The complete mitochondrial genome of Mya japonica (Jay, 1857 Myida:myidae).

The soft-shell clam Mya japonica (Jay, 1857) is a commercially important fishery resource. In this study, we identified the complete mitochondrial genome of M. japonica and performed a phylogenetic analysis to explore its genetic relationship with Mya arenaria. The genome is 21,396 bp in length and contains 13 protein-coding genes (PCGs), 23 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 5 D-Loop control regions. The atp8 gene was annotated in Myidae for the first time. Notably, the genome contains an additional trnM, consistent with M. arenaria. The length of the cox2 gene is 1,947 bp, which is 513 bp longer than that in M. arenaria. Its base composition is 29.14% A, 37.26% T, 10.89% C, and 22.71% G. Phylogenetic analysis based on 12 PCGs and 2 rRNAs indicates that M. japonica and M. arenaria form a sister group. In this study, the identification and phylogenetic analysis of the complete mitochondrial genome of M. japonica provide significant information for future taxonomic and evolutionary research of the genus Mya.

Deciphering the functional assembly of microbial communities driven by heavy metals in the tidal soils of Hangzhou Bay.

Understanding the interaction between heavy metals and soil microbiomes is essential for maintaining ecosystem health and functionality in the face of persistent human-induced challenges. This study investigated the complex relationships between heavy metal contamination and the functional characteristics of soil microbial communities in the tidal soils of Hangzhou Bay, a region experiencing substantial environmental pressure due to its proximity to densely populated and industrialized regions. The north-shore sampling site showed moderate contaminations (mg/kg) of total arsenic (16.61 ± 1.13), cadmium (0.3 ± 0.05), copper (31.28 ± 1.23), nickel (37.44 ± 2.74), lead (34.29 ± 5.99), and zinc (120.8 ± 5.96), which are 1.29-2.94 times higher than the geochemical background values in Hangzhou Bay and adjacent areas. In contrast, the south-shore sampling site showed slightly higher levels of total arsenic (13.76 ± 1.35) and cadmium (0.13 ± 0.02) than the background values. Utilizing metagenomic sequencing, we decoded microbial functional genes essential for nitrogen, phosphorus, sulfur, and methane biogeochemical cycles. Although soil available nickel content was relatively low at 1 mg/kg, it exhibited strong associations with diverse microbial genes and biogeochemical pathways. Four key genes-hxlB, glpX, opd, and phny-emerged as pivotal players in the interactions with available nickel, suggesting the adaptability of microbial metabolic responses to heavy metal. Additionally, microbial genera such as Gemmatimonas and Ilumatobacter, which harbored diverse functional genes, demonstrated potential interactions with soil nickel. These findings highlight the importance of understanding heavy metal-soil microbiome dynamics for effective environmental management strategies in the tidal soils of Hangzhou Bay, with the goal of preserving ecosystem health and functionality amidst ongoing anthropogenic challenges.

Green control for inhibiting Rhizopus oryzae growth by stress factors in forage grass factory.

The forage grass factory could break through the restrictions of land resources, region and climate to achieve efficient production throughout the year by accurate and intelligent management. However, due to its closed environment, mold outbreaks in the forage grass factory were severe, significantly affecting barley production. In this study, 9 contaminated barley tissues were collected and 45 strains were isolated in forage grass factory. After ITS sequencing, 45 strains were all identified as Rhizopus oryzae. Through stress factor assays, R. oryzae growth was seriously hindered by low concentration of sodium nitrate, high pH value and ozone water treatment. High pH and ozone water affected growth mainly by altering membrane integrity of R. oryzae. Sodium nitrate inhibited the growth of R. oryzae mainly by affecting the amount of sporulation. Low concentration of sodium nitrate and ozone water did not affect the growth of barley. High concentrations of sodium nitrate (100 mM) and pH values (8-8.5) inhibited barley growth. Among them, ozone water had the most obvious inhibition effect on R. oryzae. Large-scale ozone water treatment in the forage grass factory had also played a role in restoring barley production. Taken together, the green techonology to control mold disease and maintain the safety of forage through different physicochemical methods was selected, which was of considerable application value in animal husbandry.

