A transcriptional Regulator Gar Regulates the Growth and Virulence of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight, one of the most devastating diseases of rice. Pathogenic bacteria possess numerous transcriptional regulators to participate in the regulation of cellular processes. Here, we identified a transcriptional regulator Gar (PXO_RS11965) that is involved in regulating the growth and virulence of Xoo. Notably, the knockout of gar in Xoo enhanced bacterial virulence to the host rice. RNA-sequencing analysis and quantitative ß-glucuronidase (GUS) assay indicated that Gar positively regulates the expression of a σ54 factor rpoN2. Further experiments confirmed that overexpression of rpoN2 restored the phenotypic changes caused by gar deletion. Our research revealed that Gar influences bacterial growth and virulence by positively regulating the expression of rpoN2.

Population genomics and pathotypic evaluation of the bacterial leaf blight pathogen of rice reveals rapid evolutionary dynamics of a plant pathogen.

The Xanthomonas oryzae pv. oryzae (Xoo) is a bacterial pathogen causing bacterial blight disease in rice, resulting in significant yield reductions of up to 50% in rice production. Despite its serious threat to food production globally, knowledge of its population structure and virulence evolution is relatively limited. In this study, we employed whole-genome sequencing to explore the diversity and evolution of Xoo in the main rice-growing areas of China over the past 30 years. Using phylogenomic analysis, we revealed six lineages. CX-1 and CX-2 primarily contained Xoo isolates from South China, while CX-3 represented Xoo isolates from North China. Xoo isolates belonging to CX-5 and CX-6 were the most prevalent across all studied areas, persisting as dominant lineages for several decades. Recent sporadic disease outbreaks were primarily caused by Xoo isolates derived from the two major lineages, CX-5 and CX-6, although Xoo isolates from other lineages also contributed to these outbreaks. The lineage and sub-lineage distributions of Xoo isolates were strongly correlated with their geographical origin, which was found to be mainly determined by the planting of the two major rice subspecies, indica and japonica. Moreover, large-scale virulence testing was conducted to evaluate the diversity of pathogenicity for Xoo. We found rapid virulence evolution against rice, and its determinant factors included the genetic background of Xoo, rice resistance genes, and planting environment of rice. This study provides an excellent model for understanding the evolution and dynamics of plant pathogens in the context of their interactions with their hosts, which are shaped by a combination of geographical conditions and farming practices. The findings of this study may have important implications for the development of effective strategies for disease management and crop protection in rice production systems.

Directed evolution of glyphosate oxidase and a chemiluminescence system for glyphosate detection: A comprehensive practical laboratory experiment on biotechnology.

This article describes a comprehensive practical laboratory method for developing an enzyme to more easily measure glyphosate levels in solution. Through this article, undergraduate students of biology majors can conduct research experiments in critical fields by utilizing various techniques, such as chemiluminescence (CL) biosensors with engineered enzymes and are guided in molecular biology laboratories. A glyphosate oxidase mutant library was constructed by DNA shuffling, and a glyphosate oxidase variant with increased glyphosate degradation activity was selected by using a high-throughput screening assay. Following protein overexpression in Escherichia coli (DE3) and purification by affinity chromatography, the glyphosate oxidase variant protein combined with luminol-H2 O2 reaction was constructed as a new CL biosensor for detecting glyphosate in soils.

Transcriptional regulator Sar regulates the multiple secretion systems in Xanthomonas oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a notorious plant pathogen that causes leaf blight of rice cultivars. The pathogenic bacteria possess numerous transcriptional regulators to regulate various biological processes, such as pathogenicity in the host plant. Our previous study identified a new master regulator PXO_RS20790 that is involved in pathogenicity for Xoo against the host rice. However, the molecular functions of PXO_RS20790 are still unclear. Here, we demonstrate that transcriptional regulator Sar (PXO_RS20790) regulates multiple secretion systems. The RNA-sequencing analysis, bacterial one-hybrid assay, and electrophoretic mobility shift assay revealed that Sar enables binding of the promoters of the T1SS-related genes, the avirulence gene, raxX, and positively regulates these genes' expression. Meanwhile, we found that Sar positively regulated the T6SS-1 clusters but did not regulate the T6SS-2 clusters. Furthermore, we revealed that only T6SS-2 is involved in interbacterial competition. We also indicated that Sar could bind the promoters of the T3SS regulators, hrpG and hrpX, to activate these two genes' transcription. Our findings revealed that Sar is a crucial regulator of multiple secretion systems and virulence.

