The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region

The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine-rich protein SCRE1, which is an essential virulence effector in U. virens. When SCRE1 was heterologously expressed in Magnaporthe oryzae, the protein was secreted and translocated into plant cells during infection. SCRE1 suppresses the immunity-associated hypersensitive response in the nonhost plant Nicotiana benthamiana. Induced expression of SCRE1 in rice also inhibits pattern-triggered immunity and enhances disease susceptibility to rice bacterial and fungal pathogens. The immunosuppressive activity is localized to a small peptide region that contains an important ‘cysteine-proline-alanine-arginine-serine’ motif. Furthermore, the scre1 knockout mutant generated using the CRISPR/Cas9 system is attenuated in U. virens virulence to rice, which is greatly complemented by the full-length SCRE1 gene. Collectively, this study indicates that the effector SCRE1 is able to inhibit host immunity and is required for full virulence of U. virens.     

A high‐throughput BAC end analysis protocol (BAC‐anchor) for profiling genome assembly and physical mapping

Traditional approaches for sequencing insertion ends of bacterial artificial chromosome (BAC) libraries are laborious and expensive, which are currently some of the bottlenecks limiting a better understanding of the genomic features of auto‐ or allopolyploid species. Here, we developed a highly efficient and low‐cost BAC end analysis protocol, named BAC‐anchor, to identify paired‐end reads containing large internal gaps. Our approach mainly focused on the identification of high‐throughput sequencing reads carrying restriction enzyme cutting sites and searching for large internal gaps based on the mapping locations of both ends of the reads. We sequenced and analysed eight libraries containing over 3 200 000 BAC end clones derived from the BAC library of the tetraploid potato cultivar C88 digested with two restriction enzymes, Cla I and Mlu I. About 25% of the BAC end reads carrying cutting sites generated a 60–100 kb internal gap in the potato DM reference genome, which was consistent with the mapping results of Sanger sequencing of the BAC end clones and indicated large differences between autotetraploid and haploid genotypes in potato. A total of 5341 Cla I‐ and 165 Mlu I‐derived unique reads were distributed on different chromosomes of the DM reference genome and could be used to establish a physical map of target regions and assemble the C88 genome. The reads that matched different chromosomes are especially significant for the further assembly of complex polyploid genomes. Our study provides an example of analysing high‐coverage BAC end libraries with low sequencing cost and is a resource for further genome sequencing studies.     

Reporter mice for isolating and auditing cell type‐specific extracellular vesicles in vivo

Extracellular vesicles (EVs) are abundant, lipid‐enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type‐specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR‐Cas9‐mediated genome editing to generate mice bearing a CD63‐emGFP^{loxP/stop/loxP} knock‐in cassette that enables the specific labeling of circulating CD63⁺ vesicles from any cell type when crossed with lineage‐specific Cre recombinase driver mice. As proof‐of‐principle, we have crossed these mice with Cdh5‐Cre^ERT2 mice to generate CD63^emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma‐purified EVs contain a subpopulation of emGFP⁺ vesicles that are derived from the endothelium, co‐express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR‐126, miR‐30c, and miR‐125b). This new mouse strain should be a useful genetic tool for generating cell type‐specific, CD63⁺ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.