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The MAP kinase high osmolarity glycerol 1 (Hog1) plays a central role in responding to external oxidative stress in budding yeast Saccchromyces cerevisiae. However, the downstream responsive elements regulated by Hog1 remain poorly understood. In this study, we report that a Sporisorium scitamineum ortholog of Hog1, named as SsHog1, induced transcriptional expression of a putative cytochrome P450 oxidoreductase encoding gene SsCPR1, to antagonize oxidative stress. We found that upon exposure to hydrogen peroxide (H2 O2 ), SsHog1 underwent strikingly phosphorylation, which was proved to be critical for transcriptional induction of SsCPR1. Loss of SsCPR1 led to hypersensitive to oxidative stress similar as the sshog1Δ mutant did, but was resistant to osmotic stress, which is different from the sshog1Δ mutant. On the other hand, overexpression of SsCPR1 in the sshog1Δ mutant could partially restore its ability of oxidative stress tolerance, which indicated that the Hog1 MAP kinase regulates the oxidative stress response specifically through cytochrome P450 (SsCpr1) pathway. Overall, our findings highlight a novel MAPK signaling pathway mediated by Hog1 in regulation of the oxidative stress response via the cytochrome P450 system, which plays an important role in host-fungus interaction. This article is protected by copyright. All rights reserved.

Bacteria can withstand various types of environmental osmostress. A sudden rise in osmostress affects bacterial cell growth that is countered by activating special genes. The change of osmostress is generally a slow process under the natural environment. However, the collective response of bacteria to low osmostress remains unknown. This study revealed that the deletion of

Many prokaryotes and eukaryotes utilize two-component signaling pathways to counter environmental stress and regulate virulence genes associated with infection. In this study, we identified and characterized a conserved histidine kinase (SsSln1), which is the sensor of the two-component system of Sln1–Ypd1–Ssk1 in Sporisorium scitamineum. SsSln1 null mutant exhibited enhanced mating and virulence capabilities in S. scitamineum, which is opposite to what has been reported in Candida albicans. Further investigations revealed that the deletion of SsSLN1 enhanced SsHog1 phosphorylation and nuclear localization and thus promoted S. scitamineum mating. Interestingly, SsSln1 and cAMP/PKA signaling pathways antagonistically regulated the transcription of pheromone-responsive transcription factor SsPrf1, for regulating S. scitamineum mating and virulence. In short, the study depicts a novel mechanism in which the cross-talk between SsSln1 and cAMP/PKA pathways antagonistically regulates mating and virulence by balancing the transcription of the SsPRF1 gene in S. scitamineum.

Significance Bacterial infection has been extensively investigated; however, little is known about how bacterial pathogens timely shut down infecting machinery after successful infections. Here, a previously unknown sucrose–SghR/SghA–SAG–SA signaling axis was identified which controls the timing to shut off bacterial virulence expression and fine-tune host immune response. Sucrose, salicylic acid (SA), and its storage form SAG are small chemicals produced in plants whereas SghR is a bacterial sensor of sucrose and SghA is a bacterial enzyme that releases SA from SAG. Given that SA is an imperative signaling molecule in defense against a variety of microbial pathogens, these results depict a previously unknown 2-way chemical signaling cross-talk during microbe–host coevolution and shed mechanistic insights into host–bacteria interaction., It is highly intriguing how bacterial pathogens can quickly shut down energy-costly infection machinery once successful infection is established. This study depicts that mutation of repressor SghR increases the expression of hydrolase SghA in Agrobacterium tumefaciens, which releases plant defense signal salicylic acid (SA) from its storage form SA β-glucoside (SAG). Addition of SA substantially reduces gene expression of bacterial virulence. Bacterial vir genes and sghA are differentially transcribed at early and later infection stages, respectively. Plant metabolite sucrose is a signal ligand that inactivates SghR and consequently induces sghA expression. Disruption of sghA leads to increased vir expression in planta and enhances tumor formation whereas mutation of sghR decreases vir expression and tumor formation. These results depict a remarkable mechanism by which A. tumefaciens taps on the reserved pool of plant signal SA to reprogram its virulence upon establishment of infection.

