Analysis of Risk Factors for Pulmonary Infections During Radiotherapy in Lung Cancer Patients.

Objective: To investigate the risk factors for lung infection in lung cancer patients undergoing radiotherapy. Methods: We selected 142 patients with lung cancer who underwent radiotherapy at our hospital from January 2020 to June 2021. The patients were divided into groups according to whether they had pulmonary infection during radiotherapy in our hospital, which was infected group (n=44) and the uninfected group (n=98), respectively. To observe the incidence of lung infection in lung cancer patients during radiotherapy. The distribution of pathogenic bacteria in patients with pulmonary infection was observed. Clinical data of the two groups were collected and compared. The risk factors of lung cancer patients complicated with lung infection were analyzed by binary Logistic regression. Results: All patients with lung cancer complicated with lung infection underwent relevant examination, and the results showed that they were all complicated infections, and the composition ratio of Klebsiella pneumoniae was the highest (31.82%), followed by Staphylococcus, Pseudomonas, and fungi, which accounted for 27.27%, 22.73%, and 18.18%, respectively. Binary Logistic regression analysis showed that age ≥60 years old, smoking history ≥30 years, radiotherapy duration of combined drug regimen > 2 weeks, pathogenic bacteria combined infection, albumin content < 30 g/L were risk factors for lung cancer patients during radiotherapy. Conclusion: Age ≥60 years old, smoking history ≥30 years old, radiotherapy duration of combined drug regimen > 2 weeks, pathogenic bacteria combined infection, albumin content < 30 g/L are the risk factors for lung cancer patients during radiotherapy. Clinical prevention and intervention should be based on the aforementioned independent risk factors to decrease the incidence of lung infections, thereby enhancing patient prognosis.

Methylation entropy landscape of Chinese long-lived individuals reveals lower epigenetic noise related to human healthy aging.

The transition from ordered to noisy is a significant epigenetic signature of aging and age-related disease. As a paradigm of healthy human aging and longevity, long-lived individuals (LLI, >90 years old) may possess characteristic strategies in coping with the disordered epigenetic regulation. In this study, we constructed high-resolution blood epigenetic noise landscapes for this cohort by a methylation entropy (ME) method using whole genome bisulfite sequencing (WGBS). Although a universal increase in global ME occurred with chronological age in general control samples, this trend was suppressed in LLIs. Importantly, we identified 38,923 genomic regions with LLI-specific lower ME (LLI-specific lower entropy regions, for short, LLI-specific LERs). These regions were overrepresented in promoters, which likely function in transcriptional noise suppression. Genes associated with LLI-specific LERs have a considerable impact on SNP-based heritability of some aging-related disorders (e.g., asthma and stroke). Furthermore, neutrophil was identified as the primary cell type sustaining LLI-specific LERs. Our results highlight the stability of epigenetic order in promoters of genes involved with aging and age-related disorders within LLI epigenomes. This unique epigenetic feature reveals a previously unknown role of epigenetic order maintenance in specific genomic regions of LLIs, which helps open a new avenue on the epigenetic regulation mechanism in human healthy aging and longevity.

Revealing Potential Genes Affecting Flocculation and/or Viability of Saccharomyces pastorianus by Comparative Genomic Analysis.

Yeast flocculation and viability are critical factors in beer production. Adequate flocculation of yeast at the end of fermentation helps to reduce off-flavors and cell separation, while high viability is beneficial for yeast reuse. In this study, we used comparative genomics to analyze the genome information on Saccharomyces pastorianus W01, and its spontaneous mutant W02 with appropriate weakened flocculation ability (better off-flavor reduction performance) and unwanted decreased viability, to investigate the effect of different gene expressions on yeast flocculation or/and viability. Our results indicate that knockout of CNE1, CIN5, SIN3, HP-3, YPR170W-B, and SCEPF1_0274000100 and overexpression of CNE1 and ALD2 significantly decreased the flocculation ability of W01, while knockout of EPL1 increased the flocculation ability of W01. Meanwhile, knockout of CIN5, YPR170W-B, OST5, SFT1, SCEPF1_0274000100, and EPL1 and overexpression of SWC3, ALD2, and HP-2 decreased the viability of W01. CIN5, EPL1, SCEPF1_0274000100, ALD2, and YPR170W-B have all been shown to affect yeast flocculation ability and viability.

