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Fetal growth restriction (FGR) is characterized by impaired fetal growth and dysregulated lipid metabolism. Extracellular vesicles (EVs) have been proved playing a crucial role in transporting biomolecules from the mother to the fetus. However, the mechanisms underlying cargo sorting and loading into trophoblastic EVs remain elusive. This study focuses on examining how the essential fatty acid regulator, peroxisome proliferator-activated receptor gamma (PPARγ), is sorted and loaded into EVs originating from trophoblasts. We conducted proteomic analysis on placenta-derived EVs from normal and FGR pregnancies. Interactions between PPARγ and coat protein complex I (COPI) subunit were evaluated using co-immunoprecipitation and bioinformatics simulation. Molecular dynamics simulations were conducted to identify critical binding sites between β’-coat protein complex I (β’-COP), a subunit of COPI, and PPARγ. lentivirus-mediated knockout and overexpression techniques were employed to elucidate the role of β’-COP in PPARγ loading into EVs. Our findings demonstrate that PPARγ protein levels are significantly decreased in EVs from FGR placentas. β’-COP subunit directly interacts with PPARγ in trophoblasts, mediating its sorting into early endosomes and multivesicular bodies for EVs incorporation. Knockout of β’-COP impaired PPARγ loading into EVs. Molecular dynamics simulations identified critical binding sites for the interaction between β’-COP and PPARγ. Mutation of these sites significantly weakened the β’-COP-PPARγ interaction and reduced PPARγ levels in trophoblastic EVs. In conclusion, β’-COP mediates sorting and loading of PPARγ into trophoblastic EVs. This study provides insights into regulating EVs cargo loading and potential strategies for targeted cargo delivery from the maternal to the fetal circulation.

• During plant-pathogenic fungi and host plants interactions, numerous pathogen-derived proteins are secreted resulting in the activation of the unfolded protein response (UPR) pathway. For efficient trafficking of secretory proteins, including those important in disease progression, the cytoplasmic coat protein complex II (COPII) exhibits a multifunctional role whose elucidation remains limited. • Here, we discovered that the COPII cargo receptor MoErv29 functions as a target of MoHac1, a previously identified transcription factor of the UPR pathway. In Magnaporthe oryzae, deletion of MoERV29 severely affected the vegetative growth, conidiation and biotrophic invasion of the fungus in susceptible rice hosts. • We demonstrated that MoErv29 is required for the delivery of secreted proteins through recognition and binding of the amino-terminal tripeptide motifs following the signal peptide. By using bioinformatics analysis, we predicted a cargo spectrum of MoErv29 and found that MoErv29 is required for the secretion of many proteins, including extracellular laccases and apoplastic effectors. This secretion is mediated through the conventional endoplasmic reticulum–Golgi secretion pathway and is important for conferring host recognition and disease resistance. • Taken together, our results revealed how MoErv29 operates on effector secretion, and our findings provided a critical link between COPII vesicle trafficking and the UPR pathway.

Background CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. Results CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Conclusions Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

Summary Isoflavonoids, which include a variety of secondary metabolites, are derived from the phenylpropanoid pathway and are distributed predominantly in leguminous plants. These compounds play a critical role in plant–environment interactions and are beneficial to human health. Isoflavone synthase (IFS) is a key enzyme in isoflavonoid synthesis and shares a common substrate with flavanone‐3‐hydroxylase (F3H) and flavone synthase II (FNS II). In this study, CRISPR/Cas9‐mediated multiplex gene‐editing technology was employed to simultaneously target GmF3H1, GmF3H2 and GmFNSII‐1 in soya bean hairy roots and plants. Various mutation types and frequencies were observed in hairy roots. Higher mutation efficiencies were found in the T0 transgenic plants, with a triple gene mutation efficiency of 44.44%, and these results of targeted mutagenesis were stably inherited in the progeny. Metabolomic analysis of T0 triple‐mutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T3 generation homozygous triple mutants had approximately twice the leaf isoflavone content, and the soya bean mosaic virus (SMV) coat protein content was significantly reduced by one‐third after infection with strain SC7, suggesting that increased isoflavone content enhanced the leaf resistance to SMV. The isoflavone content in the seeds of T2 triple mutants was also significantly increased. This study provides not only materials for the improvement of soya bean isoflavone content and resistance to SMV but also a simple system to generate multiplex mutations in soya bean, which may be beneficial for further breeding and metabolic engineering.

The tomato chlorosis virus (ToCV) is one of the most destructive plant viruses affecting tomato crops, leading to significant agricultural losses. As an obligate parasite, ToCV depends on the macromolecular machinery of host cells for replication. The ubiquitin 26S proteasome system maintains the intracellular protein homeostasis, which is essential for plant growth and development. Our study found that the CPm protein of ToCV interacted with SlPAD1, a component of the 26S proteasome, to enhance viral infection. This interaction disrupts the binding between SlPAD1 and SlPA4, thereby impairing the 26S proteasome function. In addition, SlPAD1 and SlPA4 positively regulate plant resistance to ToCV. Our findings reveal a mechanism by which ToCV proteins facilitate infection by interfering with 26S proteasome function. CPm interacts with tomato SlPAD1 to block 26S proteasome function. Under physiological conditions, tomato SlPAD1 binds SlPA4 to form the 26S proteasome, maintaining protein homeostasis. During ToCV infection, viral CPm competitively interacts with SlPAD1, impairing proteasome function and inhibiting degradation of ubiquitinated proteins, thereby promoting viral pathogenesis.

