The vascular plant‐pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces

The mechanism of colonization of intercellular spaces by the soil‐borne and vascular plant‐pathogenic bacterium Ralstonia solanacearum strain OE1‐1 after invasion into host plants remains unclear. To analyse the behaviour of OE1‐1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1‐1 were observed under a scanning electron microscope. OE1‐1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1‐1 cells produced mushroom‐type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom‐type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1‐1. Mutation of lecM encoding a lectin, RS‐IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom‐type biofilms and virulence on tomato plants. Together, our findings indicate that OE1‐1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS‐IIL may contribute to biofilm formation by OE1‐1, which is required for OE1‐1 virulence.     

Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum

Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.     

Improved fruit α‐tocopherol,carotenoid, squalene and phytosterol contents through manipulation of Brassica juncea 3‐HYDROXY‐3‐METHYLGLUTARYL‐COA SYNTHASE1 in transgenic tomato

3‐Hydroxy‐3‐methylglutaryl‐coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild‐type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up‐regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10‐fold higher in vitro enzyme activity. Furthermore, tobacco HMGS overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE‐S359A over OE‐wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA‐derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)‐derived α‐tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS‐OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up‐regulated. In OE‐S359A tomato fruits, increased squalene and phytosterol contents over OE‐wtBjHMGS1 were attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE‐wtBjHMGS1 and OE‐S359A fruits, the up‐regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α‐tocopherol and carotenoid accumulation indicated cross‐talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health‐promoting squalene, phytosterols, α‐tocopherol and carotenoids in tomato, an edible fruit.     

Ⅲ型效应子GALA对青枯菌在两种寄主植物上致病性的影响

[目的]研究Ⅲ型效应子GALAs对青枯菌OE1-1在不同寄主植物致病性上的影响。[方法]构建青枯菌OE1-1的多种GALA缺失突变体,通过根切和叶片注射等方法研究GALAs对青枯菌OE1-1致病力和细胞内增殖能力的影响。[结果]GALA多基因缺失突变体对寄主烟草的致病力减弱,在烟草体内细菌繁殖能力较野生型明显降低,但在寄主番茄上不影响其致病性。[结论]GALA效应子对青枯菌OE1-1在烟草植株致病性上展现协同作用。     

Contribution of quorum‐sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1

Aims: To investigate roles of quorum‐sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin‐resistant variant (hereinafter DR1R). Methods and Results: The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane‐degradation and biofilm‐formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm‐formation and hexadecane‐biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild‐type cell supernatant and exogenous C₁₂‐AHL. Conclusions: The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. Significance and Impact of the Study: This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.     

应用竞争力指数分析III型效应子SKWP对青枯菌在寄主植物体内增殖能力的影响

[目的]研究Ⅲ型效应子SKWP对青枯菌OE1-1在寄主植物体内增殖能力的影响。[方法]构建青枯菌RK7197（野生型突变体,带Gm抗性）和SKWP单基因缺失突变体（带PB抗性）,通过竞争力指数分析SKWP各效应子对青枯菌OE1-1在叶片组织内增殖能力的影响。[结果]竞争力指数适合在寄主植物茄子上分析各效应子功能,6个SKWP效应子对OE1-1细菌增殖能力影响不同,SKWP4影响最明显。[结论]竞争力指数可提供一个新视野来分析SKWP各效应子对青枯菌OE1-1在寄主茄子上增殖能力的影响。     

An Amino Acid Substitution at Position 740 in σ70 of Ralstonia solanacearum Strain OE1-1 Affects Its In Planta Growth

Growth of Ralstonia solanacearum strain OE1-1 in roots after invasion is required for virulence. An Arg740Cys substitution in σ⁷⁰ of OE1-1 resulted in loss of in planta growth and virulence. The negative dominance of mutant σ⁷⁰ over the wild-type protein suggested that the amino acid substitution may affect the in planta growth of OE1-1, leading to a lack of virulence.     

