Milestones in understanding transport, sensing, and signaling of the plant nutrient phosphorus.

As an essential nutrient element, phosphorus (P) is primarily acquired and translocated as inorganic phosphate (Pi) by plant roots. Pi is often sequestered in the soil and becomes limited for plant growth. Plants have developed a sophisticated array of adaptive responses, termed P starvation responses, to cope with P deficiency by improving its external acquisition and internal utilization. Over the past 2 to 3 decades, remarkable progress has been made toward understanding how plants sense and respond to changing environmental P. This review provides an overview of the molecular mechanisms that regulate or coordinate P starvation responses, emphasizing P transport, sensing, and signaling. We present the major players and regulators responsible for Pi uptake and translocation. We then introduce how P is perceived at the root tip, how systemic P signaling is operated, and the mechanisms by which the intracellular P status is sensed and conveyed. Additionally, the recent exciting findings about the influence of P on plant-microbe interactions are highlighted. Finally, the challenges and prospects concerning the interplay between P and other nutrients and strategies to enhance P utilization efficiency are discussed. Insights obtained from this knowledge may guide future research endeavors in sustainable agriculture.

Functional characterization and transcriptional analysis of degQ of Xanthomonas campestris pathovar campestris.

High-temperature-requirement protein A (HtrA) family proteins play important roles in controlling protein quality and are recognized as virulence factors in numerous animal and human bacterial pathogens. The role of HtrA family proteins in plant pathogens remains largely unexplored. Here, we investigated the HtrA family protein, DegQ, in the crucifer black rot pathogen Xanthomonas campestris pathovar campestris (Xcc). DegQ is essential for bacterial attachment and full virulence of Xcc. Moreover, the degQ mutant strain showed increased sensitivity to heat treatment and sodium dodecyl sulfate. Expressing the intact degQ gene in trans in the degQ mutant could reverse the observed phenotypic changes. In addition, we demonstrated that the DegQ protein exhibited chaperone-like activity. Transcriptional analysis displayed that degQ expression was induced under heat treatment. Our results contribute to understanding the function and expression of DegQ of Xcc for the first time and provide a novel perspective about HtrA family proteins in plant pathogen.

The lolB gene in Xanthomonas campestris pv. campestris is required for bacterial attachment, stress tolerance, and virulence.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence. RESULTS: To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation. CONCLUSIONS: The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria.

Expression and function of clpS and clpA in Xanthomonas campestris pv. campestris.

ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in numerous regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen that can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris has several clp genes, namely, clpS, clpA, clpX, clpP, clpQ, and clpY. Among these genes, only clpX and clpP is known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex. Transcriptional analysis revealed that the expression of clpS and clpA was growth phase-dependent and affected by the growth temperature. The inactivation of clpA, but not of clpS, resulted in susceptibility to high temperature and attenuated virulence in the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 were the amino acid residues critical for ClpA function in heat tolerance. The protein expression profile shown by the clpA mutant in response to heat stress was different from that exhibited by the wild type. In summary, we characterized two clp genes (clpS and clpA) by examining their expression profiles and functions in different processes, including stress tolerance and pathogenicity. We demonstrated that clpS and clpA were expressed in a temperature-dependent manner and that clpA was required for the survival at high temperature and full virulence of X. campestris pv. campestris. This work represents the first time that clpS and clpA were characterized in Xanthomonas.

The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

BACKGROUND: Although native to North America, the invasion of the aphid-like grape phylloxera Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome of D. vitifoliae as a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture. RESULTS: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world. CONCLUSIONS: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture.

Correction to: The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

An amendment to this paper has been published and can be accessed via the original article.

