Integrated transcriptomic and metabolomic profiles reveal adaptive responses of three poplar varieties against the bacterial pathogen Lonsdalea populi.

Different poplar varieties vary in their tolerance to certain pathogens. However, knowledge about molecular regulation and critical responses of resistant poplars during pathogen infection remains scarce. To investigate adaptive responses to canker disease caused by the bacterium Lonsdalea populi, we screened three poplar varieties with contrasting tolerance, including Populus deltoides. 'Zhonglin 2025' (2025), Populus × Euramericana. '74/76' (107) and Populus tomentosa cv 'henan' (P. tomentosa). Transcriptomic analysis revealed significant changes in the expression levels of defence-related genes in different poplar varieties in response to infection, which reshaped the PTI and ETI processes. Intriguingly, photosynthesis-related genes were found to be highly expressed in the resistant variety, whereas the opposite was observed in the susceptible variety. Susceptible poplars maintained the activation of defence-related genes during early period of onset, which restricted the expression of photosynthesis-related and auxin signal-related genes. Furthermore, combined with metabolomic analysis, differences in the content of antibacterial substances and key differentially expressed genes in phenylpropane and flavonoid biosynthesis pathways were identified. Delayed induction of catechin in the susceptible variety and it's in vitro antibacterial activity were considered to be one of the important reasons for the differences in resistance to L. populi compared with the resistant variety, which is of practical interest for tree breeding. Moreover, the trade-off between growth and defence observed among the three poplar varieties during infection provides new insights into the multilevel regulatory circuits in tree-pathogen interactions.

Genomic Characterization Provides an Insight into the Pathogenicity of the Poplar Canker Bacterium Lonsdalea populi.

An emerging poplar canker caused by the gram-negative bacterium, Lonsdalea populi, has led to high mortality of hybrid poplars Populus × euramericana in China and Europe. The molecular bases of pathogenicity and bark adaptation of L. populi have become a focus of recent research. This study revealed the whole genome sequence and identified putative virulence factors of L. populi. A high-quality L. populi genome sequence was assembled de novo, with a genome size of 3,859,707 bp, containing approximately 3434 genes and 107 RNAs (75 tRNA, 22 rRNA, and 10 ncRNA). The L. populi genome contained 380 virulence-associated genes, mainly encoding for adhesion, extracellular enzymes, secretory systems, and two-component transduction systems. The genome had 110 carbohydrate-active enzyme (CAZy)-coding genes and putative secreted proteins. The antibiotic-resistance database annotation listed that L. populi was resistant to penicillin, fluoroquinolone, and kasugamycin. Analysis of comparative genomics found that L. populi exhibited the highest homology with the L. britannica genome and L. populi encompassed 1905 specific genes, 1769 dispensable genes, and 1381 conserved genes, suggesting high evolutionary diversity and genomic plasticity. Moreover, the pan genome analysis revealed that the N-5-1 genome is an open genome. These findings provide important resources for understanding the molecular basis of the pathogenicity and biology of L. populi and the poplar-bacterium interaction.

The Two-Component System DcuS-DcuR Is Involved in Virulence and Stress Tolerance in the Poplar Canker Bacterium Lonsdalea populi.

The gram-negative bacterium Lonsdalea populi causes an emerging poplar (Populus × euramericana) canker resulting in severe losses to poplar production in China and Europe. Two-component signal transduction systems play important roles in the regulation of virulence and stress responses in phytopathogenic bacteria. We identified a two-component pair (Lqp2625-Lqp2624) in L. populi, highly homologous to DcuS-DcuR of Escherichia coli. Mutants lacking DcuS or DcuR displayed normal growth while their virulence on poplar twigs was impaired. An inability to produce flagella indicated that DcuS and DcuR are involved in biofilm formation and swimming motility. Moreover, the loss of DcuS or DcuR led to increased sensitivity to oxidative stress and chloramphenicol through downregulation of genes associated with catalases and the multidrug efflux pump, suggesting that the two-component pair contributes to cellular adaptation to oxidative and antibiotic stresses. We identified key domains and putative phosphorylation sites important for virulence and stress responses. Our findings reveal the functions of DcuS-DcuR in virulence and stress responses in L. populi and provide increasing evidence that two-component systems are crucial during the infection process and stress adaptation in bacteria.