Heritability of tomato rhizobacteria resistant to Ralstonia solanacearum.

BACKGROUND: Ralstonia solanacearum (Rs) is a soilborne phytopathogen that causes bacterial wilt and substantial yield losses in many plants, such as tomatoes. A resistant tomato cultivar can recruit a beneficial microbiome from soil to resist Rs. However, whether this recruitment is inheritable from resistant parent to progeny has not been determined. RESULTS: In the present study, we investigated the rhizosphere microbiomes of tomatoes with clear kinship and different resistance against Rs. Resistant tomatoes grown with the additions of natural soil or its extract showed lower disease indexes than those grown in the sterile soil, demonstrating the importance of soil microbiome in resisting Rs. The results of 16S ribosomal RNA gene amplicon sequencing revealed that the resistant cultivars had more robust rhizosphere microbiomes than the susceptible ones. Besides, the resistant progeny HF12 resembled its resistant parent HG64 in the rhizosphere microbiome. The rhizosphere microbiome had functional consistency between HF12 and HG64 as revealed by metagenomics. Based on multi-omics analysis and experimental validation, two rhizobacteria (Sphingomonas sp. Cra20 and Pseudomonas putida KT2440) were enriched in HF12 and HG64 with the ability to offer susceptible tomatoes considerable protection against Rs. Multiple aspects were involved in the protection, including reducing the virulence-related genes of Rs and reshaping the transcriptomes of the susceptible tomatoes. CONCLUSIONS: We found promising bacteria to suppress the tomato bacterial wilt in sustainable agriculture. And our research provides insights into the heritability of Rs-resistant tomato rhizobacteria, echoing the inheritance of tomato genetic material. Video Abstract.

Precision Probiotics in Agroecosystems: Multiple Strategies of Native Soil Microbiotas for Conquering the Competitor Ralstonia solanacearum.

Ralstonia solanacearum (Rs), a soilborne phytopathogen, causes bacterial wilt disease in a broad range of hosts. Common approaches, for example, the direct reduction of the pathogen using classic single broad-spectrum probiotics, suffer from poor colonization efficiency, interference by resident microbiota, and nonnative-microorganism invasion. The soil microbiota plays an important role in plant health. Revealing the intrinsic linkage between the microbiome and the occurrence of disease and then applying it to agroecosystems for the precise control of soilborne diseases should be an effective strategy. Here, we surveyed the differences in the microbiome between healthy and diseased soils used for tomato planting across six climatic regions in China by using 16S rRNA amplicon and metagenomic sequencing. The roles of species associated with disease symptoms were further validated. Healthy soil possessed more diverse bacterial communities and more potential plant probiotics than diseased soil. Healthy soil simultaneously presented multiple strategies, including specifically antagonizing Rs, decreasing the gene expression of the type III secretion system of Rs, and competing for nutrition with Rs. Bacteria enriched in diseased samples promoted the progression of tomato bacterial wilt by strengthening the chemotaxis of pathogens. Therefore, Rs and its collaborators should be jointly combatted for disease suppression. Our research provides integrated insights into a multifaceted strategy for the biocontrol of tomato bacterial wilt based on the individual network of local microbiota. IMPORTANCE In the current work, the relationship between the soil microbiota and tomato bacterial wilt on a large scale offered us a comprehensive understanding of the disease. The delicate strategy of the microbiota in soil used for growing tomatoes to conquer the strong competitor, Rs, was revealed by microbiome research. The collaborators of Rs that coexist in a common niche with Rs strengthened our understanding of the pathogenesis of bacterial wilt. Bacteria enriched in healthy soil that antagonized pathogens with high specificity provide a novel view for ecofriendly probiotics mining. Our study offers new perspectives on soilborne-pathogen biocontrol in agroecosystems by decoding the rule of the natural ecosystem.

Elucidation of the Pathogenicity-Associated Regulatory Network in Xanthomonas oryzae pv. oryzae.

