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ABSTRACT This study investigated the change in the microbiome of tomato rhizosphere soils after the invasion of Ralstonia solanacearum and analyzed the correlation between microbes and soil physicochemical properties. Diversity analyses of the bacteria in healthy and diseased rhizosphere soil samples (HRS and DRS) revealed that HRS had a higher species diversity and were compositionally different from DRS (P ≤ 0.05). Substantial differences in the relative abundance of Actinobacteria (37.52% vs 28.96%, P ≤ 0.05) and Proteobacteria (29.20% vs 35.59%, P ≤ 0.05) were identified in HRS and DRS, respectively. Taxonomic composition analysis showed ten differentially abundant genera, and seven of them (Gaiella, Roseisolibacter, Solirubrobacter, Kribbella, Acidibacter, Actinomarinicola, and Marmoricola) are more abundant in HRS. Soil pH and enzyme activities were negatively correlated with the abundance of R. solanacearum. The contents of total nitrogen (TN), total phosphorus (TP), total potassium (TK), alkaline nitrogen (alkaline N), available phosphorus (AP), available potassium (AK), NO3—N(NN), NH4+-N (AN), and organic matter (OM) were all significantly increased in DRS. The composition and richness of protozoa in the samples show significant differences. Cephalobus, Acrobeles, Heteromita, norank_Tylenchida, and Rotylenchulus were enriched in DRS. Microbial interaction networks revealed that the HRS networks were more complex than the DRS networks. Overall, the results of this study demonstrate that healthy soil has a more complex microbial community structure and higher enzyme activity, and the invasion of R. solanacearum damages the soil microbial system.IMPORTANCEHow does the invasion of Ralstonia solanacearum affect tomato rhizosphere bacteria and protozoa? Which microbial changes can affect the growth of R. solanacearum? To date, most research studies focus on bacteria, with little research on protozoa, and even less on the synergistic effects between protozoa and bacteria. Here, we analyzed the correlation between tomato rhizosphere bacterial and protozoan communities and soil physicochemical properties during the invasion of R. solanacearum. We found that the diversity and abundance of rhizosphere microorganisms in healthy rhizosphere soil samples (HRS) were significantly higher than those in diseased rhizosphere soil samples (DRS), and there were significant changes in soil pH and enzyme activity. Overall, in this study, the analysis of microbial changes during the invasion of R. solanacearum provides a theoretical basis for the prevention and control of bacterial wilt.

The multifunctional secondary metabolites known as cyclic lipopeptides (CLPs), which are produced by a large variety of bacteria, have become a key category of plant immunity elicitors. Pseudomonas-CLPs (Ps-CLPs) are extremely diverse in structure and biological activity. However, an understanding of CLP-plant structure–function interactions currently remains elusive. Here, we identify medpeptin, a novel CLP from Pseudomonas mediterranea that consists of 22 amino acids. Medpeptin is synthesized by a non-ribosomal peptide synthase (NRPS) gene cluster and regulated by a quorum-sensing system. Further research indicates that medpeptin does not exhibit antimicrobial activity; instead, it induces plant cell death immunity and confers resistance to bacterial infection. Comparative transcriptome analysis and virus-induced gene silencing (VIGS) reveal a set of immune signaling candidates involved in medpeptin perception. Silencing of a cell-wall leucine-rich repeat extensin protein (NbLRX3) or a receptor-like protein kinase (NbRLK25)—but not BAK1 or SGT1—compromises medpeptin-triggered cell death and resistance to pathogen infection in Nicotiana benthamiana. Our findings point to a noncanonical mechanism of CLP sensing and suggest perspectives for the development of plant disease resistance.

