Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination.

Melia azedarach is a species of enormous value of pharmaceutical industries. Although the chloroplast genome of M. azedarach has been explored, the information of mitochondrial genome (Mt genome) remains surprisingly limited. In this study, we used a hybrid assembly strategy of BGI short-reads and Nanopore long-reads to assemble the Mt genome of M. azedarach. The Mt genome of M. azedarach is characterized by two circular chromosomes with 350,142 bp and 290,387 bp in length, respectively, which encodes 35 protein-coding genes (PCGs), 23 tRNA genes, and 3 rRNA genes. A pair of direct repeats (R1 and R2) were associated with genome recombination, resulting in two conformations based on the Sanger sequencing and Oxford Nanopore sequencing. Comparative analysis identified 19 homologous fragments between Mt and chloroplast genome, with the longest fragment of 12,142 bp. The phylogenetic analysis based on PCGs were consist with the latest classification of the Angiosperm Phylogeny Group. Notably, a total of 356 potential RNA editing sites were predicted based on 35 PCGs, and the editing events lead to the formation of the stop codon in the rps10 gene and the start codons in the nad4L and atp9 genes, which were verified by PCR amplification and Sanger sequencing. Taken together, the exploration of M. azedarach gap-free Mt genome provides a new insight into the evolution research and complex mitogenome architecture.

Transcriptional profiling of Xanthomonas campestris pv. campestris in viable but nonculturable state.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is an important seed-borne plant pathogenic bacteria that can cause a serious threat to cruciferous crops. Bacteria can enter into the viable but non-culturable (VBNC) state under stress conditions, and cause potential risks to agricultural production because the VBNC bacterial cells will evade culture-based detection. However, little is known about the mechanism of VBNC. Our previous study showed that Xcc could be induced into VBNC state by copper ion (Cu2+). RESULTS: Here, RNA-seq was performed to explore the mechanism of VBNC state. The results indicated that expression profiling was changed dramatically in the different VBNC stages (0 d, 1 d, 2 d and 10 d). Moreover, metabolism related pathways were enriched according to COG, GO and KEGG analysis of differentially expressed genes (DEGs). The DEGs associated with cell motility were down-regulated, whereas pathogenicity related genes were up-regulated. This study revealed that the high expression of genes related to stress response could trigger the active cells to VBNC state, while the genes involved in transcription and translation category, as well as transport and metabolism category, were ascribed to maintaining the VBNC state. CONCLUSION: This study summarized not only the related pathways that might trigger and maintain VBNC state, but also the expression profiling of genes in different survival state of bacteria under stress. It provided a new kind of gene expression profile and new ideas for studying VBNC state mechanism in X. campestris pv. campestris.

CsIVP functions in vasculature development and downy mildew resistance in cucumber.

Domesticated crops with high yield and quality are frequently susceptible to pathogen attack, whereas enhancement of disease resistance generally compromises crop yield. The underlying mechanisms of how plant development and disease resistance are coordinately programed remain elusive. Here, we showed that the basic Helix-Loop-Helix (bHLH) transcription factor Cucumis sativus Irregular Vasculature Patterning (CsIVP) was highly expressed in cucumber vascular tissues. Knockdown of CsIVP caused severe vasculature disorganization and abnormal organ morphogenesis. CsIVP directly binds to vascular-related regulators YABBY5 (CsYAB5), BREVIPEDICELLUS (CsBP), and AUXIN/INDOLEACETIC ACIDS4 (CsAUX4) and promotes their expression. Knockdown of CsYAB5 resulted in similar phenotypes as CsIVP-RNA interference (RNAi) plants, including disturbed vascular configuration and abnormal organ morphology. Meanwhile, CsIVP-RNAi plants were more resistant to downy mildew and accumulated more salicylic acid (SA). CsIVP physically interacts with NIM1-INTERACTING1 (CsNIMIN1), a negative regulator in the SA signaling pathway. Thus, CsIVP is a novel vasculature regulator functioning in CsYAB5-mediated organ morphogenesis and SA-mediated downy mildew resistance in cucumber.

Deletion of pbpC Enhances Bacterial Pathogenicity on Tomato by Affecting Biofilm Formation, Exopolysaccharides Production, and Exoenzyme Activities in Clavibacter michiganensis.