The clpX gene plays an important role in bacterial attachment, stress tolerance, and virulence in Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris is a bacterial pathogen and the causal agent of black rot in crucifers. In this study, a clpX mutant was obtained by EZ-Tn5 transposon mutagenesis of the X. campestris pv. campestris. The clpX gene was annotated to encode ClpX, the ATP-binding subunit of ATP-dependent Clp protease. The clpX mutant exhibited reduced bacterial attachment, extracellular enzyme production and virulence. Mutation of clpX also resulted in increased sensitivity to a myriad of stresses, including heat, puromycin, and sodium dodecyl sulfate. These altered phenotypes of the clpX mutant could be restored to wild-type levels by in trans expression of the intact clpX gene. Proteomic analysis revealed that the expression of 211 proteins differed not less than twofold between the wild-type and mutant strains. Cluster of orthologous group analysis revealed that these proteins are mainly involved in metabolism, cell wall biogenesis, chaperone, and signal transduction. The reverse transcription quantitative real-time polymerase chain reaction analysis demonstrated that the expression of genes encoding attachment-related proteins, extracellular enzymes, and virulence-associated proteins was reduced after clpX mutation. The results in this study contribute to the functional understanding of the role of clpX in Xanthomonas for the first time, and extend new insights into the function of clpX in bacteria.

Functional Characterization and Transcriptional Analysis of clpP of Xanthomonas campestris pv. campestris.

The caseinolytic protease (Clp) system is essential for survival under stress conditions and for virulence in several pathogenic bacteria. Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen which causes black rot disease in crucifers. In this study, the Xcc clpP gene which is annotated to encode the proteolytic core of Clp was characterized. Mutation of clpP resulted in susceptibility to high temperature and puromycin stresses. Site-directed mutagenesis revealed that S105, H130, and D179 are critical amino acid residues for ClpP function in puromycin tolerance. Inactivation of clpP also revealed an attenuation of virulence on the host plant and a reduction in the production of extracellular cellulase, mannanase, pectinase, and protease. The affected phenotypes of the clpP mutant could be complemented to wild-type levels by the intact clpP gene. Transcriptional analysis revealed that expression of clpP is induced under heat shock condition.

Lipoteichoic Acid Accelerates Bone Healing by Enhancing Osteoblast Differentiation and Inhibiting Osteoclast Activation in a Mouse Model of Femoral Defects.

Lipoteichoic acid (LTA) is a cell wall component of Gram-positive bacteria. Limited data suggest that LTA is beneficial for bone regeneration in vitro. Thus, we used a mouse model of femoral defects to explore the effects of LTA on bone healing in vivo. Micro-computed tomography analysis and double-fluorochrome labeling were utilized to examine whether LTA can accelerate dynamic bone formation in vivo. The effects of LTA on osteoblastogenesis and osteoclastogenesis were also studied in vitro. LTA treatment induced prompt bone bridge formation, rapid endochondral ossification, and accelerated healing of fractures in mice with femoral bone defects. In vitro, LTA directly enhanced indicators of osteogenic factor-induced MC3T3-E1 cell differentiation, including alkaline phosphatase activity, calcium deposition and osteopontin expression. LTA also inhibited osteoclast activation induced by receptor activator of nuclear factor-kappa B ligand. We identified six molecules that may be associated with LTA-accelerated bone healing: monocyte chemoattractant protein 1, chemokine (C-X-C motif) ligand 1, cystatin C, growth/differentiation factor 15, endostatin and neutrophil gelatinase-associated lipocalin. Finally, double-fluorochrome, dynamic-labeling data indicated that LTA significantly enhanced bone-formation rates in vivo. In conclusion, our findings suggest that LTA has promising bone-regeneration properties.

Synovial Fluid Interleukin-16 Contributes to Osteoclast Activation and Bone Loss through the JNK/NFATc1 Signaling Cascade in Patients with Periprosthetic Joint Infection.

Because of lipopolysaccharide (LPS)-mediated effects on osteoclast differentiation and bone loss, periprosthetic joint infection (PJI) caused by Gram-negative bacteria increases the risk of aseptic loosening after reimplantation. Synovial fluid interleukin-16 (IL-16) expression was higher in patients with PJI than in patients without joint infection. Thus, we explored the effects of IL-16 on bone. We investigated whether IL-16 modulates osteoclast or osteoblast differentiation in vitro. An LPS-induced bone loss mice model was used to explore the possible advantages of IL-16 inhibition for the prevention of bone loss. IL-16 directly activated p38 and c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling and increased osteoclast activation markers, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, and nuclear factor of activated T cells 1 (NFATc1). IL-16 directly caused monocytes to differentiate into TRAP-positive osteoclast-like cells through NFATc1 activation dependent on JNK/MAPK signaling. Moreover, IL-16 did not alter alkaline phosphatase activity or calcium deposition during osteoblastic differentiation. Finally, IL-16 inhibition prevented LPS-induced trabecular bone loss and osteoclast activation in vivo. IL-16 directly increased osteoclast activation through the JNK/NFATc1 pathway. IL-16 inhibition could represent a new strategy for treating infection-associated bone loss.