The basidiomycetous fungus Sporisorium scitamineum causes sugarcane smut that leads to severe economic losses in the major sugarcane growing areas in China, India and Brazil, etc. Autophagy is a conserved pathway in eukaryotes for bulk degradation and cellular recycling, and was shown to be important for fungal cell growth, development, and pathogenicity. However, physiological function of autophagy has not been studied in S. scitamineum. In this study, we identified a conserved Atg8 protein, named as SsAtg8 and characterized its function. Our results showed that autophagy was blocked in the ssatg8Δ mutant, in nitrogen starvation. The ssatg8Δ mutant formed pseudohypha frequently and was hypersensitive to oxidative stress. However, mating or filamenation was unaffected in the ssatg8Δ mutant in vitro. Overall we demonstrate that autophagy is dispensable for S. scitamineum mating/filamentation, while critical for oxidative stress tolerance and proper morphology in sporidial stage.

The plant pathogen Xanthomonas campestris pv. campestris produces diffusible signal factor (DSF) quorum sensing (QS) signals to regulate its biological functions and virulence. Our previous study showed that X. campestris pv. campestris utilizes host plant metabolites to enhance the biosynthesis of DSF family signals. However, it is unclear how X. campestris pv. campestris benefits from the metabolic products of the host plant. In this study, we observed that the host plant metabolites not only boosted the production of the DSF family signals but also modulated the expression levels of DSF-regulated genes in X. campestris pv. campestris. Infection with X. campestris pv. campestris induced changes in the expression of many sugar transporter genes in Arabidopsis thaliana. Exogenous addition of sucrose or glucose, which are the major products of photosynthesis in plants, enhanced DSF signal production and X. campestris pv. campestris pathogenicity in the Arabidopsis model. In addition, several sucrose hydrolase–encoding genes in X. campestris pv. campestris and sucrose invertase–encoding genes in the host plant were notably upregulated during the infection process. These enzymes hydrolyzed sucrose to glucose and fructose, and in trans expression of one of these enzymes, CINV1 of A. thaliana or XC_0805 of X. campestris pv. campestris, enhanced DSF signal biosynthesis in X. campestris pv. campestris in the presence of sucrose. Taken together, our findings demonstrate that X. campestris pv. campestris applies multiple strategies to utilize host plant sugars to enhance QS and pathogenicity.

In this study, we isolated an endophytic Burkholderia gladioli strain, named CGB10, from sugarcane leaves. B. gladioli CGB10 displayed strong inhibitory activity against filamentous growth of fungal pathogens, one of which is Sporisorium scitamineum that causes sugarcane smut, a major disease affecting the quality and production of sugarcane in tropical and subtropical regions. CGB10 could effectively suppress sugarcane smut under field conditions, without itself causing any obvious damage or disease, thus underscoring a great potential as a biocontrol agent (BCA) for the management of sugarcane smut. A toxoflavin biosynthesis and transport gene cluster potentially responsible for such antifungal activity was identified in the CGB10 genome. Additionally, a quorum-sensing gene cluster was identified too and compared with two close Burkholderia species, thus supporting an overall connection to the regulation of toxoflavin synthesis therein. Overall, this work describes the in vitro and field Sporisorium scitamineum biocontrol by a new B. gladioli strain, and reports genes and molecular mechanisms potentially involved.

Antibody repertoire sequencing enables researchers to acquire millions of B cell receptors and investigate these molecules at the single-nucleotide level. This power and resolution in studying humoral responses have led to its wide applications. However, most of these studies were conducted with a limited number of samples. Given the extraordinary diversity, assessment of these key features with a large sample set is demanded. Thus, we collect and systematically analyze 2,152 high-quality heavy-chain antibody repertoires. Our study reveals that 52 core variable genes universally contribute to more than 99% of each individual's repertoire; a distal interspersed preferences characterize V gene recombination; the number of public clones between two repertoires follows a linear model, and the positive selection dominates at RGYW motif in somatic hypermutations. Thus, this population-level analysis resolves some critical features of the antibody repertoire and may have significant value to the large cadre of scientists.