Main conclusionExogenous application of dsRNA molecules targeting MYMV genes offers a promising approach to effectively mitigate yellow mosaic disease in blackgram, demonstrating potential for sustainable plant viral disease management.The exogenous application of double-stranded RNA (dsRNA) molecules to control plant viral diseases is gaining traction due to its advantages over conventional methods, such as target specificity, non-polluting nature, and absence of residue formation. Furthermore, this approach does not involve genome modification. In this study, dsRNA molecules targeting the coat protein gene (dsCP) and replication initiator protein gene (dsRep) of mungbean yellow mosaic virus (MYMV) were synthesised using an in vitro transcription method. To evaluate the effectiveness of dsRNA treatment, blackgram plants exhibiting MYMV symptoms at the first trifoliate stage were subjected to exogenous application of dsRNA. Second, third, and fourth trifoliate leaves, which emerged at 7, 15, and 21 days after dsRNA application, respectively, were monitored for MYMV symptoms. Remarkably, a significant reduction in yellow mosaic disease (YMD) symptoms was observed in the newly emerged trifoliate leaves of MYMV-infected blackgram plants after treatment with dsRNA targeting both gene regions. This reduction was evident as a decrease in the intensity of yellow mosaic coverage on the leaf lamina compared to control. dsCP effectively reduced the MYMV titre in the treated plants for up to 15 days. However, dsRep demonstrated greater efficiency in conferring resistance to MYMV at 15 days post-application. These findings were supported by quantitative real-time PCR analysis, where the observed Ct values for DNA extracted from dsRep-treated plants were significantly higher compared to the Ct values of DNA from dsCP-treated plants at 15 days post-application. Similarly, higher viral copy numbers were observed in dsCP-treated plants 15 days after dsRNA treatment, in contrast to plants treated with dsRep.

Summary Rice black‐streaked dwarf virus (RBSDV), a member of the genus Fijivirus, is a devastating pathogen of crop plants. RBSDV S10 encodes a capsid protein (P10) that is an important component of the double‐layered particle. However, little information is available on the roles of RBSDV P10 in viral infection or in interactions with other viruses. Here, we demonstrate that the expression of P10 in plants alleviates the symptoms of both RBSDV and the closely related Southern rice black‐streaked dwarf virus (SRBSDV), and reduces the disease incidence, but renders the plants more susceptible to the unrelated Rice stripe virus (RSV). Further experiments suggest that P10‐mediated resistance to RBSDV and SRBSDV operates at the protein level, rather than the RNA level, and is not a result of post‐transcriptional gene silencing. Transcriptomic data reveal that the expression of P10 in plants significantly suppresses the expression of rice defence‐related genes, which may play important roles in resistance to RSV infection. After infection with RBSDV, plants are more resistant to subsequent challenge by SRBSDV, but more susceptible to RSV. Overall, these results indicate that P10 acts as an important effector in virus interactions.

• Plant pathogens exploit the extracellular matrix (ECM) to inhibit host immunity during their interactions with the host. The formation of ECM involves a series of continuous steps of vesicular transport events. • To understand how such vesicle trafficking impacts ECM and virulence in the rice blast fungus Magnaporthe oryzae, we characterised MoSwa2, a previously identified actin-regulating kinase MoArk1 interacting protein, as an orthologue of the auxilin-like clathrin uncoating factor Swa2 of the budding yeast Saccharomyces cerevisiae. • We found that MoSwa2 functions as an uncoating factor of the coat protein complex II (COPII) via an interaction with the COPII subunit MoSec24-2. Loss of MoSwa2 led to a deficiency in the secretion of extracellular proteins, resulting in both restricted growth of invasive hyphae and reduced inhibition of host immunity. Additionally, extracellular fluid (ECF) proteome analysis revealed that MoSwa2-regulated extracellular proteins include many redox proteins such as the berberine bridge enzyme-like (BBE-like) protein MoSef1. We further found that MoSef1 functions as an apoplastic virulent factor that inhibits the host immune response. • Our studies revealed a novel function of a COPII uncoating factor in vesicular transport that is critical in the suppression of host immunity and pathogenicity of M. oryzae.

Bacillus subtilis is a widely distributed aerobic Gram-positive species of bacteria. As a tool in the lab, it has the advantages of nonpathogenicity and limited likelihood of becoming drug resistant. It is a probiotic strain that can be directly used in humans and animals. It can be induced to produce spores under nutrient deficiency or other adverse conditions. B. subtilis spores have unique physical, chemical, and biochemical characteristics. Expression of heterologous antigens or proteins on the surface of B. subtilis spores has been successfully performed for over a decade. As an update and supplement to previously published research, this paper reviews the latest research on spore surface display technology using B. subtilis. We have mainly focused on the regulation of spore coat protein expression, display and application of exogenous proteins, and identification of developing research areas of spore surface display technology.

Distinct pathways for protein secretion have been revealed in plant cells, both conventional and unconventional. Advanced techniques have been developed to unveil their underlying mechanisms. Abstract Protein secretion is an essential process in all eukaryotic cells and its mechanisms have been extensively studied. Proteins with an N-terminal leading sequence or transmembrane domain are delivered through the conventional protein secretion (CPS) pathway from the endoplasmic reticulum (ER) to the Golgi apparatus. This feature is conserved in yeast, animals, and plants. In contrast, the transport of leaderless secretory proteins (LSPs) from the cytosol to the cell exterior is accomplished via the unconventional protein secretion (UPS) pathway. So far, the CPS pathway has been well characterized in plants, with several recent studies providing new information about the regulatory mechanisms involved. On the other hand, studies on UPS pathways in plants remain descriptive, although a connection between UPS and the plant defense response is becoming more and more apparent. In this review, we present an update on CPS and UPS. With the emergence of new techniques, a more comprehensive understanding of protein secretion in plants can be expected in the future.