Penicillin-binding proteins (PBPs) are considered essential for bacterial peptidoglycan biosynthesis and cell wall assembly. Clavibacter michiganensis is a representative Gram-positive bacterial species that causes bacterial canker in tomato. pbpC plays a significant role in maintaining cell morphological characteristics and stress responses in C. michiganensis. The current study demonstrated that the deletion of pbpC commonly enhances bacterial pathogenicity in C. michiganensis and revealed the mechanisms through which this occurs. The expression of interrelated virulence genes, including celA, xysA, xysB, and pelA, were significantly upregulated in △pbpC mutants. Compared with those in wild-type strains, exoenzyme activities, the formation of biofilm, and the production of exopolysaccharides (EPS) were significantly increased in △pbpC mutants. It is noteworthy that EPS were responsible for the enhancement in bacterial pathogenicity, with the degree of necrotic tomato stem cankers intensifying with the injection of a gradient of EPS from C. michiganensis. These findings highlight new insights into the role of pbpC affecting bacterial pathogenicity, with an emphasis on EPS, advancing the current understanding of phytopathogenic infection strategies for Gram-positive bacteria.

Unmarked Gene Editing in Clavibacter michiganensis Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection.

Plant-pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis, which couples the expression of cas9 and single-guide RNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site-directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the C. michiganensis rpsL gene to generate a streptomycin-resistant mutant, and complete gene knockout, in which the deletion of the C. michiganensis celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments, the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 5-fluorocytosine at 100 µg/ml, in which an unmarked katA mutant lacking the transformation vector was recovered. Compared with conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient because it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into C. michiganensis and other Clavibacter spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of Clavibacter michiganensis.

The viable but nonculturable (VBNC) state is a unique survival strategy of bacteria in response to stress conditions. It was confirmed that Clavibacter michiganensis, the causal agent of bacterial canker in tomato, could be induced into the VBNC state by exposure to CuSO4 in an oligotrophic solution. RNA-sequencing analysis was used to monitor the mechanisms of the VBNC state during CuSO4 induction in C. michiganensis. The results identified that numerous genes involved in stringent response, copper resistance, and stress resistance were upregulated, and some involved in cell division were downregulated significantly. The study investigated the importance of Rel, which is an essential enzyme in the synthesis of the molecular alarmone ppGpp, via the generation of a Δrel mutant and its complementation strain. Biological characterization revealed that deficiency of rel reduced the bacterial growth, production of exopolysaccharides, and pathogenicity as well as ppGpp production. The Δrel mutant increased the sensitivity to environmental stress, exhibiting reduced growth on minimal media and a propensity to enter the VBNC state in response to CuSO4. These findings have important implications for the understanding of survival mechanism and management of C. michiganensis and other phytopathogenic bacteria.

Role of Penicillin-Binding Proteins in the Viability, Morphology, Stress Tolerance, and Pathogenicity of Clavibacter michiganensis.

Previous research has shown that penicillin-binding proteins (PBPs), enzymes involved in peptidoglycan (PG) assembly, could play an important role during the induction of the viable but nonculturable (VBNC) state, which allows non-spore-forming bacteria to survive adverse environmental conditions. The current study found that Clavibacter michiganensis has seven PBPs. Mutant analysis indicated that deletion of either of the class B PBPs was lethal and that the class A PBPs had an important role in PG synthesis, with the ΔpbpC mutant having an altered cellular morphology that resulted in longer cells that were swollen at one end and had thinner cell walls. The ΔpbpC mutant was also found to produce mucoid colonies in solid culture and a lower final cell titer in liquid medium, as well as having high sensitivity to osmotic stress and lysozyme treatment and surprisingly high pathogenicity. The double mutant, ΔdacB/ΔpbpE, also had a slightly altered phenotype, resulting in longer cells. Further analysis revealed that both mutants had high sensitivity to copper, which resulted in quicker induction into the VBNC state. However, only the ΔpbpC mutant had significantly reduced survivorship in the VBNC state. The study also confirmed that the VBNC state significantly improved the survivorship of wild-type C. michiganensis cells in response to environmental stresses and systemically demonstrated the protective role of the VBNC state in C. michiganensis, which is an important finding regarding its epidemiology and has serious implications for disease management.

Polycistronic Artificial microRNA-Mediated Resistance to Cucumber Green Mottle Mosaic Virus in Cucumber.