Antibody repertoire refers to the totality of the superbly diversified antibodies within an individual to cope with the vast array of possible pathogens. Despite this extreme diversity, antibodies of the same clonotype, namely public clones, have been discovered among individuals. Although some public clones could be explained by antibody convergence, public clones in naïve repertoire or virus-neutralizing clones from not infected people were also discovered. All these findings indicated that public clones might not occur by random and they might exert essential functions. However, the frequencies and functions of public clones in a population have never been studied. Here, we integrated 2,449 Rep-seq datasets from 767 donors and discovered 5.07 million public clones – ~10% of the repertoire are public in population. We found 38 therapeutic clones out of 3,390 annotated public clones including anti-PD1 clones in healthy people. Moreover, we also revealed clones neutralizing SARS-CoV-2, Ebola, and HIV-1 viruses in healthy individuals. Our result demonstrated that these clones are predisposed in the human antibody repertoire and may exert critical functions during particular immunological stimuli and consequently benefit the donors. We also implemented RAPID – aRep-seqAnalysisPlatform withIntegratedDatabases, which may serve as a useful tool for others in the field.

Background Xanthomonas citri pv. citri (Xcc) is a citrus canker causing Gram-negative bacteria. Currently, little is known about the biological and molecular responses of Xcc to low temperatures. Results Results depicted that low temperature significantly reduced growth and increased biofilm formation and unsaturated fatty acid (UFA) ratio in Xcc. At low temperature Xcc formed branching structured motility. Global transcriptome analysis revealed that low temperature modulates multiple signaling networks and essential cellular processes such as carbon, nitrogen and fatty acid metabolism in Xcc. Differential expression of genes associated with type IV pilus system and pathogenesis are important cellular adaptive responses of Xcc to cold stress. Conclusions Study provides clear insights into biological characteristics and genome-wide transcriptional analysis based molecular mechanism of Xcc in response to low temperature.

Antibody repertoire sequencing (Ig-seq) has been widely used in studying humoral responses, with promising results. However, the promise of Ig-seq has not yet been fully realized, and key features of the antibody repertoire remain elusive or controversial. To clarify these key features, we analyzed 2,152 high-quality heavy chain antibody repertoires, representing 582 donors and a total of 360 million clones. Our study revealed that individuals exhibit very similar gene usage patterns for germline V, D, and J genes and that 53 core V genes contribute to more than 99% of the heavy chain repertoire. We further found that genetic background is sufficient but not necessary to determine usage of V, D, and J genes. Although gene usage pattern is not affected by age, we observed a significant sex preference for 24 V genes, 9 D genes and 5 J genes, but found no positional bias for V-D and D-J recombination. In addition, we found that the number of observed clones that were shared between any two repertoires followed a linear model and noted that the mutability of hot/cold spots and single nucleotides within antibody genes suggested a strand-specific somatic hypermutation mechanism. This population-level analysis resolves some critical characteristics of the antibody repertoire and thus may serve as a reference for research aiming to unravel B cell-related biology or diseases. The metrics revealed here will be of significant value to the large cadre of scientists who study the antibody repertoire.

