Gymnosperms, particularly conifers, exhibit a high abundance of transposable elements (TEs) in their giga-scale genomes. TEs interact both antagonistically and cooperatively with the host genome, promoting structural and genetic innovations across evolutionary lineages. However, how TEs shape the coding space of gymnosperm genomes remains a key unresolved question. Here, we present a high-quality genome assembly for the keystone conifer Platycladus orientalis, with a contig N50 of 57.54 Mb-the highest continuity reported to date-to investigate the role of TEs. Comparative genomics confirms the absence of recent whole-genome duplication and the presence of genome expansion in gymnosperms, revealing complex interactions among recurrent TE proliferation, low DNA removal rates, and DNA methylation-mediated silencing. Computational evidence indicates that TE-mediated gene duplication and pseudogenization provide a genetic basis for adaptive evolution and functional innovation, significantly shaping gene family dynamics and the emergence of species-specific genes. Additionally, TEs capture and duplicate an average of ∼400,000 coding gene fragments per gymnosperm genome, facilitating exon shuffling and triggering epigenetic conflicts between source genes and captured exon fragments. Genes from which fragments are captured (donor genes) show significantly higher levels of exon methylation than genes not captured by TEs (free genes), whereas syntenic donor genes exhibit lower levels of silencing responses than non-syntenic donor genes. This study provides valuable genomic resources and offers insights into the evolutionary patterns and principles underlying the large genome size and complexity of gymnosperms.

Hybridization is a driving force in ecological transitions and speciation, yet direct evidence linking it to adaptive differentiation in natural systems remains limited. This study evaluates the role of hybridization in the speciation of Pinus densata, a keystone forest species on the southeastern Tibetan Plateau. By creating artificial interspecific F1s and a long-term common garden experiment on the plateau, we provide in situ assessments on 44 growth and physiological traits across four seasons, along with RNA sequencing. We found significant phenotypic divergence between P. densata and its putative parental species P. tabuliformis and P. yunnanensis, with P. densata demonstrating superior growth and dynamic balance between photosynthesis and photoprotection. The F1s closely resembled P. densata in most traits. Gene expression revealed 19%-10% of 34,000 examined genes as differentially expressed in P. densata and F1s relative to mid-parent expression values. Both additive (4%) and non-additive gene actions (5%-6% in F1s, 10%-12% in P. densata) were common, while transgressive expression occurred more frequently in the stabilized natural hybrids, illustrating transcriptomic reprogramming brought by hybridization and further divergence by natural selection. We provide compelling evidence for hybridization-derived phenotypic divergence at both physiological and gene expression levels that could have contributed to the adaptation of P. densata to high plateau habitat where both parental species have low fitness. The altered physiology and gene expression in hybrids serve both as a substrate for novel ecological adaptation and as a mechanism for the initiation of reproductive isolation.

Pseudomonas plecoglossicida can cause visceral white spot disease in various species of teleost fish, leading to significant economic losses in the aquaculture industry. The PhoP/PhoQ two-component system plays a crucial role in regulating bacterial virulence and maintaining internal environmental balance. In this study, hybrid grouper (Epinephelus fuscoguttatus female x E. lanceolatus male) was used as a model to compare the pathogenic differences between the phoQ gene knockout strain (Delta phoQ), the phoQ gene complement strain (C-Delta phoQ), and the wild-type strain (NZBD9). Additionally, transcriptome sequencing was employed to explore the impact of phoQ gene deletion on the host immune response during P. plecoglossicida infection. The results showed that the cumulative survival rate of the Delta phoQ infection group was 10 % higher than that of the NZBD9 group, with significantly reduced bacterial load in the spleen and less tissue damage. Transcriptomic analysis revealed that, compared to the NZBD9 infection group, the Delta phoQ infection group induced 4453 differentially expressed genes, which were enriched in several immune-related pathways according to GO and KEGG analysis. These pathways included 22 immune system pathways, 28 signal transduction pathways, and 9 cell growth and death pathways. Further qRT-PCR analysis confirmed that phoQ gene deletion affected the expression of several immune-and inflammation-related genes, such as IL-1 beta, IL-6, CD3, C8, CCL2, MHC-II, PGRP6, and SLP2. Particularly during the infection process, the expression level of the PGRP6 gene in the Delta phoQ strain infection group was significantly higher than that in the NZBD9 strain infection group, suggesting that PGRP6 may be involved in the host's immune recognition of P. plecoglossicida. In conclusion, the phoQ gene plays an important role in the pathogenesis of P. plecoglossicida infection in hybrid grouper. The deletion of the phoQ gene significantly affects the pathogenicity of P. plecoglossicida and enhances the host's immune response, providing a theoretical basis for the potential application of the PhoP/PhoQ system in the immune defense of aquatic animals.