Analysis of lettuce transcriptome reveals the mechanism of different light/dark cycle in promoting the growth and quality.

Light/dark (L/D) cycle plays a crucial role in controlling the production and quality of vegetables. However, the mechanism of L/D cycle on vegetable growth and quality is scarce studied. To investigate the impact of L/D cycle on lettuce growth and quality, we designed three diel scenarios, including 16 hours of light and 8 hours of darkness (L16/D8), 12 hours of light and 6 hours of darkness (L12/D6), and 8 hours of light and 4 hours of darkness (L8/D4). By phenotypic analysis, we found that lettuce grew taller under the L8/D4 scenario than under L16/D8 light cycle scenarios. The physiological indexes showed that the lettuce leaves grown in the L8/D4 scenario exhibited greater enhancements in the levels of soluble protein, soluble sugar, and carotenoid content compared to the other scenarios. By comparing the expression levels under different diel scenarios (L16/D8 vs L12/D6, L16/D8 vs L8/D4, and L12/D6 vs L8/D4), we identified 7,209 differentially expressed genes (DEGs). Additionally, 3 gene modules that were closely related to L/D cycle of lettuce were selected by WGCNA analysis. The eigengenes of three gene modules were enriched in plant hormone signal transduction, sphingolipid metabolism, and nucleocytoplasmic transport pathways. Through network analysis, we identified six hub genes (CIP1, SCL34, ROPGEF1, ACD6, CcmB, and Rps4) in the three gene modules, which were dominant in plant circadian rhythms and greatly affected lettuce growth. qRT-PCR analysis confirmed the diurnal response patterns of the 6 hub genes in different treatments were significant. This study intensively enhanced our comprehension of the L/D cycle in the growth morphology, nutritional quality, and metabolic pathways of lettuce.

Light Intensity Modulates the Functional Composition of Leaf Metabolite Groups and Phyllosphere Prokaryotic Community in Garden Lettuce (Lactuca sativa L.) Plants at the Vegetative Stage.

Light intensity primarily drives plant growth and morphogenesis, whereas the ecological impact of light intensity on the phyllosphere (leaf surface and endosphere) microbiome is poorly understood. In this study, garden lettuce (Lactuca sativa L.) plants were grown under low, medium, and high light intensities. High light intensity remarkably induced the leaf contents of soluble proteins and chlorophylls, whereas it reduced the contents of leaf nitrate. In comparison, medium light intensity exhibited the highest contents of soluble sugar, cellulose, and free amino acids. Meanwhile, light intensity resulted in significant changes in the composition of functional genes but not in the taxonomic compositions of the prokaryotic community (bacteria and archaea) in the phyllosphere. Notably, garden lettuce plants under high light intensity treatment harbored more sulfur-cycling mdh and carbon-cycling glyA genes than under low light intensity, both of which were among the 20 most abundant prokaryotic genes in the leaf phyllosphere. Furthermore, the correlations between prokaryotic functional genes and lettuce leaf metabolite groups were examined to disclose their interactions under varying light intensities. The relative abundance of the mdh gene was positively correlated with leaf total chlorophyll content but negatively correlated with leaf nitrate content. In comparison, the relative abundance of the glyA gene was positively correlated with leaf total chlorophyll and carotenoids. Overall, this study revealed that the functional composition of the phyllosphere prokaryotic community and leaf metabolite groups were tightly linked in response to changing light intensities. These findings provided novel insights into the interactions between plants and prokaryotic microbes in indoor farming systems, which will help optimize environmental management in indoor farms and harness beneficial plant-microbe relationships for crop production.

A hyperspectral deep learning attention model for predicting lettuce chlorophyll content.