The basidiomycetous fungus Sporisorium scitamineum causes a serious sugarcane smut disease in major sugarcane growing areas. Sexual mating is essential for infection to the host; however, its underlying molecular mechanism has not been fully studied. In this study, we identified a conserved farnesyltransferase (FTase) β subunit Ram1 in S. scitamineum. The ram1Δ mutant displayed significantly reduced mating/filamentation, thus of weak pathogenicity to the host cane. The ram1Δ mutant sporidia showed more tolerant toward cell wall stressor Congo red compared to that of the wild-type. Transcriptional profiling showed that Congo red treatment resulted in notable up-regulation of the core genes involving in cell wall integrity pathway in ram1Δ sporidia compared with that of WT, indicating that Ram1 may be involved in cell wall integrity regulation. In yeast the heterodimeric FTase is responsible for post-translational modification of Ras (small G protein) and a-factor (pheromone). We also identified and characterized two conserved Ras proteins, Ras1 and Ras2, respectively, and a MAT-1 pheromone precursor Mfa1. The ras1Δ, ras2Δ and mfa1Δ mutants all displayed reduced mating/filamentation similar as the ram1Δ mutant. However, both ras1Δ and ras2Δ mutants were hypersensitive to Congo red while the mfa1Δ mutant was the same as wild-type. Overall our study displayed that RAM1 plays an essential role in S. scitamineum mating/filamentation, pathogenicity, and cell wall stability.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important bacterial diseases of citrus, impacting both plant growth and fruit quality. Identifying and elucidating the roles of genes associated with pathogenesis has aided our understanding of the molecular basis of citrus-bacteria interactions. However, the complex virulence mechanisms of X. citri subsp. citri are still not well understood. In this study, we characterized the role of PhoP in X. citri subsp. citri using a phoP deletion mutant, &Delta, phoP. Compared with wild-type strain XHG3, &Delta, phoP showed reduced motility, biofilm formation, as well as decreased production of cellulase, amylase, and polygalacturonase. In addition, the virulence of &Delta, phoP on citrus leaves was significantly decreased. To further understand the virulence mechanisms of X. citri subsp. citri, high-throughput RNA sequencing technology (RNA-Seq) was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 1017 differentially-expressed genes (DEGs), of which 614 were up-regulated and 403 were down-regulated in &Delta, phoP. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that the DEGs were enriched in flagellar assembly, two-component systems, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion systems. Our results showed that PhoP activates the expression of a large set of virulence genes, including 22 type III secretion system genes and 15 type III secretion system effector genes, as well as several genes involved in chemotaxis, and flagellar and histidine biosynthesis. Two-step reverse-transcription polymerase chain reaction analysis targeting 17 genes was used to validate the RNA-seq data, and confirmed that the expression of all 17 genes, except for that of virB1, decreased significantly. Our results suggest that PhoP interacts with a global signaling network to co-ordinate the expression of multiple virulence factors involved in modification and adaption to the host environment during infection.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important bacterial diseases of citrus, impacting both plant growth and fruit quality. Identifying and elucidating the roles of genes associated with pathogenesis has aided our understanding of the molecular basis of citrus-bacteria interactions. However, the complex virulence mechanisms of X. citri subsp. citri are still not well understood. In this study, we characterized the role of PhoP in X. citri subsp. citri using a phoP deletion mutant, ΔphoP. Compared with wild-type strain XHG3, ΔphoP showed reduced motility, biofilm formation, as well as decreased production of cellulase, amylase, and polygalacturonase. In addition, the virulence of ΔphoP on citrus leaves was significantly decreased. To further understand the virulence mechanisms of X. citri subsp. citri, high-throughput RNA sequencing technology (RNA-Seq) was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 1017 differentially-expressed genes (DEGs), of which 614 were up-regulated and 403 were down-regulated in ΔphoP. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that the DEGs were enriched in flagellar assembly, two-component systems, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion systems. Our results showed that PhoP activates the expression of a large set of virulence genes, including 22 type III secretion system genes and 15 type III secretion system effector genes, as well as several genes involved in chemotaxis, and flagellar and histidine biosynthesis. Two-step reverse-transcription polymerase chain reaction analysis targeting 17 genes was used to validate the RNA-seq data, and confirmed that the expression of all 17 genes, except for that of virB1, decreased significantly. Our results suggest that PhoP interacts with a global signaling network to co-ordinate the expression of multiple virulence factors involved in modification and adaption to the host environment during infection.

The life cycle of the sugarcane smut fungus Sporisorium scitamineum is a multistep process. Haploid sporidia of compatible (MAT-1 versus MAT-2) mating types fuse to generate pathogenic dikaryotic hyphae to infect the host. Within the host tissues, diploid teliospores are formed and induce a characteristic sorus that looks like a black whip. The diploid teliospores germinate to form haploid sporidia by meiosis. In order to monitor fungal development throughout the whole life cycle, we expressed the green fluorescent protein (GFP) and red fluorescent protein (RFP) in S. scitamineum MAT-1 and MAT-2 sporidia, respectively. Observation by epifluorescence microscope showed that conjugation tube formation and sporidia fusion occurred at 4 to 8 h, and formation of dikaryotic filaments was detected at 12 h after mating. The resultant teliospores, with diffused GFP and RFP, underwent meiosis as demonstrated by septated hypha with single fluorescent signal. We demonstrated that GFP- and RFP-tagged strains can be used to study the life cycle development of the fungal pathogen S. scitamineum, including the sexual mating and meiosis events. This dual-color imaging system would be a valuable tool for investigation of biotic and abiotic factors that might affect the fungal life cycle development and pathogenesis.