Characterization of Cetobacterium somerae CPU-CS01 isolated from the intestine of healthy crucian carp (Carassius auratus) as potential probiotics against Aeromonas hydrophila infection.

Cetobacterium somerae is a commensal bacterium for many fish species. However, research on C. somerae has been limited so far, and its function and beneficial potential require to be further investigated. The objective of this study was to evaluate the probiotic properties of C. somerae CPU-CS01 isolated from the intestinal contents of crucian carp (Carassius auratus). Hemolytic activity, antibiotic susceptibility, acid tolerance, bile salt tolerance, free radical scavenging, and enzyme production properties were tested for in vitro. Caenorhabditis elegans and zebrafish (Danio rerio) model were used to evaluate the antioxidant and anti-infective effects of C. somerae CPU-CS01 in vivo. Our results showed that C. somerae CPU-CS01 had no hemolytic activity, it produced cellulase, amylase, and survived at low pH (2.0-3.0) and in the presence of bile salts. The cell-free culture supernatant (CFCS) of C. somerae CPU-CS01 possessed DPPH radical, hydroxyl radical, and superoxide anion scavenging activity. C. elegans fed with C. somerae CPU-CS01 were more resistant to hydrogen peroxide-induced oxidative stress and Aeromonas hydrophila infection. In addition, zebrafish-fed diets containing C. somerae CPU-CS01 showed improved survival after A. hydrophila infection. Based on these results, the positive probiotic properties of C. somerae CPU-CS01 isolated from the intestinal contents of crucian carp make it a potential candidate for probiotic.

Response of Saccharomyces cerevisiae var. diastaticus to nerol: Evaluation of antifungal potential by inhibitory effect and proteome analyses.

Saccharomyces cerevisiae var. diastaticus (S. diastaticus) is a major spoilage yeast in brewing. In the present research, the antifungal properties of nerol and the proteome response of S. diastaticus were studied. Results showed nerol can inhibit cell budding and delay yeast fermentation in a dose-depended manner. After 3 d of treatment with 0.25 mg·mL-1 nerol, intracellular ROS levels increased 1.66-fold (P < 0.01), and the cells with damaged membrane increased to 23.2 %. Quantitative proteomic profiles utilizing a capillary-HPLC-MS/MS technology revealed that proteins involved in the metabolism of fermentable sugars were up-regulated in S. diastaticus cells treated with nerol, indicating nerol treatment altered the metabolite pattern of fermentable sugars. Proteins associated with the cell membrane biogenesis, heat shock proteins, amino acid biosynthesis, and glutathione metabolism were similarly up-regulated. These findings revealed the mechanism of nerol-induced yeast cell damage as well as the detoxification response of yeast cells.

Effects of Transcranial Direct Current Stimulation and Neuromuscular Joint Facilitation on Upper Limb Motor Disorders for Stroke Patients.