Xanthomonas is a notorious plant pathogen causing serious diseases in hundreds of plant hosts. Xanthomonas species are equipped with an array of signal transduction systems that regulate gene expression to survive in various harsh environments and successfully infect hosts. Although certain pathogenicity-associated regulators have been functionally characterized, signal transduction systems always function as a regulatory network which remains to be elucidated in Xanthomonas This study used a systematic approach to characterize all identified pathogenicity-associated regulators in Xanthomonas oryzae pv. oryzae (Xoo), including a transcriptional regulator with unknown function, and their interactive regulatory network. RNA sequencing was used in elucidating the patterns of the 10 pathogenicity-associated regulators identified. Results revealed that each pathogenicity-associated regulator has cross talk with others and all these regulators function as a regulatory network, with VemR and PXO_RS20790 being the master pathogenicity-associated regulators and HrpX being the final executant. Moreover, regulome analysis showed that numerous genes other than genes in pathogenicity islands are finely regulated within the regulatory network. Given that most of the pathogenicity-associated regulators are conserved in Xanthomonadales, our findings suggest a global network of gene regulation in this evolutionarily conserved pathogen. In conclusion, our study provides essential basic information about the regulatory network in Xoo, suggesting that this complicated regulatory network is one of the reasons for the robustness and fitness of Xanthomonas spp.IMPORTANCE The host plant infection process of pathogenic bacteria is a coordinating cellular behavior, which requires dynamic regulation at several levels in response to variations in host plants or fluctuations in the external environment. As one of the most important genera of plant-pathogenic bacteria, Xanthomonas has been studied as a model. Although certain pathogenicity-associated regulators have been functionally characterized, interactions among them remain to be elucidated. This study systematically characterized pathogenicity-associated regulators in Xoo and revealed that cross talk exists among pathogenicity-associated regulators and function as a regulatory network in which a hierarchy exists among the regulators. Our study elucidated the landscape of the pathogenicity-associated regulatory network in Xanthomonas, promoting understanding of the infection process of pathogenic bacteria.

Engineered glyphosate oxidase coupled to spore-based chemiluminescence system for glyphosate detection.

The extensive and intensive utilization of glyphosate (Glyp) caused public concerns on the potential risk of environment and health resulted from the chemical residues. Therefore, the development of a high-selective, low-cost and easy-operation Glyp detection methods is highly desired. Screening highly selective enzymes by directed evolution is important in practical applications. Herein, a glyphosate oxidase (GlypO) preferring substrate Glyp to produce H2O2 was obtained via directed evolution from glycine oxidase obtained from Bacillus cereus (BceGO). The catalytic efficiency, specificity constant, and affinity enhancement factor of GlypO toward Glyp were increased by 2.85 × 103-fold; 2.25 × 105-fold; and 9.64 × 104-fold, respectively, compared with those of BceGO. The catalytic efficiency toward glycine decreased by 78.60-fold. The spores of Bacillus subtilis (B. subtilis) effectively catalyzed luminol-H2O2 reaction to create excellent chemiluminescence (CL) signal because CotA-laccase exists on their surface. Based on these findings, a new CL biosensor via coupling to biological reaction system was presented for Glyp detection. The CL biosensor exhibited several advantages, such as eco-friendliness, low cost, high selectivity and sensitivity, and good practical application prospects for environmental pollution control.

In Silico Analysis Highlights the Diversity and Novelty of Circular Bacteriocins in Sequenced Microbial Genomes.

Consumer demand for "fresh food" with no chemical preservatives has prompted researchers to pay more attention to natural antimicrobial peptides such as bacteriocins. Nisin is currently the most widely used food biopreservative among the bacteriocins; however, its applications are restricted due to its low stability at neutral and alkaline pH values. Circular bacteriocins have potent antimicrobial activity against foodborne pathogens, show exceptional stability, and have great potential to be developed as biopreservatives. Here, we take advantage of the precursor peptides of 15 reported circular bacteriocins to devise an in silico approach to identify potential circular bacteriocins in sequenced microbial genomes. A total of nearly 7,000 putative precursor peptides were identified from 86 species of bacteria and further classified into 28 groups based on their amino acid similarity. Among the groups, 19 showed low similarity (less than 50%) to any known precursor peptide of circular bacteriocins. One novel circular bacteriocin in group 11, cerecyclin, showed the highest identity (34%) to the known circular bacteriocin enterocin NKR-5-3B and was selected for verification. Cerecyclin showed antimicrobial activity against several Gram-positive bacteria, inhibited the outgrowth of Bacillus cereus spores, and did not exhibit hemolysis activity. Moreover, it showed 4-fold- to 8-fold-higher antimicrobial activity against B. cereus and Listeria monocytogenes than nisin A. Cerecyclin also had increased stability compared to nisin A under neutral or alkaline conditions. This work not only identified a promising food biopreservative but also provided a rich source for novel circular bacteriocins.IMPORTANCE Circular bacteriocins are promising biopreservatives, and it is important to identify more novel circular bacteriocins to enhance the current arsenal of antimicrobials. In this study, we used an in silico approach to identify a large number of novel circular bacteriocins and classified these bacteriocins into 28 groups rather than the 2 groups that were described in previous studies. Nineteen groups were novel and had low similarity (less than 50%) to any known precursor peptides of circular bacteriocins; this finding greatly expands the awareness of the novelty and diversity of circular bacteriocins. A novel circular bacteriocin which we named cerecyclin was identified in the B. cereus group; this circular bacteriocin had great antimicrobial activity against some foodborne pathogens and showed extreme stability. This study not only identified a promising food biopreservative but also provided a rich source for the identification of novel circular bacteriocins and the development of new biopreservatives.