Cucumber green mottle mosaic virus (CGMMV), as a typical seed-borne virus, causes costly and devastating diseases in the vegetable trade worldwide. Genetic sources for resistance to CGMMV in cucurbits are limited, and environmentally safe approaches for curbing the accumulation and spread of seed-transmitted viruses and cultivating completely resistant plants are needed. Here, we describe the design and application of RNA interference-based technologies, containing artificial microRNA (amiRNA) and synthetic trans-acting small interfering RNA (syn-tasiRNA), against conserved regions of different strains of the CGMMV genome. We used a rapid transient sensor system to identify effective anti-CGMMV amiRNAs. A virus seed transmission assay was developed, showing that the externally added polycistronic amiRNA and syn-tasiRNA can successfully block the accumulation of CGMMV in cucumber, but different virulent strains exhibited distinct influences on the expression of amiRNA due to the activity of the RNA-silencing suppressor. We also established stable transgenic cucumber plants expressing polycistronic amiRNA, which conferred disease resistance against CGMMV, and no sequence mutation was observed in CGMMV. This study demonstrates that RNA interference-based technologies can effectively prevent the occurrence and accumulation of CGMMV. The results provide a basis to establish and fine-tune approaches to prevent and treat seed-based transmission viral infections.

Artificial microRNA-mediated resistance to cucumber green mottle mosaic virus in Nicotiana benthamiana.

MAIN CONCLUSION: We describe a Nicotiana benthamiana system for rapid identification of artificial microRNA (amiRNA) to control cucumber green mottle mosaic virus (CGMMV) disease. Although artificial miRNA technology has been used to control other viral diseases, it has not been applied to reduce severe cucumber green mottle mosaic virus (CGMMV) disease and crop loss in the economically important cucurbits. We used our system to identify three amiRNAs targeting CGMMV RNA (amiR1-CP, amiR4-MP and amiR6-Rep) and show that their expression reduces CGMMV replication and disease in virus-infected plants. This work streamlines the process of generating amiRNA virus-resistant crops and can be broadly applied to identify active antiviral amiRNAs against a broad spectrum of viruses to control disease in diverse crops.

Expression profiling and regulatory network of cucumber microRNAs and their putative target genes in response to cucumber green mottle mosaic virus infection.

Cucumber green mottle mosaic virus (CGMMV) is an important pathogen of cucumber (Cucumis sativus). The molecular mechanisms mediating host-pathogen interactions are likely to be strongly influenced by microRNAs (miRNAs), which are known to regulate gene expression during the disease cycle. This study focused on 14 miRNAs (miR159, miR169, miR172, miR838, miR854, miR5658, csa-miRn1-3p, csa-miRn2-3p, csa-miRn3-3p, csa-miRn4-5p, csa-miRn5-5p, csa-miRn6-3p, csa-miRn7-5p and csa-miRn8-3p) and their target genes. The data collected was used to construct a regulatory network of miRNAs and target genes associated with cucumber-CGMMV interactions, which identified 608 potential target genes associated with all of the miRNAs except csa-miRn7-5p. Five of the miRNAs (miR159, miR838, miR854, miR5658 and csa-miRn6-3p) were found to be mutually linked by target genes, while another eight (miR169, miR172, csa-miRn1-3p, csa-miRn2-3p, csa-miRn3-3p, csa-miRn4-5p, csa-miRn5-5p and csa-miRn8-3p) formed subnetworks that did not display any connectivity with other miRNAs or their target genes. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to analyze the expression levels of the different miRNAs and their putative target genes in leaf, stem and root samples of cucumber over a 42-day period after inoculation with CGMMV. A positive correlation was found between some of the miRNAs and their respective target genes, although for most, the response varied greatly depending on the time point, indicating that additional factors are likely to be involved in the interaction between cucumber miRNAs and their target genes. Several miRNAs, including miR159 and csa-miRn6-3p, were linked to target genes that have been associated with plant responses to disease. A model linking miRNAs, their targets and downstream biological processes is proposed to indicate the roles of these miRNAs in the cucumber-CGMMV pathosystem.

Variation in Streptomycin Resistance Mechanisms in Clavibacter michiganensis.