Proteomic identification of potential target proteins regulated by the SCFFbp1‐mediated proteolysis pathway in Fusarium oxysporum

F‐box proteins function in the recruitment of proteins for SCF ubiquitination and proteasome degradation. Here, we studied the role of Fbp1, a nonessential F‐box protein of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. The Δfbp1 mutant showed a significant delay in the production of wilt symptoms on tomato plants and was impaired in invasive growth on cellophane membranes and on living plant tissue. To search for target proteins recruited by Fbp1, a combination of sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight (MALDI‐TOF/TOF) was used to compare proteins in mycelia of the wild‐type and Δfbp1 mutant. The proteomic approach identified 41 proteins differing significantly in abundance between the two strains, 17 of which were more abundant in the Δfbp1 mutant, suggesting a possible regulation by proteasome degradation. Interestingly, several of the identified proteins were related to vesicle trafficking. Microscopic analysis revealed an impairment of the Δfbp1 strain in directional growth and in the structure of the Spitzenkörper, suggesting a role of Fbp1 in hyphal orientation. Our results indicate that Fbp1 regulates protein turnover and pathogenicity in F. oxysporum.     

c‐di‐AMP modulates Listeria monocytogenes central metabolism to regulate growth,antibiotic resistance and osmoregulation

Cyclic diadenosine monophosphate (c‐di‐AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall‐active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the β‐lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl‐CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100‐fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c‐di‐AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down‐stream enzymes suppressed ΔdacA phenotypes. These data suggested that c‐di‐AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild‐type bacteria.     

Quorum Sensing Contributes to Seed‐to‐Seedling Transmission of Acidovorax citrulli on Watermelon

Based on the observation that Acidovorax citrulli switches from saprobic to pathogenic growth for seed‐to‐seedling transmission of bacterial fruit blotch of cucurbits (BFB), we hypothesized that quorum sensing (QS) was involved in the regulation of this process. Using aacI (luxI homologue) and aacR (luxR homologue) mutants of AAC00‐1, we investigated the role of QS in watermelon seed colonization and seed‐to‐seedling transmission of BFB. aacR and aacI mutants of AAC00‐1 colonized germinating watermelon seed at wild‐type levels; however, BFB seed‐to‐seedling transmission was affected in a cell density‐dependent manner. There were no significant differences in BFB seedling transmission between watermelon seed infiltrated with approximately 1 × 10⁶ CFU of AAC00‐1, the aacR or aacI deletion mutants (95.2, 94.9 and 98.3% BFB incidence, respectively). In contrast, when seed inoculum was reduced to approximately 1 × 10³ CFU/seed, BFB seed‐to‐seedling transmission declined to 34.3% for the aacI mutant, which was significantly less than the wild type (78.6%). Interestingly, BFB seed‐to‐seedling transmission for the aacR mutant was not significantly different to the wild‐type strain. These data suggest that QS plays a role in regulation of genes involved in seed‐to‐seedling transmission of BFB.     

The autophagy‐related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea

Autophagy, a ubiquitous intracellular degradation process, is conserved from yeasts to humans. It serves as a major survival function during nutrient depletion stress and is crucial for correct growth and differentiation. In this study, we characterized an atg1 orthologue Bcatg1 in the necrotrophic plant pathogen Botrytis cinerea. Quantitative real‐time polymerase chain reaction (qRT‐PCR) assays showed that the expression of BcATG1 was up‐regulated under carbon or nitrogen starvation conditions. BcATG1 could functionally restore the survival defects of the yeast ATG1 mutant during nitrogen starvation. Deletion of BcATG1 (ΔBcatg1) inhibited autophagosome accumulation in the vacuoles of nitrogen‐starved cells. ΔBcatg1 was dramatically impaired in vegetative growth, conidiation and sclerotial formation. In addition, most conidia of ΔBcatg1 lost the capacity to form the appressorium infection structure and failed to penetrate onion epidermis. Pathogenicity assays showed that the virulence of ΔBcatg1 on different host plant tissues was drastically impaired, which was consistent with its inability to form an appressorium. Moreover, lipid droplet accumulation was significantly reduced in the conidia of ΔBcatg1, but the glycerol content was increased. All of the defects of ΔBcatg1 were complemented by re‐introduction of an intact copy of the wild‐type BcATG1 into the mutant. These results indicate that BcATG1 plays a critical role in numerous developmental processes and is essential to the pathogenesis of B. cinerea.