Context: Approximately 1.5- to 2-million new patients suffer from stroke annually in China. 60% of patients suffering from stroke will sustain different degrees of upper limb dysfunction at six months after onset. Recovery of upper limb function after stroke is of great significance in improving patients' quality of life. Objective: The study intended to explore the rehabilitative the effects of transcranial direct current stimulation combined with neuromuscular joint therapy on the rehabilitation of patients with upper-limb motor disorders after strokes to provide new ideas for rehabilitative treatment. Design: The study was a paired control test. Setting: The study took place in the Department of Rehabilitation Medicine at Heping Hospital of Changzhi Medical College in Changzhi, Shanxi, China. Participants: Participants were 80 stroke patients with upper-limb motor disorders who were treated at the hospital between January 2020 and December 2020. Intervention: According to the natural grouping method, the research team divided participants into an intervention group (n = 42) and a control group (n = 38). The control group received transcranial direct-current stimulation, and the intervention group received transcranial direct-current stimulation combined with neuromuscular joint therapy. Outcome Measures: The measurements included the scores on the Fugl-Meyer Assessment (FMA) scale, the Action Research Arm Test (ARAT), activities of daily living (ADL), and National Institutes of Health Stroke Scale (NIHSS) as well as the serum levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and superoxide dismutase (SOD). The team also measured the maximum isometric torque of flexion and extension of the elbow joint. The research team compared the differences in the scores between the groups for all variables. Results: Postintervention, the FMA, ARAT, and ADL scores, the torques of elbow flexion and extension maximum isometric contraction, the amplitude, and the serum BDNF, NGF, and SOD levels were significantly higher in the intervention group than those in the control group, while the NIHSS score and the incubation period of evoked potential were significantly lower than those in the control group. Conclusions: Transcranial direct current stimulation combined with the neuromuscular joint method demonstrated good rehabilitative effects on upper-limb movement disorders for stroke patients and significantly improved their upper-limb function and promoted recovery of nerve functions.

The catalase-peroxidase PiCP1 plays a critical role in abiotic stress resistance, pathogenicity and asexual structure development in Phytophthora infestans.

Catalase-peroxidase is a heme oxidoreductase widely distributed in bacteria and lower eukaryotes. In this study, we identified a catalase-peroxidase PiCP1 (PITG_05579) in Phytophthora infestans. PiCP1 had catalase/peroxidase and secretion activities and was highly expressed in sporangia and upregulated in response to oxidative and heat stresses. Compared with wild type, PiCP1-silenced transformants (STs) had decreased catalase activity, reduced oxidant stress resistance and damped cell wall integrity. In contrast, PiCP1-overexpression transformants (OTs) demonstrated increased tolerance to abiotic stresses and induced the upregulation of PR genes in the host salicylic acid pathway. The high concentration of PiCP1 can also induced callose deposition in plant tissue. Importantly, both STs and OTs have severely reduced sporangia formation and zoospore releasing rate, but the sporangia germination rate and type varied depending on environmental conditions. Comparative sequence analyses show that catalase-peroxidases are broadly distributed and highly conserved among soil-borne plant parasitic oomycetes, but not in freshwater-inhabiting or strictly plants-inhabiting oomycetes. In addition, we found that silencing PiCP1 downregulated the expression of PiCAT2. These results revealed the important roles of PiCP1 in abiotic stress resistance, pathogenicity and in regulating asexual structure development in response to environmental change. Our findings provide new insights into catalase-peroxidase functions in eukaryotic pathogens.

LysM protein BdLM1 of Botryosphaeria dothidea plays an important role in full virulence and inhibits plant immunity by binding chitin and protecting hyphae from hydrolysis.

Botryosphaeria dothidea infects hundreds of woody plants and causes a severe economic loss to apple production. In this study, we characterized BdLM1, a protein from B. dothidea that contains one LysM domain. BdLM1 expression was dramatically induced at 6 h post-inoculation in wounded apple fruit, strongly increased at 7 d post-inoculation (dpi), and peaked at 20 dpi in intact shoots. The knockout mutants of BdLM1 had significantly reduced virulence on intact apple shoots (20%), wounded apple shoots (40%), and wounded apple fruit (40%). BdLM1 suppressed programmed cell death caused by the mouse protein BAX through Agrobacterium-mediated transient expression in Nicotiana benthamiana, reduced H2O2 accumulation and callose deposition, downregulated resistance gene expression, and promoted Phytophthora nicotianae infection in N. benthamiana. Moreover, BdLM1 inhibited the active oxygen burst induced by chitin and flg22, bound chitin, and protected fungal hyphae against degradation by hydrolytic enzymes. Taken together, our results indicate that BdLM1 is an essential LysM effector required for the full virulence of B. dothidea and that it inhibits plant immunity. Moreover, BdLM1 could inhibit chitin-triggered plant immunity through a dual role, i.e., binding chitin and protecting fungal hyphae against chitinase hydrolysis.

Temperature and Humidity Regulate Sporulation of Corynespora cassiicola That Is Associated with Pathogenicity in Cucumber (Cucumis sativus L.).