Clavibacter michiganensis is the causal agent of bacterial canker of tomato, which causes significant economic losses because of the lack of resistant tomato varieties. Chemical control with streptomycin or cupric bactericides is the last defensive line in canker disease management. Streptomycin is an aminoglycoside antibiotic that inhibits protein synthesis and targets the 30S ribosomal protein RpsL. Streptomycin has been used to control multiple plant bacterial diseases. However, identification and characterization of streptomycin resistance in C. michiganensis have remained unexplored. In this study, a naturally occurring C. michiganensis strain TX-0702 exhibiting spontaneous streptomycin resistance was identified, with a minimum inhibitory concentration of 128 µg/ml. Additionally, an induced streptomycin-resistant strain BT-0505-R was generated by experimental evolution of the sensitive C. michiganensis strain BT-0505. Genome sequencing and functional analyses were used to identify the genes conferring resistance. A point mutation at the 128th nucleotide in the rpsL gene of strain BT-0505-R is responsible for conferring streptomycin resistance. However, in TX-0702, resistance is not attributed to mutation of rpsL, streptomycin inactivation enzymes, or multidrug efflux pumps. The mechanism of resistance in TX-0702 is independent of previously reported bacterial loci. Taken together, these data highlight diverse mechanisms used by a Gram-positive plant pathogenic bacterium to confer antibiotic resistance.

Roles of Genotype-Determined Mycotoxins in Maize Seedling Blight Caused by Fusarium graminearum.

Fusarium graminearum is an important causal agent of maize seedling blight. The species includes several chemotypes that produce various forms of deoxynivalenol (DON) and nivalenol (NIV). To understand the effects and roles of F. graminearum mycotoxins on maize seedling blight occurring at Zhang Ye of Gansu, China, 23 isolates of F. graminearum were collected and characterized. A PCR assay showed all 23 isolates belonged to the 15-acetyldeoxynivalenol (15-ADON) genotype. This was also confirmed by production of both DON and 15-ADON in either rice culture medium or maize seedling roots, detected by high performance liquid chromatography and mass spectrometry. In maize seedling roots, 15-ADON dominated at 6 days post inoculation (dpi) and DON was the main mycotoxin at 12 dpi. The biomass of F. graminearum doubled from 6 to 12 dpi, and was positively correlated with virulence of the isolates. Both mycotoxins affected maize root vitality, but 15-ADON had a greater effect than DON. ALDH9 and MDH, two dehydrogenase synthesis genes in maize, showed a lower relative expression in 15-ADON treatments than in DON treatments. It indicated that both mycotoxins affected seed germination and root development, with 15-ADON being more destructive. Under scanning electron microscopy and transmission electron microscopy, root hair formation and development were delayed by DON, but completely inhibited by 15-ADON. 15-ADON caused cell shrinkage, loose cellular structure, and widened intercellular spaces; it also destroyed organelles and caused plasmolysis, and eventually ruptured cell membranes causing cell death. DON did not affect cell morphology and arrangement, but altered the morphology of organelles, forming concentric membranous bodies and a large amount of irregular lipid droplets. Thus, both mycotoxins contributed to symptom expression of maize seedling blight, but 15-ADON was more destructive than DON.

Quantitative proteomics identifies 38 proteins that are differentially expressed in cucumber in response to cucumber green mottle mosaic virus infection.

BACKGROUND: Since it was first reported in 1935, Cucumber green mottle mosaic virus (CGMMV) has become a serious pathogen in a range of cucurbit crops. The virus is generally transmitted by propagation materials, and to date no effective chemical or cultural methods of control have been developed to combat its spread. The current study presents a preliminary analysis of the pathogenic mechanisms from the perspective of protein expression levels in an infected cucumber host, with the objective of elucidating the infection process and potential strategies to reduce both the economic and yield losses associated with CGMMV. METHODS: Isobaric tags for relative and absolute quantitation (iTRAQ) technology coupled with liquid chromatography-tandem mass spectrometric (LC-MS/MS) were used to identify the differentially expressed proteins in cucumber plants infected with CGMMV compared with mock-inoculated plants. The functions of the proteins were deduced by functional annotation and their involvement in metabolic processes explored by KEGG pathway analysis to identify their interactions during CGMMV infection, while their in vivo expression was further verified by qPCR. RESULTS: Infection by CGMMV altered both the expression level and absolute quantity of 38 proteins (fold change >0.6) in cucumber hosts. Of these, 23 were found to be up-regulated, while 15 were down-regulated. Gene ontology (GO) analysis revealed that 22 of the proteins had a combined function and were associated with molecular function (MF), biological process (BP) and cellular component (CC). Several other proteins had a dual function with 1, 7, and 2 proteins being associated with BP/CC, BP/MF, CC/MF, respectively. The remaining 3 proteins were only involved in MF. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 18 proteins that were involved in 13 separate metabolic pathways. These pathways were subsequently merged to generate three network diagrams illustrating the interactions between the different pathways, while qPCR was used to track the changes in expression levels of the proteins identified at 3 time points during CGMMV infection. Taken together these results greatly expand our understanding of the relationships between CGMMV and cucumber hosts. CONCLUSIONS: The results of the study indicate that CGMMV infection significantly changes the physiology of cucumbers, affecting the expression levels of individual proteins as well as entire metabolic pathways. The bioinformatic analysis also identified several pathogenesis-related (PR) proteins that could be useful in the development of disease-resistant plants.

