) were the main causes of species-specific responses to infection by Psa. Using weighted gene co-expression network analysis, we identified species-specific expressed key lncRNAs which were closely related to plant immune response and signal transduction. Our results illustrate that different kiwifruit species employ multiple different plant immunity layers to fight against Psa infection, which causes distinct responses. We also discovered that lncRNAs might affect kiwifruit responses to Psa infection, indicating that both protein-coding regions and noncoding regions can affect kiwifruit response to Psa infection.

Rapid radiations of both kiwifruit hybrid lineages and their parents shed light on a two-layer mode of species diversification.

Reticulate speciation caused by interspecific hybridization is now recognized as an important mechanism in the creation of biological diversity. However, depicting the patterns of phylogenetic networks for lineages that have undergone interspecific gene flow is challenging. Here we sequenced 25 taxa representing natural diversity in the genus Actinidia with an average mapping depth of 26× on the reference genome to reconstruct their reticulate history. We found evidence, including significant gene tree discordance, cytonuclear conflicts, and changes in genome-wide heterozygosity across taxa, collectively supporting extensive reticulation in the genus. Furthermore, at least two separate parental species pairs were involved in the repeated origin of the hybrid lineages, in some of which a further phase of syngameon was triggered. On the basis of the elucidated hybridization relationships, we obtained a highly resolved backbone phylogeny consisting of taxa exhibiting no evidence of hybrid origin. The backbone taxa have distinct demographic histories and are the product of recent rounds of rapid radiations via sorting of ancestral variation under variable climatic and ecological conditions. Our results suggest a mode for consecutive plant diversification through two layers of radiations, consisting of the rapid evolution of backbone lineages and the formation of hybrid swarms derived from these lineages.

Identification of Circular RNAs in Kiwifruit and Their Species-Specific Response to Bacterial Canker Pathogen Invasion.

Research studies have recently focused on circle RNAs (circRNAs) in relation to their regulatory functions in animals. However, the systematic identification of circRNAs in plants, especially non-model plants, is limited. In addition, raw report on the prediction of the potential role of circRNAs in plant response to pathogen invasion is currently available. We conducted the systematic identification of circRNAs from four materials originating from three species belonging to genus Actinidia under different situations using ribosomal RNA (rRNA) depleted RNA-Seq data. A total of 3,582 circRNAs were identified in Actinidia, of which 64.01, 21.44, and 14.55% were intergenic circRNAs, exonic circRNAs, and intronic circRNAs, respectively. Tissue-specific expression of circRNAs was observed in kiwifruit, and a species-specific response was detected when infected with Pseudomonas syringae pv. actinidiae (Psa), which is the causative agent of kiwifruit bacterial canker disease. Furthermore, we found that both exonic and intronic circRNAs were significantly positively correlated to parent protein-coding genes, and intronic circRNAs are a class of highly remarkable regulators the parent genes comparing to that of exonic circRNAs. Expression and weighted gene co-expression network analysis (WGCNA) identified a set of circRNAs that were closely associated with plant defense response. The findings of the presents study suggest that circRNAs exhibit tissue- and species-specific expression, as well as play an important role in plant immune response.

Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.