The Caenorhabditis elegans CUB-like-domain containing protein RBT-1 functions as a receptor for Bacillus thuringiensis Cry6Aa toxin.

Plant-parasitic nematodes cause huge agricultural economic losses. Two major families of Bacillus thuringiensis crystal proteins, Cry5 and Cry6, show nematicidal activity. Previous work showed that binding to midgut receptors is a limiting step in Cry toxin mode of action. In the case of Cry5Ba, certain Caenorhabditis elegans glycolipids were identified as receptors of this toxin. However, the receptors for Cry6 toxin remain unknown. In this study, the C. elegans CUB-like-domain containing protein RBT-1, released by phosphatidylinositol-specific phospholipase C (PI-PLC), was identified as a Cry6Aa binding protein by affinity chromatography. RBT-1 contained a predicted glycosylphosphatidylinositol (GPI) anchor site and was shown to locate in lipid rafts in the surface of the midgut cells. Western ligand blot assays and ELISA binding analysis confirmed the binding interaction between Cry6Aa and RBT-1 showing high affinity and specificity. In addition, the mutation of rbt-1 gene decreased the susceptibility of C. elegans to Cry6Aa but not that of Cry5Ba. Furthermore, RBT-1 mediated the uptake of Cry6Aa into C. elegans gut cells, and was shown to be involved in triggering pore-formation activity, indicating that RBT-1 is required for the interaction of Cry6Aa with the nematode midgut cells. These results support that RBT-1 is a functional receptor for Cry6Aa.

Enrichment of potentially beneficial bacteria from the consistent microbial community confers canker resistance on tomato.

The soil microbiota interacts with plants closely and exerts strong influences on plant health and productivity. However, the relationship between the microbiota and the bacterial canker of tomato that is caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is still unclear. In order to establish causal relationship between the microbiota and plant phenotypes, the microbial communities of 49 tomato samples (including 15 cultivars) with different canker symptoms collected from the greenhouse in Gansu province, China were investigated via 16S ribosomal RNA sequencing. Roots exhibited a strong filter effect in the process of root colonization by microorganisms according to the α-diversity and the separation patterns of the microbiota in bulk soil, rhizosphere and endosphere. In addition, the gradually decreased cluster extent from bulk soil to endosphere indicating the selective effect of tomato on microbiota. Although the composition of the microbiota is similar, the potential beneficial bacteria and functions (e.g. antibiotics production, pollution degradation, nutrition acquisition) enriched in the rhizosphere and endosphere of healthy samples compared to those in the diseased ones. Furthermore, more robust networks occurred in the rhizosphere and endosphere of healthy samples compared to the diseased ones. Our research provided substantial evidence that although the plant genotype is the dominant factor of phenotype, the rhizosphere and endosphere microbiota, as part of phytobiomes or holobiont, could contribute to the host's phenotype. This causal relationship between microbiota and host phenotypes could guide us in rationally designing novel synthetic communities (SynComs) for tomato canker biocontrol in the near future.

Endophyte Bacillus subtilis evade plant defense by producing lantibiotic subtilomycin to mask self-produced flagellin.