Novosphingobium fluoreni sp. nov., isolated from rice seeds.

A yellow-pigmented, Gram-stain-negative, rod-shaped, non-spore-forming bacterium designated strain HLJ-RS18(T), which could degrade fluorene, was isolated from rice seeds collected from Heilongjiang Province, China. Similarities of full-length of 16S rRNA gene sequences between strain HJL-RS18(T) and the type strains of the genus Novosphingobium with validly published names ranged from 93.8 to 97.1%. Phylogenetic analysis with maximum-likelihood and neighbour-joining methods revealed that strain HLJ-RS18(T) belonged to genus Novosphingobium and strain HLJ-RS18(T) formed a distinct clade to Novosphingobium chloroacetimidivorans BUT-14(T) (96.9% similarity based on 16S rRNA gene). DNA-DNA hybridization of HLJ-RS18(T) and BUT-14(T) showed a low relatedness value of 22.4 ± 0.9%, which indicated that strain HLJ-RS18(T) represents a novel species of the genus Novosphingobium. The genomic DNA G+C content of strain HLJ-RS18(T) was 62 mol%. Ubiquinone Q-10 was the major respiratory quinone. Spermidine was the predominant polyamine. Polar lipids consisted mainly of aminophospholipid, phosphatidylglycerol, phosphatidylethanolamine, phospholipid and sphingoglycolipid. The predominant fatty acid composition of HLJ-RS18(T) were summed 8 (C(18:1)ω7c and/or C(18:1)ω6c, 61.5%), C(16 : 0) (14.2%), summed 3 (C(16:1)ω7c and/or C(16:1)ω6c, 13.5%) and C14:0 2-OH (6.8%). Phylogenetic analysis, DNA-DNA hybridization, chemotaxonomic data and phenotypic characteristics support the conclusion that HLJ-RS18(T) represents a novel species within the genus Novosphingobium. Therefore, we propose the species Novosphingobium fluoreni sp. nov. with HLJ-RS18(T) ( = DSM 27568(T) = ACCC19180(T)) as the type strain.

The complete mitochondrial genome of Aglaia odorata, insights into its genomic structure and RNA editing sites.

Aglaia odorata, native to Guangdong, Guangxi, and Hainan provinces in China, has long been utilized as an herbal remedy in ancient China. In this study, we assembled and annotated the complete mitochondrial genome (mitogenome) of A. odorata, which spans a total length of 537,321 bp. Conformation of the A. odorata recombination was verified through PCR experiments and Sanger sequencing. We identified and annotated 35 protein-coding genes (PCGs), 22 tRNA genes, and 3 rRNA genes within the mitogenome. Analysis of repeated elements revealed the presence of 192 SSRs, 29 pairs of tandem repeats, and 333 pairs of dispersed repeats in the A. odorata mitogenome. Additionally, we analyzed codon usage and mitochondrial plastid DNAs (MTPTs). Twelve MTPTs between the plastome and mitogenome of A. odorata were identified, with a combined length of 2,501 bp, accounting for 0.47% of the mitogenome. Furthermore, 359 high-confidence C to U RNA editing sites were predicted on PCGs, and four selected RNA editing sites were specially examined to verify the creation of start and/or stop codons. Extensive genomic rearrangement was observed between A. odorata and related mitogenomes. Phylogenetic analysis based on mitochondrial PCGs were conducted to elucidate the evolutionary relationships between A. odorata and other angiosperms.

Identification and Pathogenicity of Pestalotioid Species on Alpinia oxyphylla in Hainan Province, China.