ABSTRACT The core endophytes of plants are regarded as promising resources in future agroecosystems. How they affect the assembly of rice-related bacterial communities after early inoculation remains unclear. Here, we examined bacterial communities across 148 samples, including bulk and rhizosphere soils, sterilized roots, stems, and seeds at the seedling, tillering, booting, and maturity stages. Tissue cultured rice seedlings were inoculated with Xathomonas sacchari JR3-14, a core endophytic bacterium of rice seeds, before transplanting. The results revealed that α-diversity indices were significantly enhanced in the root and stem endosphere at the seedling stage. β-diversity was altered at most plant developmental stages, except for the root and stem at the booting stage. Network complexity consequently increased in the root and stem across rice growth stages, other than the stem endosphere at the booting stage. Four abundant beneficial bacterial taxa, Bacillus, Azospira, Azospirillum, and Arthrobacter, were co-enriched during the early growth stage. Infer Community Assembly Mechanisms by Phylogenetic-bin-based null model analysis revealed a higher relative contribution of drift and other eco-evolutionary processes mainly in root compartments across all growth stages, but the opposite pattern was observed in stem compartments. IMPORTANCE Endophytic bacteria are regarded as promising environmentally friendly resources to promote plant growth and plant health. Some of microbes from the seed are able to be carried over to next generation, and contribute to the plant’s ability to adapt to new environments. However, the effects of early inoculation with core microbes on the assembly of the plant microbiome are still unclear. In our study, we demonstrate that early inoculation of the rice seed core endophytic bacterium Xanthomonas sacchari could alter community diversity, enhance complexity degree of network structure at most the growth stages, and enrich beneficial bacteria at the seedling stage of rice. We further analyzed the evolutionary processes caused by the early inoculation. Our results highlight the new possibilities for research and application of sustainable agriculture by considering the contribution of seed endophytes in crop production and breeding.

Abstract Background Plants and their associated microbiota constitute an assemblage of species known as holobionts. The plant seed microbiome plays an important role in nutrient uptake and stress attenuation. However, the core vertically transmitted endophytes remain largely unexplored. Results To gain valuable insights into the vertical transmission of rice seed core endophytes, we conducted a large-scale analysis of the microbiomes of two generations of six different rice varieties from five microhabitats (bulk soil, rhizosphere, root, stem, and seed) from four geographic locations. We showed that the microhabitat rather than the geographic location and rice variety was the primary driver of the rice microbiome assemblage. The diversity and network complexity of the rice-associated microbiome decreased steadily from far to near the roots, rice exterior to interior, and from belowground to aboveground niches. Remarkably, the microbiomes of the roots, stems, and seeds of the rice interior compartments were not greatly influenced by the external environment. The core bacterial endophytes of rice were primarily comprised of 14 amplicon sequence variants (ASVs), 10 of which, especially ASV_2 (Pantoea) and ASV_48 (Xanthomonas), were identified as potentially vertically transmitted taxa because they existed across generations, were rarely present in exterior rice microhabitats, and were frequently isolated from rice seeds. The genome sequences of Pantoea and Xanthomonas isolated from the parental and offspring seeds showed a high degree of average nucleotide and core protein identity, indicating vertical transmission of seed endophytes across generations. In silico prediction indicated that the seed endophytes Pantoea and Xanthomonas possessed streamlined genomes with short lengths, low-complexity metabolism, and various plant growth-promoting traits. We also found that all strains of Pantoea and Xanthomonas exhibited cellulase activity and produced indole-3-acetic acid. However, most strains exhibited insignificant antagonism to the major pathogens of rice, such as Magnaporthe oryzae and X. oryzae pv. oryzae. Conclusion Overall, our study revealed that microhabitats, rather than site-specific environmental factors or host varieties, shape the rice microbiome. We discovered the vertically transmitted profiles and keystone taxa of the rice microbiome, which led to the isolation of culturable seed endophytes and investigation of their potential roles in plant-microbiome interactions. Our results provide insights on vertically transmitted microbiota and suggest new avenues for improving plant fitness via the manipulation of seed-associated microbiomes. Video Abstract