Pseudomonas plecoglossicida is a significant pathogen in aquaculture, capable of infecting various teleost fish and causing visceral white spot disease, which leads to substantial economic losses. The PhoP/PhoQ two-component system plays a central role in virulence regulation in Gram-negative bacteria; however, its specific function in P. plecoglossicida remains unclear. To elucidate the role of the phoP gene in the pathogenicity of this bacterium, we used hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) as an infection model to compare the pathogenicity and host immune responses among the wild-type (NZBD9), phoP deletion (ΔphoP), and complemented (C-ΔphoP) strains. In artificial infection experiments, the ΔphoP strain exhibited significantly attenuated virulence, with an LD50 value of 4.082 × 104, which was 13.7 times higher than that of the wild-type strain. Furthermore, the deletion of phoP reduced bacterial load in the spleen and alleviated histopathological damage in the spleen, head kidney, and intestine. RNA-seq analysis of spleen tissue at 4 days post-infection identified 1768 differentially expressed genes (DEGs), including 704 up-regulated and 1064 down-regulated genes. GO and KEGG enrichment analyses revealed that these DEGs were primarily involved in immune-related processes and pathways, including TLR signaling, NF-κB activation, PI3K-Akt signaling, cytokine-cytokine receptor interaction, and cell adhesion molecules. Temporal expression profiling via qRT-PCR demonstrated that key immune genes (including tlr1, tlr5, tlr13, traf5, nf-κb, mcp1, cd3, and il10) were generally down-regulated or exhibited delayed and weakened activation in ΔphoP-infected fish, indicating a subdued and dysregulated immune response. These results suggest that the phoP gene is essential for maintaining full virulence in P. plecoglossicida, modulating host immunity by suppressing critical inflammatory and immune signaling pathways. Our study provides novel insights into the role of the PhoP/PhoQ system in host-pathogen interactions and lays a theoretical foundation for developing targeted control strategies against P. plecoglossicida infection in aquaculture.

In May 2022, a significant mortality event involving pearl gentian groupers (Epinephelus fuscoguttatus female x E. lanceolatus male) was reported at an aquaculture farm in Zhangzhou, China, under water conditions of approximately 25 degrees C, pH 8.0, and salinity 32 parts per thousand. A highly pathogenic bacterial strain, EFL-2201, was isolated from the spleen of a diseased fish. The outbreak was traced back to Vibrio harveyi through a series of scientific methods including isolation, identification, and regression infection. In vitro assays indicated that the conditions during the outbreak were conducive to the proliferation, motility, biofilm formation, and chemotaxis of EFL-2201. Whole-genome sequencing of the strain revealed that mechanisms related to adhesion, secretion, toxins, and invasion significantly contributed to its pathogenicity. Temporal and spatial bacterial load of EFL-2201 in various tissues of the infected groupers indicated that bacterial loads peaked at 24 h post-infection (hpi). The spleen, which had the highest bacterial load among all tissues, was subjected to transcriptome sequencing at 24 hpi, revealing significant enrichment in pathways related to immunity, cell cycle, and red blood cell oxygen transport. In conclusion, this study unveiled the pathogenic mechanisms of V. harveyi and immune response mechanisms of pearl gentian grouper spleens to infection, laying a theoretical foundation for the prevention and control of V. harveyi-induced disease.