BACKGROUND: The phenotypic traits of leaves are the direct reflection of the agronomic traits in the growth process of leafy vegetables, which plays a vital role in the selection of high-quality leafy vegetable varieties. The current image-based phenotypic traits extraction research mainly focuses on the morphological and structural traits of plants or leaves, and there are few studies on the phenotypes of physiological traits of leaves. The current research has developed a deep learning model aimed at predicting the total chlorophyll of greenhouse lettuce directly from the full spectrum of hyperspectral images. RESULTS: A CNN-based one-dimensional deep learning model with spectral attention module was utilized for the estimate of the total chlorophyll of greenhouse lettuce from the full spectrum of hyperspectral images. Experimental results demonstrate that the deep neural network with spectral attention module outperformed the existing standard approaches, including partial least squares regression (PLSR) and random forest (RF), with an average R2 of 0.746 and an average RMSE of 2.018. CONCLUSIONS: This study unveils the capability of leveraging deep attention networks and hyperspectral imaging for estimating lettuce chlorophyll levels. This approach offers a convenient, non-destructive, and effective estimation method for the automatic monitoring and production management of leafy vegetables.

Genome-Wide Identification and Characterization of Effector Candidates with Conserved Motif in Falciphora oryzae.

Microbes employ effectors to disrupt immune responses and promote host colonization. Conserved motifs including RXLR, LFLAK-HVLVxxP (CRN), Y/F/WxC, CFEM, LysM, Chitin-bind, DPBB_1 (PNPi), and Cutinase have been discovered to play crucial roles in the functioning of effectors in filamentous fungi. Nevertheless, little is known about effectors with conserved motifs in endophytes. This research aims to discover the effector genes with conserved motifs in the genome of rice endophyte Falciphora oryzae. SignalP identified a total of 622 secreted proteins, out of which 227 were predicted as effector candidates by EffectorP. By utilizing HMM features, we discovered a total of 169 effector candidates with conserved motifs and three novel motifs. Effector candidates containing LysM, CFEM, DPBB_1, Cutinase, and Chitin_bind domains were conserved across species. In the transient expression assay, it was observed that one CFEM and one LysM activated cell death in tobacco leaves. Moreover, two CFEM and one Chitin_bind inhibited cell death induced by Bax protein. At various points during the infection, the genes' expression levels were increased. These results will help to identify functional effector proteins involving omics methods using new bioinformatics tools, thus providing a basis for the study of symbiosis mechanisms.

Symbiotic relationship of Comaster schlegelii (Crinoidea: Comatulidae) And Gymnolophus obscura (Ophiuroidea: Ophiotrichidae) derived from stable isotope and fatty acid analyses.

Coral reef community exhibit high species diversity and a broad range of biological relationships including widespread symbiosis and complex food utilization patterns. In our study, we investigated the symbiotic relationship between the commonly crinoid host Comaster schlegelii and its ophiuroid obligatory symbiont Gymnolophus obscura. Using a combination of fatty acid biomarkers and stable isotopic compositions, we explored differences in their organic matter utilization strategies and nutritional relationships. The result of stable isotopes revealed that G. obscura had higher δ15N values than its crinoid host. Particulate organic matter and phytoplankton were identified as the primary food sources for both species, however C. schlegelii showed a higher proportional contribution from benthic microalgae. Fatty acid markers showed that C. schlegelii was more dependent on benthic microalgae such as diatoms, and less on debritic organic matter and bacteria than G. obscura. Elevated δ15N values of G. obscura and similar food source contribution rates between the host and symbiont suggest that ophiuroid feeds on materials filtered by crinoids and have similar diet to the host. Our results provide insights into the symbiotic patterns of crinoids and ophiuroids, while also supplying foundational data on how symbiotic reef species select organic matter utilization strategies to adapt to their environment.

Design, Synthesis, and Pharmacological Evaluation of Isoindoline Analogues as New HPK1 Inhibitors.