ABSTRACT The bacterial plant pathogen Pseudomonas syringae deploys a type III secretion system (T3SS) to deliver effector proteins into plant cells to facilitate infection, for which many effectors have been characterized for their interactions. However, few T3SS Hrp (hypersensitive response and pathogenicity) proteins from the T3SS secretion apparatus have been studied for their direct interactions with plants. Here, we show that the P. syringae pv. tomato DC3000 T3SS protein HrpP induces host cell death, suppresses pattern-triggered immunity (PTI), and restores the effector translocation ability of the hrpP mutant. The hrpP-transgenic Arabidopsis lines exhibited decreased PTI responses to flg22 and elf18 and enhanced disease susceptibility to P. syringae pv. tomato DC3000. Transcriptome analysis reveals that HrpP sensing activates salicylic acid (SA) signaling while suppressing jasmonic acid (JA) signaling, which correlates with increased SA accumulation and decreased JA biosynthesis. Both yeast two-hybrid and bimolecular fluorescence complementation assays show that HrpP interacts with mitogen-activated protein kinase kinase 2 (MKK2) on the plant membrane and in the nucleus. The HrpP truncation HrpP1–119, rather than HrpP1–101, retains the ability to interact with MKK2 and suppress PTI in plants. In contrast, HrpP1–101 continues to cause cell death and electrolyte leakage. MKK2 silencing compromises SA signaling but has no effect on cell death caused by HrpP. Overall, our work highlights that the P. syringae T3SS protein HrpP facilitates effector translocation and manipulates plant immunity to facilitate bacterial infection. IMPORTANCE The T3SS is required for the virulence of many Gram-negative bacterial pathogens of plants and animals. This study focuses on the sensing and function of the T3SS protein HrpP during plant interactions. Our findings show that HrpP and its N-terminal truncation HrpP1–119 can interact with MKK2, promote effector translocation, and manipulate plant immunity to facilitate bacterial infection, highlighting the P. syringae T3SS component involved in the fine-tuning of plant immunity.

IntroductionThe black shank disease seriously affects the health of tobacco plants. Conventional control methods have limitations in terms of effectiveness or economic aspects and cause public health concerns. Thus, biological control methods have come into the field, and microorganisms play a key role in suppressing tobacco black shank disease.MethodsIn this study, we examined the impact of soil microbial community on black shank disease basing on the structural difference of bacterial communities in rhizosphere soils. We used Illumina sequencing to compare the bacterial community diversity and structure in different rhizosphere soil samples in terms of healthy tobacco, tobacco showing typical black shank symptoms, and tobacco treated with the biocontrol agent, Bacillus velezensis S719.ResultsWe found that Alphaproteobacteria in the biocontrol group, accounted for 27.2% of the ASVs, was the most abundant bacterial class among three groups. Heatmap and LEfSe analyses were done to determine the distinct bacterial genera in the three sample groups. For the healthy group, Pseudomonas was the most significant genus; for the diseased group, Stenotrophomonas exhibited the strongest enrichment trend, and Sphingomonas showed the highest linear discriminant analysis score, and was even more abundant than Bacillus; for the biocontrol group, Bacillus, and Gemmatimonas were the largely distributed genus. In addition, co-occurrence network analysis confirmed the abundance of taxa, and detected a recovery trend in the network topological parameters of the biocontrol group. Further functional prediction also provided a possible explanation for the bacterial community changes with related KEGG annotation terms.DiscussionThese findings will improve our knowledge of plant-microbe interactions and the application of biocontrol agents to improve plant fitness, and may contribute to the selection of biocontrol strains.

Abstracts Recent evidence suggests that CD147 serves as a novel receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Blocking CD147 via anti-CD147 antibody could suppress the in vitro SARS-CoV-2 replication. Meplazumab is a humanized anti-CD147 IgG2 monoclonal antibody, which may effectively prevent SARS-CoV-2 infection in coronavirus disease 2019 (COVID-19) patients. Here, we conducted a randomized, double-blinded, placebo-controlled phase 1 trial to evaluate the safety, tolerability, and pharmacokinetics of meplazumab in healthy subjects, and an open-labeled, concurrent controlled add-on exploratory phase 2 study to determine the efficacy in COVID-19 patients. In phase 1 study, 59 subjects were enrolled and assigned to eight cohorts, and no serious treatment-emergent adverse event (TEAE) or TEAE grade ≥3 was observed. The serum and peripheral blood C max and area under the curve showed non-linear pharmacokinetic characteristics. No obvious relation between the incidence or titer of positive anti-drug antibody and dosage was observed in each cohort. The biodistribution study indicated that meplazumab reached lung tissue and maintained >14 days stable with the lung tissue/cardiac blood–pool ratio ranging from 0.41 to 0.32. In the exploratory phase 2 study, 17 COVID-19 patients were enrolled, and 11 hospitalized patients were involved as concurrent control. The meplazumab treatment significantly improved the discharged (P = 0.005) and case severity (P = 0.021), and reduced the time to virus negative (P = 0.045) in comparison to the control group. These results show a sound safety and tolerance of meplazumab in healthy volunteers and suggest that meplazumab could accelerate the recovery of patients from COVID-19 pneumonia with a favorable safety profile.