Evolutionary radiation, a pivotal aspect of macroevolution, offers valuable insights into evolutionary processes. The genus Pinus is the largest genus in conifers with c.$$ c. $$ 90% of the extant species emerged in the Miocene, which signifies a case of rapid diversification. Despite this remarkable history, our understanding of the mechanisms driving radiation within this expansive genus has remained limited. Using exome capture sequencing and a fossil-calibrated phylogeny, we investigated the divergence history, niche diversification, and introgression among 13 closely related Eurasian species spanning climate zones from the tropics to the boreal Arctic. We detected complex introgression among lineages in subsection Pinus at all stages of the phylogeny. Despite this widespread gene exchange, each species maintained its genetic identity and showed clear niche differentiation. Demographic analysis unveiled distinct population histories among these species, which further influenced the nucleotide diversity and efficacy of purifying and positive selection in each species. Our findings suggest that radiation in the Eurasian pines was likely fueled by interspecific recombination and further reinforced by their adaptation to distinct environments. Our study highlights the constraints and opportunities for evolutionary change, and the expectations of future adaptation in response to environmental changes in different lineages.

Hybrid genomes usually harbor asymmetrical parental contributions. However, it is challenging to infer the functional significance of asymmetrical retention of parental alleles in hybrid populations of conifer trees. Here we investigated the diversity in the glutathione S-transferase (GST) gene family in a hybrid pine Pinus densata and its parents (Pinus tabuliformis and Pinus yunnanensis). Plant GSTs play major roles in protecting plants against biotic and abiotic stresses. In this study, 19 orthologous groups of GST genes were identified and cloned from these three species. We examined their expression in difFerent tissues, and then purified the corresponding proteins to characterize their enzymatic activities and specificities toward difFerent substrates. We found that among the 19 GST orthologous groups, divergence in gene expression and in enzymatic activities toward difFerent substrates was prevalent. P. densata preferentially retained P. yunnanensis-like GSTs for 17 out of the 19 gene loci. We determined the first GST crystal structure from conifer species at a resolution of 2.19 A. Based on this structure, we performed site-directed mutagenesis to replace amino acid residuals in difFerent wild-types of GSTs to understand their functional impacts. Reciprocal replacement of amino acid residuals in native GSTs of P. densata and P. tabuliformis demonstrated significant changes in enzyme functions and identified key sites controlling GSTs activities. This study illustrates an approach to evaluating the functional significance of sequence variations in conifer genomes. Our study also sheds light on plausible mechanisms for controlling the selective retention of parental alleles in the P. densata genome.

Pseudomonas plecoglossicida, a gram-negative bacterium, is the main pathogen of visceral white-point disease in marine fish, responsible for substantial economic losses in the aquaculture industry. The FliL protein, involved in torque production of the bacterial flagella motor, is essential for the pathogenicity of a variety of bacteria. In the current study, the fliL gene deletion strain (ΔfliL), fliL gene complement strain (C-ΔfliL), and wild-type strain (NZBD9) were compared to explore the influence of the fliL gene on P. plecoglossicida pathogenicity and its role in host immune response. Results showed that fliL gene deletion increased the survival rate (50%) and reduced white spot disease progression in the hybrid groupers. Moreover, compared to the NZBD9 strain, the ΔfliL strain was consistently associated with lower bacterial loads in the grouper spleen, head kidney, liver, and intestine, coupled with reduced tissue damage. Transcriptomic analysis identified 2 238 differentially expressed genes (DEGs) in the spleens of fish infected with the ΔfliL strain compared to the NZBD9 strain. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the DEGs were significantly enriched in seven immune system-associated pathways and three signaling molecule and interaction pathways. Upon infection with the ΔfliL strain, the toll-like receptor (TLR) signaling pathway was activated in the hybrid groupers, leading to the activation of transcription factors (NF-κB and AP1) and cytokines. The expression levels of proinflammatory cytokine-related genes IL-1β, IL-12B, and IL-6 and chemokine-related genes CXCL9, CXCL10, and CCL4 were significantly up-regulated. In conclusion, the fliL gene markedly influenced the pathogenicity of P. plecoglossicida infection in the hybrid groupers. Notably, deletion of fliL gene in P. plecoglossicida induced a robust immune response in the groupers, promoting defense against and elimination of pathogens via an inflammatory response involving multiple cytokines.

Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.