Zeamines are family of potent antibiotics and virulence determinants produced by the rice foot rot bacterial pathogen Dickeya zeae. So zeamines are important for the pathogenesis of D. zeae and development of new strategies against this devastating disease. In this study, we show that production of zeamines is positively modulated by ZmsO, which is a conserved Sfp-type phosphopantetheinyl thransferase (PPTase) associated with post-translational activation of fatty acid synthases and polyketide synthases. Deletion of zmsO significantly abolished zeamines production and attenuated the virulence of D. zeae without affect the growth rate. The zmsO gene is located at the upstream of zmsA and zmsK in the same gene cluster. Consistent with its role in post-translational modification, deletion of zmsO did not affect the transcriptional expression of zmsA and zmsK. In trans expression of the pcpS gene from Pseudomonas aeruginosa, which encodes a Sfp-type PPTase, in the zmsO deletion mutant could also fully restore the zeamines production and bacterial virulence, establishing the important role of Sfp-type PPTase in the zeamines biosynthesis and pathogenicity of D. zeae.

Dickeya zeae is a causal agent of rice root rot disease. The pathogen is known to produce a range of virulence factors, including phytotoxic zeamines and extracellular enzymes, but the mechanisms of virulence regulation remain vague. In this study, we identified a SlyA/MarR family transcription factor SlyA in D. zeae strain EC1. Disruption of slyA significantly decreased zeamine production, enhanced swimming and swarming motility, reduced biofilm formation and significantly decreased pathogenicity on rice. Quantitative polymerase chain reaction (qPCR) analysis confirmed the role of SlyA in transcriptional modulation of a range of genes associated with bacterial virulence. In trans expression of slyA in expI mutants recovered the phenotypes of motility and biofilm formation, suggesting that SlyA is downstream of the acylhomoserine lactone-mediated quorum sensing pathway. Taken together, the findings from this study unveil a key transcriptional regulatory factor involved in the modulation of virulence factor production and overall pathogenicity of D. zeae EC1.

Adolescence is an important period when people need adequate nutrition for healthy growth and development, whether from food or water. Tap water, bottled water (bottled purified water, and bottled natural water) are now the three most popular drinking waters consumed by adolescents in China. However, the constituents of them differ, which may cause different long-term health effects. In order to determine which type of water is the most beneficial regarding developmental behaviors, lipid metabolism and antioxidant capacity of juvenile zebrafish, 21 dpf (days post-fertilization) zebrafish were given these three waters separately with the same feed until 90 dpf. Results showed that zebrafish in purified water had the lowest survival rate, body weight and body length, while zebrafish in natural water and tap water had similar developmental behaviors; the highest HDL-C level and the lowest TG level were found in natural water, tap water second; and zebrafish in natural water showed the best antioxidant capacity. Thus, the best outcomes were found in natural water, which had suitable pH and proper amount of minerals, tap water also showed good performance, while purified water seemed not suitable for juvenile zebrafish.

Pseudomonas aeruginosa uses a hierarchical quorum sensing (QS) network consisting of las, pqs and rhl regulatory elements to coordinate the expression of bacterial virulence genes. However, clinical isolates frequently contain loss-of-function mutations in the central las system. This motivated us to search for a mechanism that may functionally substitute las. Here we report identification of a new QS signal, IQS. Disruption of IQS biosynthesis paralyzes the pqs and rhl QS systems and attenuates bacterial virulence. Production of IQS is tightly controlled by las under normal culture conditions but is also activated by phosphate limitation, a common stressor that bacteria encounter during infections. Thus, these results have established an integrated QS system that connects the central las system and phosphate-stress response mechanism to the downstream pqs and rhl regulatory systems. Our discovery highlights the complexity of QS signaling systems and extends the gamut of QS and stress-response mechanisms.

Brevibacterium aureum DG-12, a new bacterial strain isolated from active sludge, was able to degrade and utilize cyfluthrin as a growth substrate in the mineral medium. Response surface methodology using central composite rotatable design of cultural conditions was successfully employed for optimization resulting in 88.6% degradation of cyfluthrin (50mgL(-1)) within 5days. The bacterium degraded cyfluthrin by cleavage of both the carboxylester linkage and diaryl bond to form 2,2,3,3-tetramethyl-cyclopropanemethanol, 4-fluoro-3-phenexy-benzoic acid, 3,5-dimethoxy phenol, and phenol, and subsequently transformed these compounds with a maximum specific degradation rate, half-saturation constant and inhibition constant of 1.0384day(-1), 20.4967mgL(-1), and 141.9013mgL(-1), respectively. A novel degradation pathway for cyfluthrin was proposed based on analysis of these metabolites. In addition, this strain was found capable of degrading a wide range of synthetic pyrethroid insecticides. Our results suggest that B. aureum DG-12 may be an ideal microorganism for bioremediation of the pyrethroid-contaminated environments.