Alpinia oxyphylla is a traditional Chinese medicinal plant with a medicinal history of more than 1700 years. Ring leaf blight (RLB) disease, caused by pestalotioid species, is an important disease of A. oxyphylla, seriously affecting the yield and quality of its fruits. The causal agent of RLB disease has not been systematically identified or characterized yet. In this study, thirty-six pestalotioid strains were isolated from the leaves and stems of A. oxyphylla that was collected from six cities of Hainan province, China. Based on the multi-locus phylogeny (ITS, tef-1α and tub2) and morphological characteristic analyses, seventeen species belonging to three genera (Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis) were identified, and six new species (N. baotingensis, N. oblatespora, N. olivaceous, N. oxyphylla, N. wuzhishanensis and N. yongxunensis) were described. Pathogenicity tests revealed that strains of Neopestalotiopsis species caused more severe ring leaf blight on A. oxyphylla than strains of Pestalotiopsis and Pseudopestalotiopsis under wounded inoculation conditions.

A novel double-ribonuclease toxin-antitoxin system linked to the stress response and survival of Acidovorax citrulli.

IMPORTANCE: Bacterial fruit blotch (BFB), which is caused by the seed-borne bacterium Acidovorax citrulli, is a devastating disease affecting cucurbit crops throughout the world. Although seed fermentation and treatment with disinfectants can provide effective management of BFB, they cannot completely guarantee pathogen-free seedstock, which suggests that A. citrulli is a highly stress-resistant pathogen. Toxin-antitoxin (TA) systems are common among a diverse range of bacteria and have been reported to play a role in bacterial stress response. However, there is currently much debate about the relationship between TA systems and stress response in bacteria. The current study characterized a novel TA system (Aave_1720-Aave_1719) from A. citrulli that affects both biofilm formation and survival in response to sodium hypochlorite stress. The mechanism of neutralization differed from typical TA systems as two separate mechanisms were associated with the antitoxin, which exhibited characteristics of both type II and type V TA systems. The Aave_1720-Aave_1719 system described here also constitutes the first known report of a double-ribonuclease TA system in bacteria, which expands our understanding of the range of regulatory mechanisms utilized by bacterial TA systems, providing new insight into the survival of A. citrulli in response to stress.

Carrier-Free Small Molecular Self-Assembly Based on Berberine and Curcumin Incorporated in Submicron Particles for Improving Antimicrobial Activity.

Nanocarrier-based pesticide formulations have been severely restricted in agriculture practices due to their high-cost preparation process, poor loading capacity, and toxicity issues. To overcome these issues, carrier-free small molecular self-assembled submicron particles (SMPs) with an improved photoactivated antimicrobial activity based on two natural microbicides berberine hydrochloride (BBR) and curcumin (CM) are constructed by noncovalent interactions through a simple and fast preparation process (solvent exchange method) without using any adjuvant. The results show that the optimized molar ratio of BBR to CM is 2:1 at pH 5 and 25 °C in an aqueous solution for the formation of B-C SMPs. The obtained B-C SMPs exhibit excellent physicochemical properties, such as uniform morphology (407 nm), low polydispersity index (0.283), and strong ζ-potential (+24.4 mV). The antibacterial activities of B-C SMPs against Pseudomonas syringae pv. lachrymans, Clavibater michiganensis subsp. Michiganensis, and Sclerotinia sclerotiorum are 4, 2, and 1.5 times that of B + C MIX, respectively, suggesting a synergistic antimicrobial activity based on BBR and CM incorporation in the submicron particles. The genotoxicity evaluation results show that the self-assembled B-C SMPs are harmless to plant cells. Therefore, due to rational utilization of natural resources (natural microbicides, sunlight, and oxygen), carrier-free small molecular self-assembled B-C SMPs with synergistic photoactivated antimicrobial activity developed by a simple and fast preparation process would have great potential for sustainable plant disease management.

Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation.