Southeast Asia (SEA) has seen strong climatic oscillations and fluctuations in sea levels during the Quaternary. The impact of past climate changes on the evolution and distribution of local flora in SEA is still poorly understood. Here we aim to infer how the Quaternary climate change affects the evolutionary process and range shifts in two pine species. We investigated the population genetic structure and diversity using cytoplasmic DNA markers, and performed ecological niche modeling to reconstruct the species past distribution and to project range shift under future climates. We found substantial gene flow across the continuous distribution of the subtropical Pinus yunnanensis. In contrast, the tropical Pinus kesiya showed a strong population structure in accordance with its disjunct distribution across montane islands in Indochina and the Philippines. A broad hybrid zone of the two species occurs in southern Yunnan. Asymmetric introgression from the two species was detected in this zone with dominant mitochondrial gene flow from P. yunnanensis and chloroplast gene flow from P. kesiya. The observed population structure suggests a typical postglaciation expansion in P. yunnanensis, and a glacial expansion and interglacial contraction in P. kesiya. Ecological niche modeling supports the inferred demographic history and predicts a decrease in range size for P. kesiya under future climates. Our results suggest that tropical pine species in SEA have undergone evolutionary trajectories different from high latitude species related to their Quaternary climate histories. We also illustrate the need for urgent conservation actions in this fragmented landscape.

Pseudomonas plecoglossicida is a pathogen that causes visceral white spot disease in a variety of teleosts. The protein encoded by fliP gene is involved in the assembly of bacterial flagella, which plays a vital role in bacterial pathogenicity. However, the roles of the fliP gene on the host immune response remain unclear. Here, we compared the pathogenicity of fliP gene-deleted (ΔfliP) strain, fliP gene-complemented (C-ΔfliP) strain and wild-type (NZBD9) strain of P. plecoglossicida to hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), and explored the impacts of fliP gene on the immune response of hybrid grouper to P. plecoglossicida infection by using RNA-seq. In this study, the grouper in the ΔfliP strain-infected group had a 30% higher survival rate than those in the NZBD9 strain-infected group. In addition, the deletion of fliP gene decreased bacterial load in the spleen, intestine, liver as well as head kidney of hybrid grouper and the tissues damage were weakened. Moreover, the infection of hybrid grouper spleen by the ΔfliP strain induced 1,189 differential expression genes compared with the counterpart infected by NZBD9 strain. KEGG enrichment analysis showed that 9 immune-related pathways, 5 signal transduction pathways, and 3 signaling molecules and interaction pathways were significantly enriched. qRT-PCR analysis revealed that the ΔfliP strain mainly up-regulated the expression of inflammation related genes (IL-6, IL-12, IL-1β, IL-10, CXCL8, CXCL10) and immune regulation related genes (TLR2, P65, MyD88, P85, AKT), but down-regulated the expression of cell death related genes (FoxO1, Bim, PLK2 and LDHA) during infection. Based on the above results, fliP gene contributed to the pathogenicity of P. plecoglossicida to hybrid grouper (E. fuscoguttatus ♀ × E. lanceolatus ♂), deletion of fliP gene promoted the inflammation and immune response of hybrid grouper to P. plecoglossicida infection, which accelerating host clearance of pathogen and reducing tissue damages.

As an opportunistic causative pathogen of "Visceral White Spot Disease" in several kinds of teleost, Pseudomonas plecoglossicida has caused huge economic losses to aquaculture. Flagella is the motility organ of bacteria as well as a vital virulence factor. The protein encoded by the fliP gene is involved in the formation of the flagellar type III secretion export apparatus, which plays a crucial role in flagellar assembly. In this study, the Delta fliP and C-Delta fliP strains of P. plecoglossicida were constructed to explore the biological profiles mediated by the fliP gene and its effects on the virulence of this organism. Compared to the wild type strain (NZBD9) of P. plecoglossicida, the deletion strain Delta fliP exhibited a significant impairment in swimming motility, chemotaxis, adhesion, and biofilm formation abilities. The transcriptomic analysis of the NZBD9, Delta fliP and C-Delta fliP strains revealed that the fliP gene may affect the biological profiles of P. plecoglossicida by down-regulating the expression of flagellar assembly, type 6 secretion system and bacterial chemotaxis related genes. Furthermore, the results of artificial infection showed that the virulence of Delta fliP strain to hybrid grouper (Epinephelus fuscoguttatus female x E. lanceolatus male) was attenuated, with a LD50 value of 2.5 x 104 CFU/fish. These findings demonstrate that the fliP gene contributes to the virulence of P. plecoglossicida by regulating its motility.