The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end-product of the shikimate pathway, to produce 3-hydroxybenzoic acid (3-HBA) and 4-HBA. 3-HBA and 4-HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3-HBA and 4-HBA biosynthesis via a unique mechanism with the C-terminal YjgF-like domain conferring activity for 3-HBA biosynthesis and the N-terminal FGFG motif-containing domain responsible for 4-HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate-pyruvate lyase UbiC. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria.

The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.

The Arabidopsis thaliana F-box protein CORONATINE INSENSITIVE1 (COI1) perceives jasmonate (JA) signals and subsequently targets the Jasmonate-ZIM domain proteins (JAZs) for degradation by the SCFCOI1-26S proteasome pathway to mediate various jasmonate-regulated processes, including fertility, root growth, anthocyanin accumulation, senescence, and defense. In this study, we screened JAZ-interacting proteins from an Arabidopsis cDNA library in the yeast two-hybrid system. MYB21 and MYB24, two R2R3-MYB transcription factors, were found to interact with JAZ1, JAZ8, and JAZ11 in yeast and in planta. Genetic and physiological experiments showed that the myb21 myb24 double mutant exhibited defects specifically in pollen maturation, anther dehiscence, and filament elongation leading to male sterility. Transgenic expression of MYB21 in the coi1-1 mutant was able to rescue male fertility partially but unable to recover JA-regulated root growth inhibition, anthocyanin accumulation, and plant defense. These results demonstrate that the R2R3-MYB transcription factors MYB21 and MYB24 function as direct targets of JAZs to regulate male fertility specifically. We speculate that JAZs interact with MYB21 and MYB24 to attenuate their transcriptional function; upon perception of JA signal, COI1 recruits JAZs to the SCFCOI1 complex for ubiquitination and degradation through the 26S proteasome; MYB21 and MYB24 are then released to activate expression of various genes essential for JA-regulated anther development and filament elongation.

Sporisorium scitamineum is the causal agent of sugarcane smut, which is one of the most serious constraints to global sugarcane production. S. scitamineum and Ustilago maydis are two closely related smut fungi, that are predicted to harbor similar sexual mating processes/system. To elucidate the molecular basis of sexual mating in S. scitamineum, we identified and deleted the ortholog of mating-specific U. maydis locus b, in S. scitamineum. The resultant b-deletion mutant was defective in mating and pathogenicity in S. scitamineum. Furthermore, a functional b locus heterodimer could trigger filamentous growth without mating in S. scitamineum, and functionally replace the b locus in U. maydis in terms of triggering aerial filament production and forming solopathogenic strains, which do not require sexual mating prior to pathogenicity on the host plants.

Cyhalothrin is a common environmental pollutant which poses increased risks to non-target organisms including human beings. This study reported for the first time a newly isolated strain, Bacillus thuringiensis ZS-19 completely degraded cyhalothrin in minimal medium within 72 h. The bacterium transformed cyhalothrin by cleavage of both the ester linkage and diaryl bond to yield six intermediate products. Moreover, a novel degradation pathway of cyhalothrin in strain ZS-19 was proposed on the basis of the identified metabolites. In addition to degradation of cyhalothrin, this strain was found to be capable of degrading 3-phenoxybenzoic acid, a common metabolite of pyrethroids. Furthermore, strain ZS-19 participated in efficient degradation of a wide range of pyrethroids including cyhalothrin, fenpropathrinn, deltamethrin, beta-cypermethrin, cyfluthrin and bifenthrin. Taken together, our results provide insights into the mechanism of cyhalothrin degradation and also highlight the promising potentials of B.thuringiensis ZS-19 in bioremediation of pyrethroid-contaminated environment. This is the first report of (i) degradation of cyhalothrin and other pyrethroids by B.thuringiensis, (ii) identification of 3-phenoxyphenyl acetonitrile and N-(2-isoproxy-phenyl)-4-phenoxy-benzamide as the metabolites in the degradation pathway of pyrethroids and (iii) a pathway of degradation of cyhalothrin by cleavage of both the ester linkage and diaryl bond in a microorganism.