Small alarmone hydrolases (SAHs) are alarmone metabolizing enzymes found in both metazoans and bacteria. In metazoans, the SAH homolog Mesh1 is reported to function in cofactor metabolism by hydrolyzing NADPH to NADH. In bacteria, SAHs are often identified in genomes with toxic alarmone synthetases for self-resistance. Here, we characterized a bacterial orphan SAH, i.e., without a toxic alarmone synthetase, in the phytopathogen Xanthomonas campestris pv. campestris (XccSAH) and found that it metabolizes both cellular alarmones and cofactors. In vitro, XccSAH displays abilities to hydrolyze multiple nucleotides, including pppGpp, ppGpp, pGpp, pppApp, and NADPH. In vivo, X. campestris pv. campestris cells lacking sah accumulated higher levels of cellular (pp)pGpp and NADPH compared to wild-type cells upon amino acid starvation. In addition, X. campestris pv. campestris mutants lacking sah were more sensitive to killing by Pseudomonas during interbacterial competition. Interestingly, loss of sah also resulted in reduced growth in amino acid-replete medium, a condition that did not induce (pp)pGpp or pppApp accumulation. Further metabolomic characterization revealed strong depletion of NADH levels in the X. campestris pv. campestris mutant lacking sah, suggesting that NADPH/NADH regulation is an evolutionarily conserved function of both bacterial and metazoan SAHs and Mesh1. Overall, our work demonstrates a regulatory role of bacterial SAHs as tuners of stress responses and metabolism, beyond functioning as antitoxins. IMPORTANCE Small alarmone hydrolases (SAHs) comprise a widespread family of alarmone metabolizing enzymes. In metazoans, SAHs have been reported to control multiple aspects of physiology and stress resistance through alarmone and NADPH metabolisms, but their physiological functions in bacteria is mostly uncharacterized except for a few reports as antitoxins. Here, we identified an SAH functioning independently of toxins in the phytopathogen Xanthomonas campestris pv. campestris. We found that XccSAH hydrolyzed multiple alarmones and NADPH in vitro, and X. campestris pv. campestris mutants lacking sah displayed increased alarmone levels during starvation, loss of interspecies competitive fitness, growth defects, and strong reduction in NADH. Our findings reveal the importance of NADPH hydrolysis by a bacterial SAH. Our work is also the first report of significant physiological roles of bacterial SAHs beyond functioning as antitoxins and suggests that SAHs have far broader physiological roles and share similar functions across domains of life.

Class A Penicillin-Binding Protein C Is Responsible for Stress Response by Regulation of Peptidoglycan Assembly in Clavibacter michiganensis.

The cell wall peptidoglycan of bacteria is essential for their survival and shape development. The penicillin-binding proteins (PBPs) are responsible for the terminal stage of peptidoglycan assembly. It has been shown that PBPC, a member of class A high-molecular-weight PBP, played an important role in morphology maintenance and stress response in Clavibacter michiganensis. Here, we reported the stress response strategies under viable but nonculturable (VBNC) state and revealed the regulation of peptidoglycan assembly by PBPC in C. michiganensis cells. Using atomic force microscopy imaging, we found that peptidoglycan of C. michiganensis cells displayed a relatively smooth and dense surface, whereas ΔpbpC was characterized by a "ridge-and-groove" surface, which was more distinctive after Cu2+ treatment. The peptidoglycan layer of wild type cells exhibited a significant increase in thickness and slight increase in cross-linkage following Cu2+ treatment. Compared with wild type, the thickness and cross-linkage of peptidoglycan decreased during log phase in ΔpbpC cells, but the peptidoglycan cross-linkage increased significantly under Cu2+ stress, while the thickness did not change. It is noteworthy that the above changes in the peptidoglycan layer resulted in a significant increase in the accumulation of amylase and exopolysaccharide in ΔpbpC. This study elucidates the role of PBPC in Gram-positive rod-shaped plant pathogenic bacterium in response to environmental stimuli by regulating the assembling of cell wall peptidoglycan, which is significant in understanding the survival of C. michiganensis under stress and the field epidemiology of tomato bacterial canker disease. IMPORTANCE Peptidoglycan of cell walls in bacteria is a cross-linked and meshlike scaffold that provides strength to withstand the external pressure. The increased cross-linkage in peptidoglycan and altered structure in VBNC cells endowed the cell wall more resistant to adversities. Here we systematically evaluated the stress response strategies in Gram-positive rod-shaped bacterium C. michiganensis log phase cells and revealed a significant increase of peptidoglycan thickness and slight increase of cross-linkage after Cu2+ treatment. Most strikingly, knocking-out of PBPC leads to a significant increase in cross-linking of peptidoglycan in response to Cu2+ treatment. Understanding the stress resistance mechanism and survival strategy of phytopathogenic bacteria is the basis of exploring bacterial physiology and disease epidemiology.