Polyploids recurrently emerge in angiosperms, but most polyploids are likely to go extinct before establishment due to minority cytotype exclusion, which may be specifically a constraint for dioecious plants to evolve polyploid populations. Investigations into the frequency and distribution of polyploids in natural populations is thus necessary for understanding polyploid evolution in plants. This study determined the ploidy levels of 28 populations and 351 individuals of Salix polyclona, and identified the type of polyploidy (auto- vs. allo-) using whole genome re-sequencing data. We further investigated the phylogeny, population genetic diversity and species range shifts to explore the origin and spatiotemporal evolution of the polyploid complex. Our analyses revealed a high frequency (52%) of autopolyploids in it with a clear geographic distribution confined to the western part of its range where complex mountain systems create higher levels of environmental heterogeneity. Comparisons of diploid male and female genomes suggested a female heterogametic sex-determining factor on chromosome 15, which likely also acts in the dioecious polyploids. Fossil-calibrated phylogeny showed a more recent diversification of the polyploids (ca. 2.3 Ma) than the diploid (ca. 6.2 Ma), and population demographic histories largely corroborated the geological and climatic history of the region. Our results suggest that climatic oscillations and uplift of eastern Qinghai-Tibetan Plateau and the connecting mountains may have facilitated the preservation and establishment of polyploid populations. This study provides an example of the evolution of a diploid-polyploid complex in a willow species and illustrates a role of polyploidization in mountain biodiversity.

Pseudomonas plecoglossicida is a gram-negative pathogenic bacterium that causes visceral white spot disease in several marine and aquaculture fish species, resulting in high mortality and severe financial loss. Based on previous RNA sequencing (RNA-seq), fliK gene expression is significantly up-regulated in P. plecoglossicida during infection, indicating that fliK may contribute to its bacterial pathogenicity. To investigate the role of fliK, four specific short hairpin RNAs (shRNAs) were designed and synthesized according to the fliK gene sequence, with three of the four mutants exhibiting a significant decrease in fliK gene expression in P. plecoglossicida. The shRNA-406 mutant with the maximum silencing efficiency (97.5%) was chosen for further study. Compared with the wild-type (WT) P. plecoglossicida strain, silencing fliK in the fliK-RNA interference (RNAi) strain resulted in a significant decrease in growth, motility, chemotaxis, adhesion, and biofilm formation in P. plecoglossicida. Silencing of fliK also resulted in a 95% increase in the survival rate, a 2-day delay in the onset of death, and a significant decrease in the number of white spots on the spleen surface in infected orange-spotted groupers (Epinephelus coioides). In addition, fliK gene expression and pathogen load were significantly lower in the spleens of E. coioides infected with the fliK-RNAi strain than in those infected with the WT strain of P. plecoglossicida. RNA-seq of the spleens further revealed that fliK silencing significantly regulated the immune response of E. coioides during the pathogenic process. Compared with the WT-infected group, the differentially expressed genes (DEGs) in the fliK-RNAi-infected group were enriched in 344 and 345 KEGG pathways at 3 and 5 days post infection (dpi), respectively. Among these pathways, 21 immune system-related pathways were enriched, including the natural killer (NK) cell-mediated cytotoxicity, platelet activation, and Th17 cell differentiation signaling pathways. The NK cell-mediated cytotoxicity pathway was the most significantly enriched, which may enhance the host's ability to remove pathogens and reduce inflammation. This study revealed the effects of the fliK gene in P. plecoglossicida pathogenicity and identified the main pathways involved in the immune response of E. coioides.

Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker (Larimichthys crocea) and orange-spotted grouper (Epinephelus coioides). Previously, RNA sequencing showed that P. plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection. To explore the role of flgK in P. plecoglossicida pathogenicity, RNA interference (RNAi) was performed to silence the P. plecoglossicida flgK gene, and the mutant (flgK-RNAi strain) with the best silencing efficiency (89.40%) was chosen for further study. Results showed that flgK gene silencing significantly attenuated P. plecoglossicida motility, adhesion, and biofilm formation. Compared to those fish infected with the wild-type strain of P. plecoglossicida, orange-spotted grouper infected with the flgK-RNAi strain showed a 55% increase in the survival rate and a one-day delay in time of first death, with fewer pathogens in the spleen and fewer white spots on the spleen surface. RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase (MAPK) signaling pathway was related to multiple immune-related pathways. Furthermore, arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways. These findings suggest that flgK is a virulence gene of P. plecoglossicida. Furthermore, flgK appears to be involved in the regulation of motility, adhesion, and biofilm formation in P. plecoglossicida, as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P. plecoglossicida infection.

Pseudomonas plecoglossicida is an important pathogen in aquaculture and causes serious economic losses. Our previous study indicated that znuA gene might play an important role in the pathogenicity of P. plecoglossicida. Five shRNAs were designed and synthesized to silence the znuA gene of P. plecoglossicida. Two of the five mutants of P. plecoglossicida exhibited significant reduction in the expression level of znuA mRNA with different efficiencies. The mutant with the highest silencing efficiency of 89.2% was chosen for further studies. Intrapleural injection of the znuA-RNAi strain at a dose of 10(5) cfu/fish did not cause the death of Epinephelus coioides, and no significant signs were observed at the spleen surface of infected E. coioides, while the counterpart E. coioides infected by the same dose of wild-type strain of P. plecoglossicida all died in 5 days post-infection (dpi). The expression of znuA gene of znuA-RNAi strain in E. coioides was always lower than that in wild-type strain of P. plecoglossicida. The pathogen load in the early stage of infection was higher than that in the later stage of infection. Although the infection of the znuA-RNAi strain of P. plecoglossicida could induce the production of antibodies in E. coioides, it failed to produce a good immune protection against the infection of wild-type strain of P. plecoglossicida. Compared with the transcriptome data of E. coioides infected by the wild-type strain of P. plecoglossicida, the transcriptome data of E. coioides infected by the znuA-RNAi strain of P. plecoglossicida have altered significantly. Among them, KEGG enrichment analysis showed that the focal adhesion pathway was significantly enriched and exhibited the largest number of 302 DEMs (differentially expressed mRNAs). These results showed that the immune response of E. coioides to P. plecoglossicida infection was significantly affected by the RNAi of znuA gene.

Pseudomonas plecoglossicida is the causative agent of “visceral white spot disease” in cultured fish and has resulted in serious economic losses. tonB gene plays a crucial role in the uptake of nutrients from the outer membranes in Gram-negative bacteria. The previous results of our lab showed that the expression of tonB gene of P. plecoglossicida was significantly upregulated in the spleens of infected Epinephelus coioides . To explore the effect of tonB gene on the virulence of P. plecoglossicida and the immune response of E. coioides, tonB gene of P. plecoglossicida was knocked down by RNAi; and the differences between the wild-type strain and the tonB -RNAi strain of P. plecoglossicida were investigated. The results showed that all of the four mutants of P. plecoglossicida exhibited significant decreases in mRNA of tonB gene, and the best knockdown efficiency was 94.0%; the survival rate of E. coioides infected with the tonB -RNAi strain was 20% higher than of the counterpart infected with the wild strain of P. plecoglossicida . Meanwhile, the E. coioides infected with the tonB -RNAi strain of P. plecoglossicida carried less pathogens in the spleen and less white spots on the surface of the spleen; compared with the wild-type strain, the motility, chemotaxis, adhesion, and biofilm formation of the tonB -RNAi strain were significantly attenuated; the transcriptome data of E. coioides infected with the tonB -RNAi strain were different from the counterpart infected with the wild strain of P. plecoglossicida ; the antigen processing and presentation pathway and the complement and coagulation cascade pathway were the most enriched immune pathways. The results indicated that tonB was a virulence gene of P. plecoglossicida ; tonB gene was involved in the regulation of motility, chemotaxis, adhesion, and biofilm formation; tonB gene affected the immune response of E. coioides to P. plecoglossicida infection.