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

The widely used insecticide fenpropathrin in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals, yet little is known about the kinetic and metabolic behaviors of this pesticide. This study reports the degradation kinetics and metabolic pathway of fenpropathrin in Bacillus sp. DG-02, previously isolated from the pyrethroid-manufacturing wastewater treatment system. Up to 93.3% of 50 mg L(-1) fenpropathrin was degraded by Bacillus sp. DG-02 within 72 h, and the degradation rate parameters qmax, Ks, and Ki were determined to be 0.05 h(-1), 9.0 mg L(-1), and 694.8 mg L(-1), respectively. Analysis of the degradation products by gas chromatography-mass spectrometry led to identification of seven metabolites of fenpropathrin, which suggest that fenpropathrin could be degraded first by cleavage of its carboxylester linkage and diaryl bond, followed by degradation of the aromatic ring and subsequent metabolism. In addition to degradation of fenpropathrin, this strain was also found to be capable of degrading a wide range of synthetic pyrethroids including deltamethrin, λ-cyhalothrin, β-cypermethrin, β-cyfluthrin, bifenthrin, and permethrin, which are also widely used insecticides with environmental contamination problems with the degradation process following the first-order kinetic model. Bioaugmentation of fenpropathrin-contaminated soils with strain DG-02 significantly enhanced the disappearance rate of fenpropathrin, and its half-life was sharply reduced in the soils. Taken together, these results depict the biodegradation mechanisms of fenpropathrin and also highlight the promising potentials of Bacillus sp. DG-02 in bioremediation of pyrethroid-contaminated soils.

Dickeya zeae is the causal agent of rice foot rot and maize stalk rot diseases, which could cause severe economic losses. The pathogen is known to produce two phytotoxins known as zeamine and zeamine II which are also potent antibiotics against both gram-positive and gram-negative bacteria pathogens. Zeamine II is a long-chain aminated polyketide and zeamine shares the same polyketide structure as zeamine II, with an extra valine derivative moiety conjugated to the primary amino group of zeamine II. In this study, we have identified a gene designated as zmsK encoding a putative nonribosomal peptide synthase (NRPS) by screening of the transposon mutants defective in zeamine production. Different from most known NRPS enzymes, which are commonly multidomain proteins, ZmsK contains only a condensation domain. High-performance liquid chromatography and mass spectrometry analyses showed that the ZmsK deletion mutant produced only zeamine II but not zeamine, suggesting that ZmsK catalyzes the amide bond formation by using zeamine II as a substrate to generate zeamine. We also present evidence that a partially conserved catalytic motif within the condensation domain is critical for zeamine production. Furthermore, we show that deletion of zmsK substantially decreased the total antimicrobial activity and virulence of D. zeae. Our findings provide a new insight into the biosynthesis pathway of zeamines and the virulence mechanisms of the bacterial pathogen D. zeae.

The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end-product of the shikimate pathway, to produce 3-hydroxybenzoic acid (3-HBA) and 4-HBA. 3-HBA and 4-HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3-HBA and 4-HBA biosynthesis via a unique mechanism with the C-terminal YjgF-like domain conferring activity for 3-HBA biosynthesis and the N-terminal FGFG motif-containing domain responsible for 4-HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate-pyruvate lyase UbiC. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria.

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis -2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF − mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.

Betaine homocysteine S-methyltransferase (BHMT, EC 2.1.1.5) is a key enzyme in the methionine cycle and is highly expressed in the liver. Despite its important biochemical function, it is not known whether BHMT plays a role during organ development. In this report, we showed that early in development of zebrafish before endoderm organogenesis, bhmt is first expressed in the yolk syncytial layer and then after liver formation becomes a liver-enriched gene. By using the anti-bhmt morpholinos that deplete the Bhmt, we found that in morphant embryos, several endoderm-derived organs, including liver, exocrine pancreas, and intestine are hypoplastic. Strikingly, the number of β-cells in the pancreatic islet was increased rather than reduced in the morphant. Additional studies showed that Bhmt depletion elevates the sonic hedgehog (shh) transcript level in the morphant, whereas Bhmt-depletion in the Shh-deficient mutant syu failed to rescue the isletless phenotype. These molecular and genetic data strongly suggest that Shh functions downstream of Bhmt to promote β-cell development. Therefore, although there are still many intriguing questions to be answered, our finding may identify a novel function for Bhmt involving modulation of Shh signaling to control β-cell development.