Microbes can enter into healthy plants as endophytes and confer beneficial functions. The entry of commensal microbes into plants involves penetrating plant defense. Most mechanisms about overcoming plant defense are focused on adapted pathogens, while the mechanism involved in beneficial endophyte evades plant defense to achieve harmonious commensalism is unclear. Here, we discover a mechanism that an endophyte bacterium Bacillus subtilis BSn5 reduce to stimulate the plant defensive response by producing lantibiotic subtilomycin to bind self-produced flagellin. Subtilomycin bind with flagellin and affect flg22-induced plant defense, by which means promotes the endophytic colonization in A. thaliana. Subtilomycin also promotes the BSn5 colonization in a distinct plant, Amorphophallus konjac, where the BSn5 was isolated. Our investigation shows more independent subtilomycin/-like producers are isolated from distinct plants. Our work unveils a common strategy that is used for bacterial endophytic colonization.

Single Amino Acid Substitution in Homogentisate Dioxygenase Affects Melanin Production in Bacillus thuringiensis.

Bacillus thuringiensis formulation losing its activity under field conditions due to UV radiation and photoprotection of B. thuringiensis based on melanin has attracted the attention of researchers for many years. Here, a single amino acid substitution (G272E) in homogentisate 1,2-dioxygenase was found to be responsible for pigment overproduction in B. thuringiensis BMB181, a derivative of BMB171. Disrupting the gene encoding homogentisate dioxygenase in BMB171 induced the accumulation of the homogentisic acid and provoked an increased pigment formation. To gain insights into homogentisate 1,2-dioxygenase in B. thuringiensis, we constructed a total of 14 mutations with a single amino acid substitution, and six of the mutant proteins were found to affect the melanin production when substituted by alanine. This study provides a new way to construct pigment-overproducing strains by impairing the homogentisate dioxygenase with a single mutation in B. thuringiensis, and the findings will facilitate a better understanding of this enzyme.

Bacillus thuringiensis produces the lipopeptide thumolycin to antagonize microbes and nematodes.

Bacillus thuringiensis has been widely used as a bio-insecticide. However, novel biological activities other than insect toxicity of B. thuringiensis are still underestimated. In this study, a new lipopeptide biosynthesis gene cluster in B. thuringiensis BMB171 was discovered by genome mining and verified by reverse genetics. Thumolycin, the lipopeptide synthesized by this gene cluster, was then isolated and purified. Mass spectrum analysis revealed the molecular mass of thumolycin is 696.51 Da with the predicted molecular formula of C38H64N8O4. Further bioactivities assay showed that thumolycin endowed B. thuringiensis BMB171 with broad spectrum antimicrobial and nematocidal activities.

Isolation and characterization of a novel phage Xoo-sp2 that infects Xanthomonas oryzae pv. oryzae.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a serious bacterial disease in rice-growing regions worldwide. Phage therapy has been proposed as a potential measure to treat bacterial infections. In this study, a novel phage, Xoo-sp2, which infects Xoo was isolated from soil. The characteristics of Xoo-sp2, including the morphology, one-step growth curve and host range, were analysed. The genome of phage Xoo-sp2 was sequenced and annotated. The results demonstrated that Xoo-sp2 is a siphovirus and has a broad lytic spectrum, infecting 9 out of 10 representative Xoo strains. Genome analysis showed that the Xoo-sp2 genome consists of a linear double-stranded DNA molecule of length 60 370 bp. Annotation of the whole genome indicated that Xoo-sp2 encodes 79 putative open reading frames (ORFs). Comparative genomics analysis of Xoo-sp2 showed that it shares significant similarity only with Pseudomonas and Stenotrophomonas phages (with maximum identity reaching 80 % along 69 % of the genome), and thus represents a novel Xanthomonas phage. Xoo-sp2 significantly inhibited Xoo growth in liquid culture. An experiment with potted plants indicated that Xoo-sp2 could efficiently control BLB in living rice. In summary, our work characterized a novel Xanthomonas phage and demonstrated its potential as a prophylactic agent in the control of BLB in rice.

Activation of PhoBR under phosphate-rich conditions reduces the virulence of Xanthomonas oryzae pv. oryzae.