Cucumber target leaf spot, caused by Corynespora cassiicola, is an emerging disease with a high incidence that causes severe damage to cucumbers on a global scale. Therefore, efforts need to be undertaken to limit the spread and infection of this pathogen, preferably by using environmentally friendly methods. In this study, the effects of temperature and moisture on the sporulation of C. cassiicola were investigated in vitro and in vivo. The novelty of our study refers to the observation of spore production and size as well as the revelation of a correlation between spore size and virulence. On potato dextrose agar (PDA) and cucumber-leaf extract agar (CEA), temperature played a critical role in spore production, which was strongly influenced by both temperature and moisture on detached leaves and cucumber seedlings. Maximum spore production was found at 30 °C on PDA and 25 °C on CEA, cucumber detached leaves and living plants. Lower spore productions were observed with a stepwise change of 5 °C. In addition, the largest spore production was found at 100% relative humidity (RH) in comparison to the other tested moisture. Moreover, moisture was found to be the most important factor affecting spore size, accounting for 83.09-84.86% of the total variance in length and 44.72-73.10% of the total variance in width. The longest-narrowest spores were formed at 100% RH, and the shortest-widest spores were formed at 75% RH. Furthermore, the result showed that larger spores of C. cassiicola were more virulent and small spores were avirulent. Our findings will contribute to the development of new strategies for the effective alleviation and control of cucumber target leaf spot.

An updated network meta-analysis of EGFR-TKIs and combination therapy in the first-line treatment of advanced EGFR mutation positive non-small cell lung cancer.

Objectives: Tyrosine kinase inhibitors (TKIs) are a standard care option in patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutation. TKI-based combination treatment modes show encouraging outcomes. However, it remains unknown which is the optimal treatment as the first-line regimen for these patients on overall survival (OS). Materials and methods: Randomized controlled trials and meeting abstracts that investigated EGFR-TKIs alone or in combination as front-line care for patients with NSCLC were systematically searched in relevant databases and reviewed. Fixed and random effects network meta-analysis models were used to estimate progression-free survival (PFS), OS, overall response rate, and grade three and higher adverse events (AEs). Surface under the cumulative ranking curves (SUCRAs) were used to rank treatment effects. Results: Eighteen studies covering six treatments and involving a total of 4389 patients were included in this network meta-analysis. On OS, the top three treatment were first-generation EGFR-TKIs (1G EGFR-TKIs) plus chemotherapy (SUCRA, 88.1%), osimertinib (SUCRA, 65.8%) and second-generation EGFR-TKIs (2GEGFR-TKIs) (SUCRA, 63.3%). On PFS, the top three treatments were osimertinib (SUCRA, 96.0%), 1G EGFR-TKIs plus chemotherapy (SUCRA, 67.1%), and 1G EGFR-TKIs plus antiangiogenesis (SUCRA, 48.2%). Two types of TKI-based combination therapy have significantly higher risk of grade three and higher AEs than TKI alone. Conclusion: 1G EGFR-TKIs plus chemotherapy and osimertinib seem to be the two better options as first-line care in advanced NSCLC patients with EGFR-mutation. Osimertinib caused the lowest incidence of AEs. However, TKIs-based combination therapy significantly increased AEs.

Telomere length is maternally inherited and associated with lipid metabolism in Chinese population.

Telomere is a unique DNA-protein complex which covers the ends of chromosomes to avoid end fusion and maintain the stability and integrity of chromosomes. Telomere length (TL) shortening has been linked to aging and various age-related diseases in humans. Here we recruited a total of 1031 Chinese individuals aged between 12 and 111 years, including 108 families with parents and their offspring. DNA was extracted from peripheral white blood cells and TL was measured by quantitative PCR (qPCR). We explored the associations of TL with age, gender and clinical variables, and tested the parental effects on TL variation. First, we found that TL was shortened with age, however, TL was better maintained in females than males. Second, there was a robust association of TL between mother and offspring, but not between father and their offspring. In addition, TL was inversely associated with visceral fat index in females, and positively associated with apolipoprotein A levels. Knockdown of the key genes for lipid metabolism (PNPLA2 and CPT1) shortened the TL in HepG2 cells. These findings indicate that TL is maternally inherited, and impairment of lipid metabolism may contribute to the TL shortening in the Chinese population.