Abstract Strain S150 was isolated from the tobacco rhizosphere as a plant growth‐promoting rhizobacterium. It increased plant fresh weight significantly and lateral root development, and it antagonized plant pathogenic fungi but not phytobacteria. Further tests showed that strain S150 solubilized organic phosphate and produced ammonia, siderophore, protease, amylase, and cellulase, but it did not produce indole‐3‐acetic acid. Using morphology, physiological characteristics, and multi‐locus sequence analysis, strain S150 was identified as Pseudomonas koreensis. The complete genome of strain S150 was sequenced, and it showed a single circular chromosome of 6,304,843 bp with a 61.09% G + C content. The bacterial genome contained 5,454 predicted genes that occupied 87.7% of the genome. Venn diagrams of the identified orthologous clusters of P. koreensis S150 with the other three sequenced P. koreensis strains revealed up to 4,167 homologous gene clusters that were shared among them, and 21 orthologous clusters were only present in the genome of strain S150. Genome mining of the bacterium P. koreensis S150 showed that the strain possessed 10 biosynthetic gene clusters for secondary metabolites, which included four clusters of non‐ribosomal peptide synthetases (NRPSs) involved in the biosynthesis of cyclic lipopeptides (CLPs). One of the NRPSs possibly encoded lokisin, a cyclic lipopeptide produced by fluorescent Pseudomonas. Genomic mutation of the lokA gene, which is one of the three structural NRPS genes for lokisin in strain S150, led to a deficiency in fungal antagonism that could be restored fully by gene complementation. The results suggested that P. koreensis S150 is a novel plant growth‐promoting agent with specific cyclic lipopeptides and contains a lokisin‐encoding gene cluster that is dominant against plant fungal pathogens.

Background: Mitochondrial DNA (mtDNA) content measured by different techniques cannot be compared between studies, and age- and tissue-related control values are hardly available. In the present study, we aimed to establish the normal reference range of mtDNA copy number in the Chinese population. Methods: Two healthy cohorts of 200 Chinese minors (0.1–18.0 years) and 200 adults (18.0–88.0 years) were recruited. Then, they were further categorized into eight age groups. The absolute mtDNA copy number per cell was measured by a quantitative real-time polymerase chain reaction. We subsequently used this range to evaluate mtDNA content in four patients (0.5–4.0 years) with molecularly proven mitochondrial depletion syndromes (MDSs) and 83 cases of mitochondrial disease patients harboring the m.3243A>G mutation. Results: The reference range of mtDNA copy number in peripheral blood was 175–602 copies/cell (mean: 325 copies/cell) in minors and 164–500 copies/cell (mean: 287 copies/cell) in adults. There was a decreasing trend in mtDNA copy number in blood with increasing age, especially in 0–2-year-old and >50-year-old donors. The mean mtDNA copy number level among the mitochondrial disease patients with m.3243A>G mutation was significantly higher than that of healthy controls. The mtDNA content of POLG, DGUOK, TK2, and SUCLA2 genes in blood samples from MDS patients was reduced to 25%, 38%, 32%, and 24%, respectively. Conclusions: We primarily establish the reference intervals of mtDNA copy number, which might contribute to the clinical diagnosis and monitoring of mitochondrial disease.