The two-component signal transduction system PhoBR regulates the adaptation to phosphate limitation and the virulence of many animal bacterial pathogens. However, PhoBR in phytopathogens has rarely been investigated. In this study, we found that PhoBR in Xanthomonas oryzae pv. oryzae (Xoo), the pathogen of rice bacterial leaf blight, also regulates the adaptation to phosphate starvation. Unexpectedly, rice leaves infected by the phoBR-deleted mutant and wild-type PXO99A showed similar lesions, indicating that PhoBR is unnecessary for the virulence of Xoo. phoBR was found to be silenced during host infection, whereas artificially constitutive PhoBR expression attenuated virulence on host rice and growth in phosphate-rich media. RNA-sequencing (RNA-seq) was then performed to investigate the global effect caused by constitutive PhoBR activation. RNA-seq and further experiments revealed that the PhoBR regulon in Xoo comprised a wide range of genes. Nutrient transport and metabolism readjustments that resulted from PhoBR regulon activation may be responsible for growth attenuation. Our findings suggest that growth reduction regulated by PhoBR is a fitness cost of adaptation to phosphate starvation. PhoBR in Xoo is activated under phosphate-limited conditions, which could exist in epiphytic and saprophytic surviving phases, and is strictly repressed within phosphate-rich host plants to minimize fitness costs.

Comparative Genomics of Bacillus thuringiensis Reveals a Path to Specialized Exploitation of Multiple Invertebrate Hosts.

Understanding the genetic basis of host shifts is a key genomic question for pathogen and parasite biology. The Bacillus cereus group, which encompasses Bacillus thuringiensis and Bacillus anthracis, contains pathogens that can infect insects, nematodes, and vertebrates. Since the target range of the essential virulence factors (Cry toxins) and many isolates is well known, this group presents a powerful system for investigating how pathogens can diversify and adapt to phylogenetically distant hosts. Specialization to exploit insects occurs at the level of the major clade and is associated with substantial changes in the core genome, and host switching between insect orders has occurred repeatedly within subclades. The transfer of plasmids with linked cry genes may account for much of the adaptation to particular insect orders, and network analysis implies that host specialization has produced strong associations between key toxin genes with similar targets. Analysis of the distribution of plasmid minireplicons shows that plasmids with orf156 and orf157, which carry genes encoding toxins against Lepidoptera or Diptera, were contained only by B. thuringiensis in the specialized insect clade (clade 2), indicating that tight genome/plasmid associations have been important in adaptation to invertebrate hosts. Moreover, the accumulation of multiple virulence factors on transposable elements suggests that cotransfer of diverse virulence factors is advantageous in terms of expanding the insecticidal spectrum, overcoming insect resistance, or through gains in pathogenicity via synergistic interactions between toxins.IMPORTANCE Population genomics have provided many new insights into the formation, evolution, and dynamics of bacterial pathogens of humans and other higher animals, but these pathogens usually have very narrow host ranges. As a pathogen of insects and nematodes, Bacillus thuringiensis, which produces toxins showing toxicity to many orders of insects and other invertebrates, can be used as a model to study the evolution of pathogens with wide host ranges. Phylogenomic analysis revealed that host specialization and switching occur at the level of the major clade and subclade, respectively. A toxin gene co-occurrence network indicates that multiple toxins with similar targets were accumulated by the same cell in the whole species. This accumulation may be one of the strategies that B. thuringiensis has used to fight against host resistance. This kind of formation and evolution of pathogens represents a different path used against multiple invertebrate hosts from that used against higher animals.

Dissimilar Crystal Proteins Cry5Ca1 and Cry5Da1 Synergistically Act against Meloidogyne incognita and Delay Cry5Ba-Based Nematode Resistance.

Cry proteins of Bacillus thuringiensis (Bt) have been successfully used as biopesticides and in transgenic crops throughout the world. However, resources against the most serious agricultural pathogens, plant root-knot nematodes, are limited. The genomes of several highly nematicidal virulent Bt strains from our laboratory have been sequenced, facilitating the identification of novel Cry proteins and other virulence factors. We identified two novel Cry proteins, Cry5Ca1 and Cry5Da1, that exhibit high toxicity against Meloidogyne incognita Using the Caenorhabditis elegans model, the two Cry5 toxins were shown to negatively affect nematode life span, fertility, and survival. The 50% lethal concentrations (LC50s) of Cry5Ca1 and Cry5Da1 were 57.22 µg/ml and 36.69 µg/ml, respectively. Moreover, a synergistic effect (synergism factor, 1.61 to 2.04) was observed for nematicidal toxicity of Cry5Ca1 and Cry5Da1, which is accordant with the phylogenetic results suggesting that domain II of the two novel Cry5 toxins evolved into two independent clades. Through comparison of the depressed degree of toxicity in the ß-methylgalactoside detoxification test, we found that the novel toxin Cry5D possesses a different galactose-binding epitope; meanwhile, the finding that Cry5D does not share a motif (GXXXE) in the corresponding loop of domain II with Cry5B could explain the different galactose binding performance. Additionally, low-level cross-resistance of C. elegans bre mutant strains was evident between Cry5B and Cry5D. These results suggest that Cry5D can be used as an alternative to delay the potential resistance of nematodes to Cry5B.IMPORTANCE Although proper gene resources for Bt crops against the most serious agricultural pathogens, plant root-knot nematodes, are limited, we have identified two novel nematicidal toxins, Cry5Ca1 and Cry5Da1, against M. incognita, which have supplied more gene candidates for Bt crops designed against nematodes. Moreover, the association of the dissimilarity between Cry5Da1 and Cry5Ba1 and their low cross-resistance can be attributed not only to a low sequence similarity of domain II but also to the structural difference of the key motif and receptor-binding epitope in the loops. This association facilitates the selection of a proper candidate for the prospective design of pyramided Bt crops that can delay potential resistance.