SCFAs improve disease resistance via modulate gut microbiota, enhance immune response and increase antioxidative capacity in the host.

To evaluate the effects of dietary short chain fatty acids (SCFAs) on the intestinal health and innate immunity in crucian carp, a six-week feeding trial was carried out with following treatments: basal diet (BD), basal diet supplementation with 1% sodium acetate (BDSA), basal diet supplementation with 1% sodium propionate (BDSP) and basal diet supplementation with 1% sodium butyrate (BDSB). The results showed dietary BDSA, BDSP and BDSB could protect the host against oxidative stress by improving the activity of certain antioxidative enzymes (T-SOD, GSH-Px and CAT). Additionally, dietary SCFAs could enhance mucosal and humoral immune responses by improving certain innate immune parameters in serum and skin mucus productions (IgM, ACH50 and T-SOD). Furthermore, dietary BDSA and BDSP could up-regulate the expression of immune related genes (TNF-α, TGF-ß and IL-8) and tight junction protein genes (occludin and ZO-1). Dietary BDSB could also elevate the expression of IL-8, TGF-ß, ZO-1 and Occludin in the midgut. Although dietary differences of SCFAs didn't alter the α-diversity of the intestinal flora, they altered the core microbiota. Finally, the challenge trial showed that dietary basal diet supplementation with SCFAs could protect zebrafish against Aeromonas hydrophila. These results suggest that dietary SCFAs could improve innate immunity, modulate gut microbiota and increase disease resistance in the host, which indicated the potential of SCFAs as immunostimulants in aquaculture.

Test-Retest Reliability of a New Device Versus a Long-Arm Goniometer to Evaluate Knee Proprioception.

CONTEXT: Many methods used to evaluate knee proprioception have shortcomings that limit their use in clinical settings. Based on an inexpensive 3D camera, a new portable device was recently used to evaluate the joint position sense (JPS) of the knee joint. However, the test-retest reliability of the new method remains unclear. This study aimed to evaluate the test-retest reliability of the new device and a long-arm goniometer for assessing knee JPS, and to compare the variability of the 2 methods. DESIGN: Prospective observational study of the test-retest reliability of knee JPS measurements. METHODS: Twenty-one healthy adults were tested in 2 sessions with a 1-week interval. Three target knee flexion angles (30°, 45°, and 60°) were reproduced in each session. Target and reproduced angles were measured with both methods. Intraclass correlation coefficients, standard error of the measurement, and Bland-Altman plots were used to quantify test-retest reliability. Paired t tests were used to compare knee JPS (absolute error of the target-reproduced angle) between the methods. RESULTS: The new device (good to excellent intraclass correlation coefficients .74-.80; standard error of the measurement 0.52°-0.61°) demonstrated better test-retest reliability than the goniometer (poor to fair intraclass correlation coefficients .23-.43; standard error of the measurement 0.89°-2.07°) and better test-retest agreement (respective mean differences for the 30°, 45°, and 60° knee angles: 0.11°, 0.13°, and 0.41° for the new system; 0.84°, 1.52°, and 1.18° for the goniometer). The measurements (absolute errors of the target-reproduced angles) with the goniometer were significantly greater than those with the new device (P < .05); the SDs of repeated measurements with the goniometer (1.50°-2.41°) were greater than with the new device (1.08°-1.38°). CONCLUSIONS: Given that the new device has good reliability and sufficient precision, it is the better alternative for evaluating knee JPS. Goniometers should be used with caution to assess knee JPS.

Photoredox-Catalyzed Difunctionalization of Unactivated Olefins for Synthesizing Lactam-Substituted gem-Difluoroalkenes.