Functional characterization and proteomic analysis of lolA in Xanthomonas campestris pv. campestris.

BACKGROUND: The gram-negative Xanthomonas campestris pv. campestris is the pathogenic bacterium that causes black rot disease in crucifers. The virulence determinants of this bacterium include extracellular enzymes, exopolysaccharides, and biofilm formation. Here, one transposon mutant of X. campestris pv. campestris strain 17 that affects biofilm formation was isolated, and subsequent analyses led to the identification of the lolA gene, which encodes an outer membrane lipoprotein chaperone. RESULTS: The lolA mutant exhibited significant reductions in bacterial attachment, extracellular enzyme production, virulence, and tolerance in the presence of myriad membrane-perturbing agents. These phenotypic changes of the mutant could be complemented to the wild-type level through the intact lolA gene. Proteomic analysis revealed that 109 proteins were differentially expressed after lolA mutation. These differentially expressed proteins were categorized in various functional groups and were mainly associated with the membrane component, were involved in transport, and contained receptor activity. Through reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, deletion of lolA was determined to have caused significantly reduced expression of genes that encode the major extracellular enzymes, the biofilm-related proteins, and the virulence-related proteins. The RT-qPCR analysis also indicated that the expression of several genes that encode putative outer membrane lipoproteins and TonB-dependent receptors was reduced after lolA mutation. CONCLUSIONS: This is the first report to define the lolA gene as a virulence factor and to contribute to the functional understanding of, and provide new information concerning, the role of lolA in Xanthomonas. Furthermore, the results of this study provide and extend new insights into the function of lolA in bacteria.

WxcX is involved in bacterial attachment and virulence in Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. Here, one EZ-Tn5 transposon mutant of Xcc, altered in bacterial attachment, was isolated. Further analysis revealed that the transposon was inserted in the wxcX gene (encodes a hypothetical protein) of the transposon mutant. Sequence analysis revealed that WxcX is highly conserved in Xanthomonas, but none has been characterized. In this study, it was indicated that mutation of wxcX resulted in enhanced bacterial attachment, reduced virulence on the host cabbage, and increased sensitivity to sodium dodecyl sulfate. The affected phenotypes of the wxcX mutant could be complemented to wild-type levels by the intact wxcX gene. Site-directed mutagenesis revealed that E408 and E411 are critical amino acid residues for WxcX function in bacterial attachment. Taken together, our results demonstrate the roles of wxcX in attachment, virulence, and tolerance to sodium dodecyl sulfate in Xanthomonas for the first time.

Functional characterization and transcriptional analysis of icd2 gene encoding an isocitrate dehydrogenase of Xanthomonas campestris pv. campestris.

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate. In the genome of Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, two putative IDH genes, icd1 and icd2, have been annotated. Their physiological roles in X. campestris pv. campestris are unclear. In this study, the icd2 gene from X. campestris pv. campestris was characterized in detail. We demonstrated genetically that icd2 gene encodes a functional IDH, and is involved in virulence as well as bacterial attachment. Furthermore, the icd2 transcription initiation site was mapped at nucleotide G, 127 nucleotide upstream of the icd2 translation start codon. In addition, promoter analysis revealed that icd2 expression exhibits a distinct expression profile under different culture conditions, is subjected to catabolite repression, and is affected by acetate. This is the first time that the function and transcription of icd2 have been characterized in the crucifer pathogen X. campestris pv. campestris.