Molecular Aspects of an Emerging Poplar Canker Caused by Lonsdalea populi.

The Gram-negative bacterium Lonsdalea populi causes a lethal disease known as bark canker on Populus × euramericana in China and Europe. Typical symptoms of bark canker include an abundant white-colored fluid, which oozes from the infected tissues. The availability of the genomic sequence of the bacterium provided the necessary resource to launch genome-scale investigations into the mechanisms fundamental to pathogenesis. Functional analyses of a diverse group of genes encoding virulence factors and components of signaling pathways indicate that successful bark infection depends on specific responses by the pathogen to various stresses, including oxidative stress. Although physiology of resistance is well studied, the molecular processes underlying the defense responses and the genetic basis of resistance to L. populi and in other poplar species remain largely unknown. Control of the disease has relied on chemical measures. Due to the genetic amenability of Lonsdalea and poplar, this pathosystem will become an important model system to unravel molecular mechanisms of bacterial pathogenicity on woody plants. Increased understanding of pathogenesis and signaling in the interaction will facilitate the management of this kind of poplar canker.

[MarR family transcription regulator HpaR and XC0449 coordinately regulate the virulence of Xanthomonas campestris pv. campestris].

MarR family transcription regulators are ubiquitous among bacteria and archaea. They extensively control multiple cellular processes and elaborately regulate the expression of genes involved in virulence, stress response and antibiotics at translational level. In Xanthomonas campestris pv. campestris, insertional inactivation of MarR family transcription regulator HpaR (XC2827) resulted in significantly decrease in virulence and increase in the production of the extracellular proteases. Here, we reported that the genome of Xcc 8004 encodes nine MarR family transcription regulators. The MarR family transcription regulators, HpaR (XC2827) and XC0449, were heterologous expressed and purified. In vitro MST and Pull-down assay confirmed the physical interaction between HpaR and XC0449. Phenotypical assay determined that deletion of XC0449 resulted in substantial virulence attenuation. In vitro EMSA, in vivo qRT-PCR and GUS activity assay identified that HpaR and XC0449 coordinately act as the transcriptional activator to regulate the expression of the virulence-associated gene XC0705, and eventually control the bacterial virulence and the production of extracellular proteases.

A Large-Scale Mutational Analysis of Two-Component Signaling Systems of Lonsdalea quercina Revealed that KdpD-KdpE Regulates Bacterial Virulence Against Host Poplar Trees.

Poplar, which is a dominant species in plant communities distributed in the northern hemisphere, is commonly used as a model plant in forestry studies. Poplar production can be inhibited by infections caused by bacteria, including Lonsdalea quercina subsp. populi, which is a gram-negative bacterium responsible for bark canker disease. However, the molecular basis of the pathogenesis remains uncharacterized. In this study, we annotated the two-component signal transduction systems (TCSs) encoded by the L. quercina subsp. populi N-5-1 genome and identified 18 putative histidine kinases and 24 response regulators. A large-scale mutational analysis revealed that 19 TCS genes regulated bacterial virulence against poplar trees. Additionally, the deletion of kdpE or overexpression of kdpD resulted in almost complete loss of bacterial virulence. We observed that kdpE and kdpD formed a bi-cistronic operon. KdpD exhibited autokinase activity and could bind to KdpE (Kd = 5.73 ± 0.64 µM). Furthermore, KdpE is an OmpR family response regulator. A chromatin immunoprecipitation sequencing analysis revealed that KdpE binds to an imperfect palindromic sequence within the promoters of 44 genes, including stress response genes Lqp0434, Lqp3037, and Lqp3270. A comprehensive analysis of TCS functions may help to characterize the regulation of poplar bark canker disease.

PsMPK1, an SLT2-type mitogen-activated protein kinase, is required for hyphal growth, zoosporogenesis, cell wall integrity, and pathogenicity in Phytophthora sojae.