Herein, the synthesis of lactam-substituted gem-difluoroalkenes has been developed through a photoredox-catalyzed radical cascade reaction. This developed photoredox-catalyzed, Brønsted base-assisted intramolecular 5-exo-trig cyclization/intermolecular radical addition/ß-fluoride elimination reaction offers a simple method for producing lactam, carbamate, or urea-substituted gem-difluoroalkenes with good functional group tolerance and high yields.

Quantifying Airborne Dispersal Route of Corynespora cassiicola in Greenhouses.

Target leaf spot (TLS), caused by Corynespora cassiicola, is an emerging and high-incidence disease that has spread rapidly on the global scale. Aerospores released by infected plants play a significant role in the epidemiology of cucumber TLS disease; however, no data exist concerning the infectiousness and particle size of C. cassiicola aerospores, and the experimental evidence for the aerospores transmission was lacking. In the present study, highly effective approaches to collect and quantify aerospores were developed for exposure chamber and greenhouse studies. Quantifiable levels of C. cassiicola aerospores were detected in 27 air samples from nine naturally infested greenhouses, ranging from 198 to 5,969 spores/m3. The C. cassiicola strains isolated from air samples were infective to healthy cucumber plants. Exposure chambers were constructed to study the characteristics of C. cassiicola aerospores released by artificially infested cucumber plants. The particle size of C. cassiicola ranged predominately from 2.1 to 4.7 µm, accounting for 71.97% of the total amount. In addition, the transmission dynamics of C. cassiicola aerospores from donor cucumber plants to recipient cucumber plants were confirmed in exposure chambers and greenhouses. The concentration of C. cassiicola aerospores was positively associated with cucumber TLS disease severity. This study suggested that aerospore dispersal is an important route for the epidemiology of plant fungal disease, and these data will contribute to the development of new strategies for the effective alleviation and control of plant diseases.

miRNA-451 regulates the NF-κB signaling pathway by targeting IKKß to inhibit glioma cell growth.

Glioblastoma multiforme (GBM) is associated with a poor prognosis, and effective treatments are lacking. Our previous studies have shown that miRNA-451 is closely related to the development and progression of glioma. miRNA-451 is a tumor suppressor whose expression is negatively correlated with the WHO grades of gliomas, but its specific mechanism is still unclear. Research shows that NF-κB is highly expressed in early malignant glioma, and thus, the NF-κB signaling pathway has become an important target for the treatment of malignant glioma. Activation of IKK is a critical step in the activation of the classical NF-κB pathway. By performing a bioinformatics analysis, we found that IKKß is a potential direct target of miRNA-451 in glioma. In this study, we transfected lentivirus expressing miRNA-451 to test the effect of miRNA-451 overexpression on malignant glioma cell lines and confirmed that IKKß is a target gene of miRNA-451 by luciferase assay. By targeting IKKß, MTT, cell invasion and wound-healing assays showed that cell proliferation, cell invasion and migration were significantly suppressed in the LV-miRNA-451 group. Western blotting results showed that the expression levels of IKKß, p-p65, MMP-2, MMP-9, Cyclin D1, p16 and PCNA were significantly decreased in the LV-miRNA-451 group. In vivo, miRNA-451 significantly decreased glioma cell growth, and the survival of BALB/c-A nude mice was significantly prolonged. Immunohistochemistry showed that p-p65, Cyclin D1 and Ki67 expression was significantly reduced in the LV-miRNA-451 group. Taken together, these results suggest that miRNA-451 could regulate the NF-κB signaling pathway by targeting IKKß, which inhibits glioma cell growth in vitro and in vivo. Therefore, this study may provide novel insight into miRNA-451-targeted therapy for glioma.

miR-451 suppresses EMT and metastasis in glioma cells.