Hematopoietic progenitor kinase 1 (HPK1) is an important negative regulator in T-cell receptor signaling and as a promising key target for immunotherapy. Herein, based on the reported HPK1 inhibitor 2 featuring an isofuranone component, a structural optimization approach was conducted leading to several series of derivatives characterized by containing an isoindoline structural motif. Compound 49 was identified as a new potent HPK1 inhibitor with an IC50 value of 0.9 nM, more potent than compound 2 (5.5 nM). It also has an improved IV profile in rats and enhanced aqueous solubility. It effectively inhibited pSLP76 and reinvigorated T-cell receptor (TCR) signaling, promoting T-cell function and cytokine production both in naïve and antigen-specific T cells. Furthermore, compound 49 reversed the inhibition on T-cell activity mediated by classic immunosuppressive factors in the tumor microenvironment (TME). In the murine CT-26 tumor model, this compound reinvigorated the T cell and synergistically enhanced the antitumor efficacy of anti-PD1 at a well-tolerant dosage.

Estimation of rice seedling growth traits with an end-to-end multi-objective deep learning framework.

In recent years, rice seedling raising factories have gradually been promoted in China. The seedlings bred in the factory need to be selected manually and then transplanted to the field. Growth-related traits such as height and biomass are important indicators for quantifying the growth of rice seedlings. Nowadays, the development of image-based plant phenotyping has received increasing attention, however, there is still room for improvement in plant phenotyping methods to meet the demand for rapid, robust and low-cost extraction of phenotypic measurements from images in environmentally-controlled plant factories. In this study, a method based on convolutional neural networks (CNNs) and digital images was applied to estimate the growth of rice seedlings in a controlled environment. Specifically, an end-to-end framework consisting of hybrid CNNs took color images, scaling factor and image acquisition distance as input and directly predicted the shoot height (SH) and shoot fresh weight (SFW) after image segmentation. The results on the rice seedlings dataset collected by different optical sensors demonstrated that the proposed model outperformed compared random forest (RF) and regression CNN models (RCNN). The model achieved R2 values of 0.980 and 0.717, and normalized root mean square error (NRMSE) values of 2.64% and 17.23%, respectively. The hybrid CNNs method can learn the relationship between digital images and seedling growth traits, promising to provide a convenient and flexible estimation tool for the non-destructive monitoring of seedling growth in controlled environments.

SYK-mediated epithelial cell state is associated with response to c-Met inhibitors in c-Met-overexpressing lung cancer.

Genomic MET amplification and exon 14 skipping are currently clinically recognized biomarkers for stratifying subsets of non-small cell lung cancer (NSCLC) patients according to the predicted response to c-Met inhibitors (c-Metis), yet the overall clinical benefit of this strategy is quite limited. Notably, c-Met protein overexpression, which occurs in approximately 20-25% of NSCLC patients, has not yet been clearly defined as a clinically useful biomarker. An optimized strategy for accurately classifying patients with c-Met overexpression for decision-making regarding c-Meti treatment is lacking. Herein, we found that SYK regulates the plasticity of cells in an epithelial state and is associated with their sensitivity to c-Metis both in vitro and in vivo in PDX models with c-Met overexpression regardless of MET gene status. Furthermore, TGF-ß1 treatment resulted in SYK transcriptional downregulation, increased Sp1-mediated transcription of FRA1, and restored the mesenchymal state, which conferred resistance to c-Metis. Clinically, a subpopulation of NSCLC patients with c-Met overexpression coupled with SYK overexpression exhibited a high response rate of 73.3% and longer progression-free survival with c-Meti treatment than other patients. SYK negativity coupled with TGF-ß1 positivity conferred de novo and acquired resistance. In summary, SYK regulates cell plasticity toward a therapy-sensitive epithelial cell state. Furthermore, our findings showed that SYK overexpression can aid in precisely stratifying NSCLC patients with c-Met overexpression regardless of MET alterations and expand the population predicted to benefit from c-Met-targeted therapy.

Purification and characterization of bacteriocin produced by a strain of Lacticaseibacillus rhamnosus ZFM216.

