Erythrocyte ghosts loaded with ' 25 1-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol . Erythrocyte-mediated microinjection of ' 25 1-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum.A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins . Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of shortlived proteins . Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h . Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone .The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination .Intracellular protein degradation is a fundamentally important process occurring in all organisms from bacteria to humans (5,20,23,24,53) . The continued breakdown and replacement of proteins allows the cell to regulate concentrations of specific enzymes as well as to alter overall protein content in response to changing physiological demands. The major areas of current study in protein degradation can be divided into three broad topics : (a) the influence of polypeptide structure on protein half-lives (20, 23), (b) the physiological regulation of protein degradative rates (5,20,24), and (c) the mechanisms by which proteins are degraded within cells (5,24,53) . Despite considerable recent progress in each ofthese areas, many ofthe major questions in this field of research remain unanswered.Microinjection offers several advantages for analysis of protein degradation over more conventional approaches in which endogenous cellular proteins are radiolabeled and their deg- radative rates determined (I1, 34, 51). Microinjection of mammalian cells in culture has been achieved using microneedles (6,55) or by inducing fusion of the recipient cell with erythrocyte ghosts containing the protein to be microinjected (6,34,51) . Most studies of protein degradation have used erythrocytemediated microinjection because sufficient numbers of cells can be microinject...

Sec4/Rab8 is one of the well-studied members of the Rab GTPase family, previous studies have shown that Sec4/Rab8 crucially promotes the pathogenesis of phytopathogens, but the upstream regulators of Rab8 are still unknown. Here, we have identified two Sec2 homologues FgSec2A and FgSec2B in devastating fungal pathogen Fusarium graminearum and investigated their functions and interactions with FgRab8 by live-cell imaging, genetic and functional analyses. Yeast two-hybrid assay shows that FgSec2A specifically interacts with FgRab8DN(N123I) and itself. Importantly, FgSec2A is required for growth, conidiation, DON production and virulence of F. graminearum. Live-cell imaging shows that FgSec2A and FgSec2B are both localized to the tip region of hyphae and conidia. Both N-terminal region and Sec2 domain of FgSec2A are essential for its function, but not for localization, whereas the C-terminal region is important for its polarized localization. Furthermore, constitutively active FgRab8CA(Q69L) partially rescues the defects of ΔFgsec2A. Consistently, FgSec2A is required for the polarized localization of FgRab8. Finally, FgSec2A and FgSec2B show partial functions, but FgSec2A does not interact and co-localize with FgSec2B. Taken together, these results indicate that FgSec2A acts as a FgRab8 guanine nucleotide exchange factor and is necessary for polarized growth, DON production and pathogenicity in F. graminearum.

Fusarium graminearum is a fungal pathogen that causes Fusarium head blight (FHB) in wheat and barley. Autophagy is a highly conserved vacuolar degradation pathway essential for cellular homeostasis in which Atg9 serves as a multispanning membrane protein important for generating membranes for the formation of phagophore assembly site. However, the mechanism of autophagy or autophagosome formation in phytopathogens awaits further clarifications. In this study, we identified and characterized the Atg9 homolog (FgAtg9) in F. graminearum by live cell imaging, biochemical and genetic analyses. We find that GFP-FgAtg9 localizes to late endosomes and trans-Golgi network under both nutrient-rich and nitrogen starvation conditions and also show its dynamic actin-dependent trafficking in the cell. Further targeted gene deletion of FgATG9 demonstrates that it is important for growth, aerial hyphae development, and pathogenicity in F. graminearum. Furthermore, the deletion mutant (ΔFgatg9) shows severe defects in autophagy and lipid metabolism in response to carbon starvation. Interestingly, small GTPase FgRab7 is found to be required for the dynamic trafficking of FgAtg9, and co-immunoprecipitation (Co-IP) assays show that FgAtg9 associates with FgRab7 in vivo. Finally, heterologous complementation assay shows that Atg9 is functionally conserved in F. graminearum and Magnaporthe oryzae. Taken together, we conclude that FgAtg9 is essential for autophagy-dependent development and pathogenicity of F. graminearum, which may be regulated by the small GTPase FgRab7.

Aeromonas hydrophila is the leading cause of fish borne outbreaks and also represents a major threat to human health worldwide. This bacterium harbors many virulence factors such as aerolysin, elastase, and lipase genes. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant A. hydrophila. In the current study, we aimed to determine the incidence and features of A. hydrophila in grass carp in south China. We found 63 (31.5%) A. hydrophila isolates through investigation of 200 samples from five provinces in south China. All isolates were analyzed for virulence genes by polymerase chain reaction (PCR), antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). The results showed that the positive for aerolysin, elastase, hemolysin, and lipase gene were 71.43, 46.03, 0.00, and 55.56%, respectively. Antimicrobial susceptibility results indicated that most strains were resistant to ampicillin (95.24%), tetracycline (88.89%), and streptomycin (79.37%). A lot of multiple antibiotic resistance were found. The isolates were grouped into five clusters by ERIC-PCR and shown the genetic diversity. Thus, our findings demonstrated the presence of A. hydrophila from grass carp, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of microbiological risk assessment in this bacteria. Practical applicationsInvestigating the prevalence of Aeromonas hydrophilia isolates from grass carp could improved for assuring this microbial safety on food. In this study, antibiotic susceptibility, virulence genes, and molecular subtyping were selected to comprehensive analyze A. hydrophilia isolates. As grass carp are common and popular fish choices in China, therefore the continuous monitoring of this pathogens are vital to ensure the safety.

M. oryzae , the causative agent of the rice blast disease, threaten rice production worldwide. Our results revealed that T6P accumulation, caused by the disruption of MoTps2, has toxic effects on fugal development and pathogenesis in M. oryzae .