Intravitreal Delivery of rAAV2-hSyn-hRS1 Results in Retinal Ganglion Cell-Specific Gene Expression and Retinal Improvement in the Rs1-KO Mouse.

X-linked retinoschisis (XLRS) is a monogenic recessive inherited retinal disease caused by defects in retinoschisin (RS1). It manifests clinically as retinal schisis cavities and a disproportionate reduction of b-wave amplitude compared with the a-wave amplitude. Currently there is no approved treatment. In the last decade, there has been major progress in the development of gene therapy for XLRS. Previous preclinical studies have demonstrated the treatment benefits of hRS1 gene augmentation therapy in mouse models. However, outcomes in clinical trials have been disappointing, and this might be attributed to dysfunctional assembly of RS1 complexes and/or the impaired targeted cells. In this study, the human synapsin 1 gene promoter (hSyn) was used to control the expression of hRS1 to specifically target retinal ganglion cells and our results confirmed the specific expression and functional assembly of the protein. Moreover, our results demonstrated that a single intravitreal injection of rAAV2-hSyn-hRS1 results in architectural restoration of retinal schisis cavities and improvement in vision in a mouse model of XLRS. In brief, this study not only supports the clinical development of the rAAV2-hSyn-hRS1 vector in XLRS patients but also confirms the therapeutic potential of rAAV-based gene therapy in inherited retinal diseases.

Virus-Induced Gene Silencing in Diploid and Tetraploid Potato Species.

Potato is the world's fourth largest food crop and a vegetatively propagated model polyploid plant. To facilitate genomic studies in potato, here we describe detailed protocols to silence genes in both diploid potato Solanum bulbocastanum and tetraploid potato cultivars such as Maris Bard, Arran Pilot, Ancilla, and Serrana using tobacco rattle virus (TRV)- or potato virus X (PVX)-induced gene silencing (VIGS) system, respectively. The established VIGS system represents an efficient and powerful approach for functional analysis of genes involved in growth, development, metabolism, and responses to biotic and abiotic stresses in potato.

Virus-Based microRNA Silencing in Plants.

Virus-based microRNA silencing (VbMS) is an efficient, powerful, and high-throughput approach to screen and investigate the function of microRNAs (miRNAs) in plants. The VbMS system was originally developed in Nicotiana benthamiana and tomato (Solanum lycopersicum) and has been extended to various other plant species such as Arabidopsis, cotton, and wheat with different virus vectors. VbMS is generally designed to use virus vectors to direct the expression of miRNA target mimic (TM) molecules which can complementarily pair to target miRNAs and block their function. Here, we describe the TRV- and PVX-based VbMS approaches to silence endogenous miRNAs in N. benthamiana and tomato plants by Agrobacterium infiltration. This method can be further applied to other plant species using suitable virus vectors in combination with diverse TM strategies, which will facilitate functional studies of miRNAs in plants.

Endoplasmic reticulum-associated degradation mediated by MoHrd1 and MoDer1 is pivotal for appressorium development and pathogenicity of Magnaporthe oryzae.

Most secretory proteins are folded and modified in the endoplasmic reticulum (ER); however, protein folding is error-prone, resulting in toxic protein aggregation and cause ER stress. Irreversibly misfolded proteins are subjected to ER-associated degradation (ERAD), modified by ubiquitination, and degraded by the 26S proteasome. The yeast ERAD ubiquitin ligase Hrd1p and multispanning membrane protein Der1p are involved in ubiquitination and transportation of the folding-defective proteins. Here, we performed functional characterization of MoHrd1 and MoDer1 and revealed that both of them are localized to the ER and are pivotal for ERAD substrate degradation and the ER stress response. MoHrd1 and MoDer1 are involved in hyphal growth, asexual reproduction, infection-related morphogenesis, protein secretion and pathogenicity of M. oryzae. Importantly, MoHrd1 and MoDer1 mediated conidial autophagic cell death and subsequent septin ring assembly at the appressorium pore, leading to abnormal appressorium development and loss of pathogenicity. In addition, deletion of MoHrd1 and MoDer1 activated the basal unfolded protein response (UPR) and autophagy, suggesting that crosstalk between ERAD and two other closely related mechanisms in ER quality control system (UPR and autophagy) governs the ER stress response. Our study indicates the importance of ERAD function in fungal development and pathogenesis of M. oryzae.

Isopropylmalate isomerase MoLeu1 orchestrates leucine biosynthesis, fungal development, and pathogenicity in Magnaporthe oryzae.

The biosynthesis of branched-chain amino acids (BCAAs) is conserved in fungi and plants, but not in animals. The Leu1 gene encodes isopropylmalate isomerase that catalyzes the conversion of α-isopropylmalate into ß-isopropylmalate in the second step of leucine biosynthesis in yeast. Here, we identified and characterized the functions of MoLeu1, an ortholog of yeast Leu1 in the rice blast fungus Magnaporthe oryzae. The transcriptional level of MoLEU1 was increased during conidiation and in infectious stages. Cellular localization analysis indicated that MoLeu1 localizes to the cytoplasm at all stages of fungal development. Targeted gene deletion of MoLEU1 led to leucine auxotrophy, and phenotypic analysis of the generated ∆Moleu1 strain revealed that MoLeu1-mediated leucine biosynthesis was required for vegetative growth, asexual development, and pathogenesis of M. oryzae. We further observed that invasive hyphae produced by the ∆Moleu1 strain were mainly limited to the primary infected host cells. The application of exogenous leucine fully restored vegetative growth and partially restored conidiation as well as pathogenicity defects in the ∆Moleu1 strain. In summary, our results suggested that MoLeu1-mediated leucine biosynthesis crucially promotes vegetative growth, conidiogenesis, and pathogenicity of M. oryzae. This study helps unveil the regulatory mechanisms that are essential for infection-related morphogenesis and pathogenicity of the rice blast fungus.

Putative Interaction Proteins of the Ubiquitin Ligase Hrd1 in Magnaporthe oryzae.

The endoplasmic reticulum (ER) is the entry portal of the conventional secretory pathway where the newly synthesized polypeptides fold, modify, and assemble. The ER responses to the unfolded proteins in its lumen (ER stress) by triggering intracellular signal transduction pathways include the ER-associated degradation (ERAD) pathway and the unfolded protein response (UPR) pathway. In yeast and mammals, the ubiquitin ligase Hrd1 is indispensable for the ERAD pathway, and also Hrd1-mediated ERAD pathway plays a crucial role in maintaining homeostasis and metabolism of human beings. However, the underlying physiological roles and regulatory mechanism of the Hrd1-involved ERAD pathway in the plant pathogenic fungi are still unclear. Here, we identified the Hrd1 orthologous proteins from 9 different fungi and noticed that these Hrd1 orthologs are conserved. Through identification of MoHrd1 putative interacting proteins by co-immunoprecipitation assays and enrichment analysis, we found that MoHrd1 is involved in the secretory pathway, energy synthesis, and metabolism. Taken together, our results suggest that MoHrd1 is conserved among fungi and play an important role in cellular metabolism and infection-related development. Our finding helps uncover the mechanism of Hrd1-involved ERAD pathway in fungi and sheds a new light to understand the pathogenic mechanism of Magnaporthe oryzae.