The global strategy employed by Xanthomonas oryzae pv. oryzae to conquer low-oxygen tension.

Xanthomonas oryzae pv. oryzae (Xoo) is a notorious rice pathogen that causes bacterial leaf blight (BLB), a destructive rice disease. Low-oxygen tension in the xylem vessels of rice stresses Xoo during infection. In this study, differentially expressed proteins under normoxic and hypoxic conditions were identified using high-performance liquid chromatography (HPLC) coupled with LC-MS/MS to investigate the global effects of low oxygen environment on Xoo PXO99A. A statistically validated list of 187 (normoxia) and 140 (hypoxia) proteins with functional assignments was generated, allowing the reconstruction of central metabolic pathways. Ten proteins involved in aromatic amino acid biosynthesis, glycolysis, butanoate metabolism, propanoate metabolism and biological adhesion were significantly modulated under low-oxygen tension. The genes encoded by these proteins were in-frame deleted, and three of them were determined to be required for full virulence in Xoo. The contributions of these three genes to important virulence-associated functions, including extracellular polysaccharide, cell motility and antioxidative ability, are presented. BIOLOGICAL SIGNIFICANCE: To study how Xanthomonas oryzae pv. oryzae (Xoo) conquers low-oxygen tension in the xylem of rice, we identified differentially expressed proteins under normoxic and hypoxia. We found 140 proteins that uniquely expressed under the hypoxia were involved in 33 metabolism pathways. We identified 3 proteins were required for full virulence in Xoo and related to the ability of extracellular polysaccharide, cell motility, and antioxidative. This study is helpful for broadening our knowledge of the metabolism processed of Xoo in the xylem of rice.

Genetic and Biochemical Characterization of a Gene Operon for trans-Aconitic Acid, a Novel Nematicide from Bacillus thuringiensis.

trans-Aconitic acid (TAA) is an isomer of cis-aconitic acid (CAA), an intermediate of the tricarboxylic acid cycle that is synthesized by aconitase. Although TAA production has been detected in bacteria and plants for many years and is known to be a potent inhibitor of aconitase, its biosynthetic origins and the physiological relevance of its activity have remained unclear. We have serendipitously uncovered key information relevant to both of these questions. Specifically, in a search for novel nematicidal factors from Bacillus thuringiensis, a significant nematode pathogen harboring many protein virulence factors, we discovered a high yielding component that showed activity against the plant-parasitic nematode Meloidogyne incognita and surprisingly identified it as TAA. Comparison with CAA, which displayed a much weaker nematicidal effect, suggested that TAA is specifically synthesized by B. thuringiensis as a virulence factor. Analysis of mutants deficient in plasmids that were anticipated to encode virulence factors allowed us to isolate a TAA biosynthesis-related (tbr) operon consisting of two genes, tbrA and tbrB We expressed the corresponding proteins, TbrA and TbrB, and characterized them as an aconitate isomerase and TAA transporter, respectively. Bioinformatics analysis of the TAA biosynthetic gene cluster revealed the association of the TAA genes with transposable elements relevant for horizontal gene transfer as well as a distribution across B. cereus bacteria and other B. thuringiensis strains, suggesting a general role for TAA in the interactions of B. cereus group bacteria with nematode hosts in the soil environment. This study reveals new bioactivity for TAA and the TAA biosynthetic pathway, improving our understanding of virulence factors employed by B. thuringiensis pathogenesis and providing potential implications for nematode management applications.