XCC2366, A Gene Encoding A Putative TetR Family Transcriptional Regulator, is Required for Acriflavin Resistance and Virulence of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot disease in cruciferous plants. The XCC2366 gene product is annotated as a protein belonging to the TetR family of transcriptional regulators. In this study, we evaluated the function and expression of the XCC2366 gene. Mutational analysis demonstrated that XCC2366 is involved in the resistance to acriflavin and is necessary for virulence in Xcc. In addition, the XCC2366 transcription initiation site was mapped at nucleotide A, 63 nucleotide upstream of the XCC2366 translation start codon. Furthermore, transcriptional analysis revealed that the expression of XCC2366 is induced in the presence of acriflavin. Reporter assay also showed that XCC2366 regulates its own expression under acriflavin-supplemented condition. To the best of our knowledge, acriflavin resistance-related gene in the crucifer pathogen Xcc was characterized for the first time.

Characterization of genes encoding proteins containing HD-related output domain in Xanthomonas campestris pv. campestris.

The Gram-negative plant pathogen Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. The production of Xcc virulence factors is regulated by Clp and RpfF. HD-related output domain (HDOD) is a protein domain of unknown biochemical function. The genome of Xcc encodes three proteins (GsmR, HdpA, and HdpB) with an HDOD. The GsmR has been reported to play a role in the general stress response and cell motility and its expression is positively regulated by Clp. Here, the function and transcription of hdpA and hdpB were characterized. Mutation of hdpA resulted in enhanced bacterial attachment. In addition, the expression of hdpA was positively regulated by RpfF but not by Clp, subject to catabolite repression and affected by several stress conditions. However, mutational analysis and reporter assay showed that hdpB had no effect on the production of a range of virulence factors and its expression was independent of Clp and RpfF. The results shown here not only extend the previous work on RpfF regulation to show that it influences the expression of hdpA in Xcc, but also expand knowledge of the function of the HDOD containing proteins in bacteria.

GMI, an Immunomodulatory Protein from Ganoderma microsporum, Potentiates Cisplatin-Induced Apoptosis via Autophagy in Lung Cancer Cells.

Cisplatin-based therapy is common in the treatment of several types of cancers, including lung cancers. In our previous study, GMI, an immunomodulatory protein cloned from Ganoderma microsporum, induced a cytotoxic effect in lung cancer cells via autophagy. The aim of this study is to examine the role of GMI in enhancing cisplatin-mediated cell death. On the basis of MTT assay and Combination Index, GMI and cisplatin cotreatment induced a synergistic cytotoxic effect. GMI and cisplatin-induced apoptosis was determined by sub-G1, nuclear condensation, and annexin-V/propidium iodide analyses. On Western blot, expressions of γH2AX and cleaved forms of PARP, caspase-3, and caspase-7 were induced by combined treatment. Akt/mTOR pathway activity, LC3-II expression, and acidic vesicular organelle development demonstrated that cisplatin does not abolish GMI-mediated autophagy. Cyto-ID Green/hoechst 33342 double staining and time-dependent experiment indicated that GMI and cisplatin-treated A549 cells simultaneously express autophagosomes and apoptotic nuclei. To elucidate the role of autophagy in inducing apoptosis by GMI and cisplatin, chemical inhibitors and LC3 shRNA were used to inhibit autophagy. The results showed that 3-methyladenine decreases, while chloroquine increases GMI and cisplatin cotreatment-induced cleavage of caspase-7 and PARP. LC3 silencing abolished activation of apoptosis in A549 cells. Caspase inhibitors and caspase-7 silencing mitigated GMI and cisplatin-elicited cell viability inhibition and apoptosis. This is the first study to reveal the novel function of GMI in potentiating cisplatin-mediated apoptosis. GMI and cisplatin induce apoptosis via autophagy/caspase-7-dependent and survivin- and ERCC1-independent pathway. GMI may be a potential cisplatin adjuvant against lung cancer.

Alleviation of Dermatophagoides microceras-induced allergy by an immunomodulatory protein, FIP-fve, from Flammulina velutipes in mice.