Mitogen-activated protein kinases (MAPKs) play important roles in the regulation of vegetative and pathogenic growth in plant pathogens. Here, we identified an SLT2-type MAP kinase in Phytophthora sojae, PsMPK1, which was transcriptionally induced in sporulating hyphae and the early stages of infection. Silencing of PsMPK1 caused defects in growth and zoosporogenesis, and increased hyphal swellings after the induction of sporangia formation, along with increasing hypersensitivity to cell wall-degrading enzymes. Transmission electron microscopy showed that the cell wall of PsMPK1-silenced mutants was also deleteriously affected. A dark outermost layer in the cell walls disappeared in the mutants, and an additional layer of the mutant cell wall that was deposited abnormally inside an inner bright layer appeared nonhomogeneous and rough compared to the wild type. Pathogenicity assays showed that PsMPK1-silenced transformants lost their pathogenicity on susceptible soybean host plants and triggered stronger cell death. Overall, PsMPK1 is involved in growth, differentiation, cell wall integrity, and pathogenicity in P. sojae.

A Myb transcription factor of Phytophthora sojae, regulated by MAP kinase PsSAK1, is required for zoospore development.

PsSAK1, a mitogen-activated protein (MAP) kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE) profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down) in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a) no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b) direct germination of sporangia, and c) afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae.

[Advances on molecular mechanisms of plant-pathogen interactions].

Plants have established a complicated immune defense system during co-evolution with pathogens. The innate immune system of plants can be generally divided into two levels. One, named PAMP-triggered immunity (PTI), is based on the recognition of pathogen-associated molecular patterns by pattern-recognition receptors, which confers resistance to most pathogenic microbes. The other begins in cytoplasm and mainly relies on recognition of microbial effectors by plant resistance proteins in direct or indirect ways, which then initiates potent defense responses. This process, termed effector-triggered immunity (ETI), is necessary for defense against pathogens that can secret effectors to suppress the first level of immunity. Activation of these two layers of immunity in plant is based on distinguishing and recognition of "self" and "non-self" signals. Recognition of "non-self" signals can activate signal cascades, such as MAPK cascades, which will then induce defense gene expression and corresponding defense responses. In this review, we focused on underlying molecular mechanisms of plant-pathogen interactions and the latest advances of the PTI and ETI signaling network.

PsSAK1, a stress-activated MAP kinase of Phytophthora sojae, is required for zoospore viability and infection of soybean.

Mitogen-activated protein kinase (MAPK) pathways are universal and evolutionarily conserved signal transduction modules in all eukaryotic cells. In this study, PsSAK1, which encodes a stress-activated MAPK of Phytophthora sojae, was identified. PsSAK1 is highly conserved in oomycetes, and it represents a novel group of MAPK due to its pleckstrin homology domain. Reverse-transcription polymerase chain reaction analysis showed that PsSAK1 expression was upregulated in zoospores and cysts and during early infection. In addition, its expression was induced by osmotic and oxidative stress mediated by NaCl and H(2)O(2), respectively. To elucidate the function, the expression of PsSAK1 was silenced using stable transformation of P. sojae. The silencing of PsSAK1 did not impair hyphal growth, sporulation, or oospore production but severely hindered zoospore development, in that the silenced strains showed quicker encystment and a lower germination ratio than the wild type. PsSAK1-silenced mutants produced much longer germ tubes and could not colonize either wounded or unwounded soybean leaves. Our results indicate that PsSAK1 is an important regulator of zoospore development and pathogenicity in P. sojae.

GPR11, a putative seven-transmembrane G protein-coupled receptor, controls zoospore development and virulence of Phytophthora sojae.

G protein-coupled receptors (GPCRs) represent a large receptor family involved in a broad spectrum of cell signaling. To understand signaling mechanisms mediated by GPCRs in Phytophthora sojae, we identified and characterized the PsGPR11 gene, which encodes a putative seven-transmembrane GPCR. An expression analysis revealed that PsGPR11 was differentially expressed during asexual development. The highest expression level occurred in zoospores and was upregulated during early infection. PsGPR11-deficienct transformants were obtained by gene silencing strategies. Silenced transformants exhibited no differences in hyphal growth or morphology, sporangium production or size, or mating behavior. However, the release of zoospores from sporangia was severely impaired in the silenced transformants, and about 50% of the sporangia did not completely release their zoospores. Zoospore encystment and germination were also impaired, and zoospores of the transformants lost their pathogenicity to soybean. In addition, no interaction was observed between PsGPR11 and PsGPA1 with a conventional yeast two-hybrid assay, and the transcriptional levels of some genes which were identified as being negatively regulated by PsGPA1 were not clearly altered in PsGPR11-silenced mutants. These results suggest that PsGPR11-mediated signaling controls P. sojae zoospore development and virulence through the pathways independent of G protein.