The recent surge in demand for natural preservatives has ushered in a new era of research into novel bacteriocins capable of effectively combating food-borne infections. In this study, the bacteriocin from Lacticaseibacillus rhamnosus ZFM216, which has a molecular mass of 11851.9 Da, was purified using macroporous resin, gel chromatography, and reversed-phase high performance liquid chromatography. This bacteriocin could inhibit both Gram-positive and Gram-negative bacteria. It had a strong inhibitory effect on Staphylococcus aureus D48 with minimum inhibitory concentration values of 1.75 µM. Bacteriocin ZFM216 was heat stable and showed pH stability under weakly acidic conditions. It was sensitive to pepsin, proteinase K and trypsin. Electron microscopy results showed that when treated with bacteriocin ZFM216, S. aureus D48 was severely deformed, the cell structure was obviously changed, and the intracellular electrolyte leaked to the outside of the cell. Bacteriocin ZFM216 caused the ATP level of the indicator to decrease, the conductivity to sharply increase, and the transmembrane potential difference (ΔΨ) to instantaneously decrease. This research formed the basis for further development and utilization of bacteriocin ZFM216 which has potential in the food industry.

TLR Signaling Pathway Gene Polymorphisms, Gene-Gene and Gene-Environment Interactions in Allergic Rhinitis.

Background: Allergic rhinitis (AR) is a nasal inflammatory disease resulting from a complex interplay between genetic and environmental factors. The association between Toll-like receptor (TLR) signaling pathway and environmental factors in AR pathogenesis remains to be explored. This study aims to assess the genetic association of AR with single nucleotide polymorphisms (SNPs) in TLR signaling pathway, and investigate the roles of gene-gene and gene-environment interactions in AR. Methods: A total of 452 AR patients and 495 healthy controls from eastern China were enrolled in this hospital-based case-control study. We evaluated putatively functional genetic polymorphisms in TLR2, TLR4 and CD14 genes for their association with susceptibility to AR and related clinical phenotypes. Interactions between environmental factors (such as traffic pollution, residence, pet keeping) and polymorphisms with AR were examined using logistic regression. Models were stratified by genotype and interaction terms, and tested for the significance of gene-gene and gene-environment interactions. Results: In the single-locus analysis, two SNPs in CD14, rs2563298 (A/C) and rs2569191 (C/T) were associated with a significantly decreased risk of AR. Compared with the GG genotype, the GT and GT/TT genotypes of TLR2 rs7656411 (G/T) were associated with a significantly increased risk of AR. Gene-gene interactions (eg, TLR2 rs7656411, TLR4 rs1927914, and CD14 rs2563298) was associated with AR. Gene-environment interactions (eg, TLR4 or CD14 polymorphisms and certain environmental exposures) were found in AR cases, but they were not significant after Bonferroni correction. Conclusion: The genetic polymorphisms of TLR2 and CD14 and gene-gene interactions in TLR signaling pathway were associated with susceptibility to AR in this Han Chinese population. However, the present results were limited to support the association between gene-environment interactions and AR.

A New Species in Pseudophialophora From Wild Rice and Beneficial Potential.

Wild rice (Oryza granulata) is a natural resource pool containing abundant unknown endophytic fungi species. There are few reports on the endophytic fungi in wild rice. Here, one isolate recovered from wild rice roots was identified as a new species Pseudophialophora oryzae sp. nov based on the molecular phylogeny and morphological characteristics. Fluorescent protein-expressing P. oryzae was used to monitor the fungal colonization pattern. Hyphae invaded the epidermis to the inner cortex but not into the root stele. The inoculation of P. oryzae promoted the rice growth, with the growth parameters of chlorophyll content, shoot height, root length, fresh shoot weight, fresh root weight and dry weight increasing by 24.10, 35.32, 19.35, 90.00, 33.3, and 79.17%, respectively. P. oryzae induced up-regulation of nitrate transporter OsPTR9 and potassium transporter OsHAK16 by 7.28 ± 0.84 and 2.57 ± 0.80 folds, promoting nitrogen and potassium elements absorption. In addition, P. oryzae also conferred a systemic resistance against rice blast, showing a 72.65 and 75.63% control rate in sterile plates and potting conditions. This systemic resistance was mediated by the strongly up-regulated expression of resistance-related genes NAC, OsSAUR2, OsWRKY71, EL5, and PR1α. Since P. oryzae can promote rice growth, biomass and induce systemic disease resistance, it can be further developed as a new biogenic agent for agricultural production, providing a new approach for biocontrol of rice blast.