Asthma is a major public health concern. Its greatest risk factor is house dust mite (HDM). Dermatophagoides microceras (Der m) is a type of HDM, and in central Taiwan, there is approximately 80% prevalence of sensitization to Der m. FIP-fve is a fungal immunomodulatory protein (FIP) isolated from the fungus Flammulina velutipes, and exhibits anti-inflammatory properties. To investigate whether FIP-fve affects Der m-induced asthma and inflammation, we evaluated hyper-responsiveness (AHR), pathological changes, and cytokines in mice. We demonstrated that oral FIP-fve decreased Der m-induced airway AHR, airway inflammation, cell infiltration, and expression of cytokines in the bronchoalveolar lavage fluid of Balb/c mice. The results of this study suggest that FIP-fve suppresses asthma, inflammation, and respiratory pathogenesis stimulated by Der m. FIP-fve is able to maintain immunomodulatory activity even in simulated gastric fluid and intestinal fluid. FIP-fve could be a safe and stable agent for suppression of allergic asthma.

The galU gene of Xanthomonas campestris pv. campestris is involved in bacterial attachment, cell motility, polysaccharide synthesis, virulence, and tolerance to various stresses.

Uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.9) is an enzyme that catalyzes the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate. GalU is involved in virulence in a number of animal-pathogenic bacteria since its product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharide and exopolysaccharide. However, its function in Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, is unclear. Here, we characterized a galU mutant of X. campestris pv. campestris and showed that the X. campestris pv. campestris galU mutant resulted in a reduction in virulence on the host cabbage. We also demonstrated that galU is involved in bacterial attachment, cell motility, and polysaccharide synthesis. Furthermore, the galU mutant showed increased sensitivity to various stress conditions including copper sulfate, hydrogen peroxide, and sodium dodecyl sulfate. In addition, mutation of galU impairs the expression of the flagellin gene fliC as well as the attachment-related genes xadA, fhaC, and yapH. In conclusion, our results indicate involvement of galU in the virulence factor production and pathogenicity in X. campestris pv. campestris, and a role for galU in stress tolerance of this crucifer pathogen.

SsrA and SmpB have multifaceted physiological roles in the black rot pathogen Xanthomonas campestris pathovar campestris.

SsrA and SmpB are known to play important roles in translational quality control and are essential for virulence in many human and animal pathogenic bacteria. The physiological roles and contribution of SsrA and SmpB to plant pathogen are unclear. Here, we present evidence to show that ssrA and smpB are involved in pathogenesis of Xanthomonas campestris pathovar campestris, the cause of black rot diseases in crucifers. The ssrA and smpB mutants exhibited defects in bacterial attachment, cell motility, and extracellular enzyme activity. The mutation of ssrA and smpB also resulted in a reduction in temperature tolerance. These altered phenotypes of the ssrA and smpB mutants could be complemented to wild-type levels by the intact ssrA and smpB genes. This is the first demonstration of the roles of SsrA and SmpB in phytopathogen.

Transcriptional analysis and functional characterization of XCC1294 gene encoding a GGDEF domain protein in Xanthomonas campestris pv. campestris.

The nucleotide cyclic di-GMP is a second messenger in bacteria that regulates a range of cellular functions including the virulence of pathogens. GGDEF is a protein domain involved in the synthesis of cyclic di-GMP. The genome of the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 21 proteins with a GGDEF domain. Clp, a homolog of the model transcription factor Crp of Escherichia coli, is a global regulator in Xcc. The aim of this study is to identify genes encoding GGDEF domain proteins whose expression is regulated by Clp. Results of reporter assay and RT-PCR analysis suggested that Clp regulates the expression of a set of genes encoding proteins harboring GGDEF domain. The transcription initiation site of XCC1294, one of the Clp regulated gene encoding a GGDEF domain protein, was mapped. Promoter analysis and gel retardation assay indicated that the transcription of XCC1294 is positively and directly regulated by Clp. Furthermore, transcription of XCC1294 was subject to catabolite repression and affected by several stress conditions. We also showed that mutation of XCC1294 results in enhanced surface attachment. In addition, transcription of three putative adhesin genes (xadA, fhaC, and yapH) was increased in the XCC1294 mutant. Taken together, the data presented here indicate that Clp positively regulates expression of XCC1294, and that XCC1294 serves a regulator of bacterial attachment and regulates different adhesin genes expression.