The PsCZF1 gene encoding a C2H2 zinc finger protein is required for growth, development and pathogenesis in Phytophthora sojae.

The C(2)H(2) zinc finger proteins form one of the largest families of transcriptional regulators in eukaryotes. We identified a Phytophthora sojae C(2)H(2) zinc finger (PsCZF1), that is highly conserved in sequenced oomycete pathogens. In transformants of P. sojae containing the PsCZF1 promoter fused to the beta-glucuronidase (GUS) reporter gene, GUS activity was highly induced in the P. sojae oospore stage and upregulated after infection. To elucidate the function of PsCZF1, its expression was silenced by introducing anti-sense constructs into P sojae. PsCZF1-silenced transformants did not exhibit altered cell size or morphology of sporangia and hyphae; however, hyphal growth rate was reduced by around 50% in the mutants. PsCZF1-deficient mutants were also impaired in production of oospores, swimming zoospores and germinating cysts, indicating that the gene is involved in various stages of the life cycle. Furthermore, we found that PsCZF1-deficient mutants lost virulence on host soybean cultivars. Our results suggest that this oomycete-specific C(2)H(2)-type zinc finger protein plays an important role in growth, development, and pathogenesis; therefore, PsCZF1 might be an attractive oomycete-specific target for chemical fungicide screening.

Liver cell adenoma: a case report with clonal analysis and literature review.

We report a case of liver cell adenoma (LCA) in a 33-year-old female patient with special respect to its clonality status, pathogenic factors and differential diagnosis. The case was examined by histopathology, immunohistochemistry and a clonality assay based on X-chromosomal inactivation mosaicism in female somatic tissues and polymorphism at androgen receptor focus. The clinicopathological features of the reported cases from China and other countries were compared. The lesion was spherical, sizing 2 cm in its maximal dimension. Histologically, it was composed of cells arranged in cords, most of which were two-cell-thick and separated by sinusoids. Focal fatty change and excessive glycogen storage were observed. The tumor cells were round or polygonal in shape, resembling the surrounding parenchymal cells. Mitosis was not found. No portal tract, central vein or ductule was found within the lesion. The tumor tissue showed a positive reaction for cytokeratin (CK) 18, but not for CK19, vimentin, estrogen and progesterone receptors. Monoclonality was demonstrated for the lesion, confirming the diagnosis of an LCA. Clonality analysis is helpful for its distinction from focal nodular hyperplasia.

Clinical study for alleviating opiate drug psychological dependence by a method of ablating the nucleus accumbens with stereotactic surgery.

The aim of this study was to explore a new way of treating drug addiction by ablating the nucleus accumbens (NAC), which has a close relationship with drug-induced psychological dependence, using stereotactic surgery, blocking the mesocorticolimbic dopamine circuit, alleviating craving for drugs and lowering the relapse rate after detoxification. On the basis of animal experiments, stereotactic surgery was performed in 28 patients by making a lesion in the NAC bilaterally to treat opiate drug dependence. Indications, the criterion of therapeutic effect, treatment process and the therapeutic and safety evaluation index of the surgery were formulated particularly. The mean follow-up period was 15 months. Relapse has not occurred in 11 cases up till now. Drug-free time in these patients has been more than half a year in 4 cases (more than a year in 3 cases), and less than half a year in 7 cases. Relapse occurred in 15 cases after surgery. Drug-free time in these patients was more than half a year in 3 cases, between 1 month and half a year in 10 cases and less than 1 month in 2 cases. The therapeutic effect was excellent in 7 cases (26.9%), good in 10 cases (38.5%) and poor in 2 cases (7.7%). Another 7 cases were still under investigation at the time of writing. Relapse rates after surgery were 7.7, 38.5 and 57.5% within 1 month, between 1 month and half a year and after more than half a year, respectively. There were no common complications of surgery such as intracranial hematoma or infection in these patients after operation. Character type was changed slightly in 2 cases, and 4 cases suffered temporary memory loss, which did not affect their daily lives and learning function. They all recovered within 1 month. There were different degrees of effectiveness of treating drug addicts' psychological dependence by making lesions in the NAC bilaterally with stereotactic surgery. No particular complications occurred. The operation is safe and feasible. The mean follow-up time in this study was 15 months. The effectiveness was satisfactory. The relapse rate of drug addicts after detoxification was clearly reduced.