Genetic variants in Hippo pathway genes are associated with house dust mite-induced allergic rhinitis in a Chinese population.

BACKGROUND: House dust mite (HDM)-induced allergic rhinitis (AR) is a highly prevalent disease with bothersome symptoms. Genetic variants of the Hippo pathway genes play a critical role in the respiratory disease. However, no study has reported associations between variants of the Hippo pathway genes and HDM-induced AR risk. METHODS: Forty-three key genes in the Hippo pathway were selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome pathway database, and previous reported studies. A case-control study of 222 cases and 237 controls was performed to assess the associations between 121 genetic variants in these genes and HDM-induced AR risk. DNeasy Blood & Tissues Kits were used for extracting genomic DNA from the venous blood and Infinium Asian Screening Array BeadChips for performing genotyping. A logistic regression model was applied to evaluate the effects of variants on HDM-induced AR risk. The false discovery rate (FDR) method was utilized to correct for multiple testing. The receiver operating characteristic (ROC) curve was plotted to obtain the cut-off value of total IgE for the diagnosis of HDM-induced AR. Histone modification and transcription factor binding sites were visualized by UCSC genome browser. Moreover, expression qualitative trait loci (eQTL) analysis was obtained from Genotype-Tissue Expression (GTEx) database. RESULTS: We found that rs754466 in DLG5 was significantly associated with a decreased HDM-induced AR risk after FDR correction (adjusted odds ratio [OR] = 0.52, 95% confidence interval [CI] = 0.36-0.74, p = 3.25 × 10-4 , PFDR  = 3.93 × 10-2 ). The rs754466 A allele reduced the risk of HDM-induced AR in the subgroup of moderate/severe total nasal symptom score (TNSS). Furthermore, rs754466 was associated with a high mRNA expression of DLG5. Additionally, histone modification and transcription factor binding sites were rich in the region containing rs754466. CONCLUSION: Our findings indicated that rs754466 in DLG5 decreased the susceptibility to HDM-induced AR.

The Endophytic Fungus Piriformospora Indica-Assisted Alleviation of Cadmium in Tobacco.

Increasing evidence suggests that the endophytic fungus Piriformospora indica helps plants overcome various abiotic stresses, especially heavy metals. However, the mechanism of heavy metal tolerance has not yet been elucidated. Here, the role of P. indica in alleviating cadmium (Cd) toxicities in tobacco was investigated. It was found that P. indica improved Cd tolerance to tobacco, increasing Cd accumulation in roots but decreasing Cd accumulation in leaves. The colonization of P. indica altered the subcellular repartition of Cd, increasing the Cd proportion in cell walls while reducing the Cd proportion in membrane/organelle and soluble fractions. During Cd stress, P. indica significantly enhanced the peroxidase (POD) activity and glutathione (GSH) content in tobacco. The spatial distribution of GSH was further visualized by Raman spectroscopy, showing that GSH was distributed in the cortex of P. indica-inoculated roots while in the epidermis of the control roots. A LC-MS/MS-based label-free quantitative technique evaluated the differential proteomics of P. indica treatment vs. control plants under Cd stress. The expressions of peroxidase, glutathione synthase, and photosynthesis-related proteins were significantly upregulated. This study provided extensive evidence for how P. indica enhances Cd tolerance in tobacco at physiological, cytological, and protein levels.