The type III secretion system (T3SS) in many Gram-negative bacterial pathogens is regarded as the most critical virulence determinant and an attractive target for novel anti-virulence drugs. In this study, we constructed a T3SS secretion reporter containing the β-lactamase gene fused with a signal peptide sequence of the T3SS effector gene, and established a high-throughput screening system for T3SS inhibitors in the plant pathogenic bacterium Acidovorax citrulli. From a library of 12,000 chemical compounds, we identified a series of benzyloxy carbonimidoyl dicyanide (BCD) derivatives that effectively blocked T3SS-dependent β-lactamase secretion. Substitution of halogens or nitro groups at the para-position on the benzene ring contributed to an increased inhibitory activity. One representative compound, BCD03 (3,4-dichloro-benzyloxy carbonimidoyl dicyanide), dramatically reduced pathogenicity of A. citrulli on melon seedlings, and attenuated hypersensitive responses in the non-host Nicotiana tabacum caused by pathogenic bacteria A. citrulli, Xanthomonas oryzae pv. oryzae and Pseudomonas syringae pv. tomato at sub-MIC concentrations. Western blotting assay further confirmed that BCD03 inhibited effector secretion from the above bacteria via T3SS in the liquid medium. Taken together, our data suggest that BCD derivatives act as novel inhibitors of T3SS in multiple plant bacterial pathogens.

Background: Mitochondrial diseases are a group of energy metabolic disorders with multisystem involvements. Variable clinical features present a major challenge in pediatric diagnoses. We summarized the clinical spectrum of m.3243A>G mutation in Chinese pediatric patients, to define the common clinical manifestations and study the correlation between heteroplasmic degree of the mutation and clinical severity of the disease. Methods: Clinical data of one-hundred pediatric patients with symptomatic mitochondrial disease harboring m.3243A>G mutation from 2007 to 2013 were retrospectively reviewed. Detection of m.3243A>G mutation ratio was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Correlation between m.3243A>G mutation ratio and age was evaluated. The differences in clinical symptom frequency of patients with low, middle, and high levels of mutation ratio were analyzed by Chi-square test. Results: Sixty-six patients (66%) had suffered a delayed diagnosis for an average of 2 years. The most frequent symptoms were seizures (76%), short stature (73%), elevated plasma lactate (70%), abnormal magnetic resonance imaging/computed tomography (MRI/CT) changes (68%), vomiting (55%), decreased vision (52%), headache (50%), and muscle weakness (48%). The mutation ratio was correlated negatively with onset age (r = −0.470, P < 0.001). Myopathy was more frequent in patients with a high level of mutation ratio. However, patients with a low or middle level of m.3243A>G mutation ratio were more likely to suffer hearing loss, decreased vision, and gastrointestinal disturbance than patients with a high level of mutation ratio. Conclusions: Our study showed that half of Chinese pediatric patients with m.3243A>G mutation presented seizures, short stature, abnormal MRI/CT changes, elevated plasma lactate, vomiting, and headache. Pediatric patients with these recurrent symptoms should be considered for screening m.3243A>G mutation. Clinical manifestations and laboratory abnormalities should be carefully monitored in patients with this point mutation.

Abstract Background Radioresistance is one of the main obstacle limiting the therapeutic efficacy and prognosis of patients, the molecular mechanisms of radioresistance is still unclear. The purpose of this study was to identify the key genes and miRNAs and to explore their potential molecular mechanisms in radioresistant nasopharyngeal carcinoma. Methods In this study, we analysis the differentially expressed genes and microRNA based on the database of GSE48501 and GSE48502, and then employed bioinformatics to analyze the pathways and GO terms in which DEGs and DEMS target genes are involved. Moreover, Construction of protein-protein interaction network and identification of hub genes. Finally, analyzed the biological networks for validated target gene of hub miRNAs. Results A total of 373 differentially expressed genes (DEGs) and 14 differentially expressed microRNAs (DEMs) were screened out. The up-regulated gene JUN was overlap both in DEGs and publicly available studies, which was potentially targeted by three miRNAs, including hsa-miR-203, hsa-miR-24 and hsa-miR-31. Moreover, Pathway analysis showed that both up-regulated gene and DEMs target genes were enriched in TGF-beta signaling pathway, Hepatitis B, Pathways in cancer and p53 signaling pathway. Finally, we further constructed protein-protein interaction network (PPI) of DEGs and analyzed the biological networks for above mentioned common miRNAs, the result indicated that JUN was a core hub gene in PPI network, hsa-miR-24 and its target gene were significantly enriched in P53 signaling pathway. Conclusions These results might provide new clues to improve the radiosensitivity of Nasopharyngeal Carcinoma.