Phytosterol biomarkers in marine sediments are often used to indicate the source of the sedimentary organics in the ocean, and to reflect the evolution of marine ecosystem and environment. In this study, a novel method 14 simultaneous determination of common phytosterols in marine sediments by non-aqueous reversed-phase liquid chromatography-atmospheric pressure photoionization mass spectrometry ( LC-APPI-MS ) was established. First, the APPI-MS characteristics of phytosterols were clarified. Under the positive APPI mode, phytosterols tended to produce stable characteristic ion peaks such as [ M + H - H2O ](+) , [ M + H - H-2 ](+) and [ M + H - 2H(2) ](+) . In the process of APPI-MS2, abundant fragment ion peaks were produced mainly through i-heterolysis. Second, LC-APPI-MS conditions were optimized, and a reversed-phase C-18 column was used to separate phytosterols with pure methanol as mobile phase and toluene as APPI ionization dopant , which could realize high sensitivity of detection for phytosterols in sediments. Furthermore, the method showed strong matrix interference resistance ability , good linear relationship ( R-2 >= 0. 9990 ) , high recovery rate ( 86. 4% - 94. 9% ) and good repeatability ( RSDs >= 8. 3% ). Finally, the proposed method was applied to detect common phytosterols in the surface sediment of Laizhou Bay in China, and several sterols, such as brassicasterol , fucosterol and sitosterol, etc. , were detected in every sediment sample. The total concentration of sterols was 4. 21-12. 91 mu g/g, among which the concentration of sitosterol was the highest. To sum up , the developed LC-APPI-MS method was a powerful tool 14 determination of common phytosterols in marine sediments , with easy-operation and high sensitivity.

To study the roles of the exbB gene in Pseudomonas plecoglossicida during interactions with Epinephelus coioides, five short hairpin RNAs (shRNAs) were designed and synthesized to silence the exbB gene in P. plecoglossicida which resulted in significant reductions in exbB mRNA expression. The mutant with the best silencing efficiency (89.3%) was selected for further study. Silencing exbB in the exbB-RNA interference (RNAi) strain resulted in a 70% increase in the survival rate and a 3-day delay in the onset of infection in E. coioides. Silencing of the exbB gene also resulted in a significant decrease in the number of white spots on the spleen surface and in the spleen pathogen load. The results of dual RNA-seq showed that exbB silencing in P. plecoglossicida also resulted in a significant change in both the pathogen and host transcriptomes in the spleens of infected E. coioides. Comparative transcriptome analysis showed that silencing exbB caused significant changes in multiple signaling molecules and interaction- and immune system-related genes in E. coioides. Gene silencing also resulted in the differential expression of flagellar assembly and the bacterial secretion system in P. plecoglossicida during the infection period, and most of the DEGs were down-regulation. These host-pathogen interactions may make it easier for E. coioides to eliminate the exbB-RNAi strain of P. plecoglossicida, suggesting a significant decrease in the pathogenicity of this strain. These results indicated that exbB was a virulence gene of P. plecoglossicida which contributed a lot in the pathogen-host interactions with E. coioides.

Plant mitogenomes can be difficult to assemble because they are structurally dynamic and prone to intergenomic DNA transfers, leading to the unusual situation where an organelle genome is far outnumbered by its nuclear counterparts. As a result, comparative mitogenome studies are in their infancy and some key aspects of genome evolution are still known mainly from pre-genome, qualitative methods. To help address these limitations, we combined machine learning and in silico enrichment of mitochondrial-like long reads to assemble the bacterial-sized mitogenome of Norway spruce (Pinaceae: Picea abies ). We conducted comparative analyses of repeat abundance, intergenomic transfers, substitution and rearrangement rates, and estimated repeat-by-repeat homologous recombination rates. Prompted by our discovery of highly recombinogenic small repeats in P. abies , we assessed the genomic support for the prevailing hypothesis that intramolecular recombination is predominantly driven by repeat length, with larger repeats facilitating DNA exchange more readily. Overall, we found mixed support for this view: recombination dynamics were heterogeneous across vascular plants and highly active small repeats ( ca . 200 bp) were present in about a third of studied mitogenomes. As in previous studies, we did not observe any robust relationships among commonly-studied genome attributes, but we identify variation in recombination rates as a underinvestigated source of plant mitogenome diversity.