The metastasis of tumor cells is a challenge for the clinical treatment of glioma. Epithelial-mesenchymal transition (EMT) contributes to glioma cell invasiveness. Our previous study confirmed that the expression of miRNA-451, which inhibits the PI3K/Akt signaling pathway by directly targeting CAB39 and plays a repressive role in glioma, is downregulated in glioma. However, the specific mechanism of miRNA-451 regulation in glioma is unclear. In this study, we investigated whether miRNA-451 blocks the processes of EMT and metastasis in glioma cells in vivo and in vitro. By targeting CAB39, miRNA-451 likely triggers the PI3K/Akt/Snail signaling pathway to reduce glioma proliferation, invasion, migration and EMT. We used Western blotting experiments to demonstrate that overexpression of miRNA-451 significantly reduced p-AKT(Ser473), N-cadherin, Vimentin, Twist, Snail and Cyclin D1 expression and increased E-cadherin expression. We demonstrated that overexpression of miR-451 suppressed glioma cell proliferation, invasion, migration and EMT by MTT and colony formation assays, Transwell assays, wound healing assays and animal experiments. Taken together, these results suggest that miRNA-451 can reduce EMT and metastasis in glioma cells through the suppression of the PI3K/Akt/Snail signaling pathway by targeting CAB39 in vitro and in vivo. miR-451 may be a new target for glioma treatment.

The Cutinase Bdo_10846 Play an Important Role in the Virulence of Botryosphaeria dothidea and in Inducing the Wart Symptom on Apple Plant.

Botryosphaeria dothidea is a pathogen with worldwide distribution, infecting hundreds of species of economically important woody plants. It infects and causes various symptoms on apple plants, including wart and canker on branches, twigs, and stems. However, the mechanism of warts formation is unclear. In this study, we investigated the mechanism of wart formation by observing the transection ultrastructure of the inoculated cortical tissues at various time points of the infection process and detecting the expression of genes related to the pathogen pathogenicity and plant defense response. Results revealed that wart induced by B. dothidea consisted of proliferous of phelloderm cells, the newly formed secondary phellem, and the suberized phelloderm cells surrounding the invading mycelia. The qRT-PCR analysis revealed the significant upregulation of apple pathogenesis-related and suberification-related genes and a pathogen cutinase gene Bdo_10846. The Bdo_10846 knockout transformants showed reduced cutinase activity and decreased virulence. Transient expression of Bdo_10846 in Nicotiana benthamiana induced ROS burst, callose formation, the resistance of N. benthamiana to Botrytis cinerea, and significant upregulation of the plant pathogenesis-related and suberification-related genes. Additionally, the enzyme activity is essential for the induction. Virus-induced gene silencing demonstrated that the NbBAK1 and NbSOBIR1 expression were required for the Bdo_10846 induced defense response in N. benthamiana. These results revealed the mechanism of wart formation induced by B. dothidea invasion and the important roles of the cutinase Bdo_10846 in pathogen virulence and in inducing plant immunity.

Candidate Effectors from Botryosphaeria dothidea Suppress Plant Immunity and Contribute to Virulence.

Fungal effectors play important roles in host-pathogen interactions. Botryosphaeria dothidea is an ascomycetous fungus that is responsible for the diseases of hundreds of woody plant species, including apple ring rot, which seriously affects apples worldwide. However, little is known about the effectors of B. dothidea. In this study, we analyzed the B. dothidea genome and predicted 320 candidate effector genes, 124 of which were successfully amplified and cloned. We investigated the effects of these genes on plant cell death in Nicotiana benthamiana while using a transient expression system. Twenty-four hours after initial inoculation with Agrobacterium tumefaciens cells carrying candidate effectors, the infiltrated leaves were challenged with A. tumefaciens cells carrying the BAX gene. In total, 116 candidate effectors completely inhibited, while one partially inhibited, the programmed cell death (PCD) of N. benthamiana induced by BAX, whereas seven candidate effectors had no effect. We then further tested seven candidate effectors able to suppress BAX-triggered PCD (BT-PCD) and found that they all completely inhibited PCD triggered by the elicitors INF1, MKK1, and NPK1. This result suggests that these effectors were activated in order to suppress pathogen-associated molecular pattern-triggered immunity. The signal peptides of these candidate effectors exhibited secretory activity in yeast (pSUC2 vector). Moreover, the respective deletion of Bdo_11198 and Bdo_12090 significantly reduced the virulence of B. dothidea. These results suggest that these effectors play important roles in the interaction of B. dothidea with its hosts.