A Novel Bacteriocin From Lactobacillus Pentosus ZFM94 and Its Antibacterial Mode of Action.

Bacteriocins are bioactive antimicrobial peptides synthesized in the ribosome of numerous bacteria and released extracellularly. Pentocin ZFM94 produced by Lactobacillus pentosus (L. pentosus) ZFM94, isolated from infant feces with strong antibacterial activity, was purified by ammonium sulfate precipitation, dextran gel chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of the purified bacteriocin was 3,547.74 Da determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Pentocin ZFM94 exhibited broad-spectrum antimicrobial activity against tested Gram-positive and Gram-negative bacteria, and the minimal inhibitory concentrations (MICs) of Micrococcus luteus (M. luteus) 10,209, Staphylococcus aureus (S. aureus) D48, and Escherichia coli (E. coli) DH5α were 1.75, 2.00, and 2.50 µm, respectively. Pentocin ZFM94 was heat-stable (30 min at 80°C) and showed inhibitory activity over a wide pH range (5.00-7.00). It could be degraded by trypsin and pepsin, but not by amylase, lysozyme, lipase, and ribonuclease A. Fluorescence leakage assay showed that pentocin ZFM94 induced disruption of the cell membrane and caused leakage of cellular content. Furthermore, lipid II was not an antibacterial target of pentocin ZFM94. This study laid the foundation for further development and utilization of L. pentosus ZFM94 and its bacteriocin.

An efficient genetic manipulation protocol for dark septate endophyte Falciphora oryzae.

OBJECTIVE: To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae. RESULTS: F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9 M KCl solution and 10 mg mL-1 glucanase at 30 °C with shaking at 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar medium containing 1 M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots. CONCLUSIONS: This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.

Dark septate endophyte Falciphora oryzae-assisted alleviation of cadmium in rice.

Dark septate endophytes (DSEs) are the typical representatives of root endophytic fungi in heavy metal (HM)-contaminated environments. However, little is known about their roles in the HMs tolerance of hosts and the underlying mechanism. Here, we investigated the biological roles and molecular mechanisms of a DSE strain Falciphora oryzae in alleviating cadmium (Cd) toxicities in rice. It was found that F. oryzae possessed a capacity of accumulating Cd in its vacuoles and chlamydospores. During symbiosis, F. oryzae conferred improved Cd tolerance to rice, decreasing Cd accumulation in roots and translocation to shoots. F. oryzae alleviated Cd toxicity to rice by sequestering Cd in its vacuoles. Further application of F. oryzae as fertilizer in the field could reduce Cd content in rice grains. We identified a SNARE Syntaxin 1 gene through proteomics, which participated in Cd tolerance of F. oryzae by regulating chlamydospore formation and vacuole enlargement. This study provided novel insights into how the DSEs and their host plants combat Cd stress.

Isolation and Functional Analysis of Effector Proteins of Magnaporthe oryzae.

In nature, plants have evolved a myriad of preformed and induced defenses to protect themselves from microbes. Upon microbial infection, the recognition of the microbe-associated molecular patterns (MAMPs) by the pattern recognition receptors (PRRs) triggers the first stage of defense response (Dodds and Rathjen, Nat Rev Genet 11:539-548, 2010). However, in order to develop microbial delivery, effectors target PRRs for deregulating immune responses and facilitating host colonization (Thomma et al., Plant Cell 23:4-15, 2011). Here, we contribute a protocol for the screening system of Magnaporthe oryzae effectors and construct a fluorescent system to trace secretory proteins in the sheath infection samples. Using the tobacco rattle virus (TRV) system, the proteins including LysM, Chitin, Cutinase, and CFEM domains were selected and divided into two kinds according to the results of cell death induced or inhibited test in Nicotiana benthamiana. Then, candidate effectors can be deleted or overexpressed in M. oryzae. The barley or rice infection with M. oryzae, rice leaf sheath inoculation, and subcellular localization during infection can be performed to explore the functions of these effectors.