Comparative genome anatomy reveals evolutionary insights into a unique amphitriploid fish.

Triploids are rare in nature because of difficulties in meiotic and gametogenic processes, especially in vertebrates. The Carassius complex of cyprinid teleosts contains sexual tetraploid crucian carp/goldfish (C. auratus) and unisexual hexaploid gibel carp/Prussian carp (C. gibelio) lineages, providing a valuable model for studying the evolution and maintenance mechanism of unisexual polyploids in vertebrates. Here we sequence the genomes of the two species and assemble their haplotypes, which contain two subgenomes (A and B), to the chromosome level. Sequencing coverage analysis reveals that C. gibelio is an amphitriploid (AAABBB) with two triploid sets of chromosomes; each set is derived from a different ancestor. Resequencing data from different strains of C. gibelio show that unisexual reproduction has been maintained for over 0.82 million years. Comparative genomics show intensive expansion and alterations of meiotic cell cycle-related genes and an oocyte-specific histone variant. Cytological assays indicate that C. gibelio produces unreduced oocytes by an alternative ameiotic pathway; however, sporadic homologous recombination and a high rate of gene conversion also exist in C. gibelio. These genomic changes might have facilitated purging deleterious mutations and maintaining genome stability in this unisexual amphitriploid fish. Overall, the current results provide novel insights into the evolutionary mechanisms of the reproductive success in unisexual polyploid vertebrates.

Developing reverse genetics systems of northern cereal mosaic virus to reveal superinfection exclusion of two cytorhabdoviruses in barley plants.

Recently, reverse genetics systems of plant negative-stranded RNA (NSR) viruses have been developed to study virus-host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV-RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV-RFP and green fluorescent protein (GFP)-tagged barley yellow striate mosaic virus (rBYSMV-GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV-GFP and rNCMV-RFP was observed in barley leaves. Our results demonstrate that two insect-transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.

Casein Kinase 1 Regulates Cytorhabdovirus Replication and Transcription by Phosphorylating a Phosphoprotein Serine-Rich Motif.

Casein kinase 1 (CK1) family members are conserved Ser/Thr protein kinases that regulate important developmental processes in all eukaryotic organisms. However, the functions of CK1 in plant immunity remain largely unknown. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, infects cereal crops and is obligately transmitted by the small brown planthopper (SBPH; Laodelphax striatellus). The BYSMV phosphoprotein (P) exists as two forms with different mobilities corresponding to 42 kD (P42) and 44 kD (P44) in SDS-PAGE gels. Mass spectrometric analyses revealed a highly phosphorylated serine-rich (SR) motif at the C-terminal intrinsically disordered region of the P protein. The Ala-substitution mutant (PS5A) in the SR motif stimulated virus replication, whereas the phosphorylation-mimic mutant (PS5D) facilitated virus transcription. Furthermore, PS5A and PS5D associated preferentially with nucleocapsid protein-RNA templates and the large polymerase protein to provide optimal replication and transcription complexes, respectively. Biochemistry assays demonstrated that plant and insect CK1 protein kinases could phosphorylate the SR motif and induce conformational changes from P42 to P44. Moreover, overexpression of CK1 or a dominant-negative mutant impaired the balance between P42 and P44, thereby compromising virus infections. Our results demonstrate that BYSMV recruits the conserved CK1 kinases to achieve its cross-kingdom infection in host plants and insect vectors.

Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies.

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid ß-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.

Cucumber mosaic virus coat protein modulates the accumulation of 2b protein and antiviral silencing that causes symptom recovery in planta.

Shoot apical meristems (SAM) are resistant to most plant viruses due to RNA silencing, which is restrained by viral suppressors of RNA silencing (VSRs) to facilitate transient viral invasion of the SAM. In many cases chronic symptoms and long-term virus recovery occur, but the underlying mechanisms are poorly understood. Here, we found that wild-type Cucumber mosaic virus (CMVWT) invaded the SAM transiently, but was subsequently eliminated from the meristems. Unexpectedly, a CMV mutant, designated CMVRA that harbors an alanine substitution in the N-terminal arginine-rich region of the coat protein (CP) persistently invaded the SAM and resulted in visible reductions in apical dominance. Notably, the CMVWT virus elicited more potent antiviral silencing than CMVRA in newly emerging leaves of infected plants. However, both viruses caused severe symptoms with minimal antiviral silencing effects in the Arabidopsis mutants lacking host RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) or SUPPRESSOR OF GENE SILENCING 3 (SGS3), indicating that CMVWT induced host RDR6/SGS3-dependent antiviral silencing. We also showed that reduced accumulation of the 2b protein is elicited in the CMVWT infection and consequently rescues potent antiviral RNA silencing. Indeed, co-infiltration assays showed that the suppression of posttranscriptional gene silencing mediated by 2b is more severely compromised by co-expression of CPWT than by CPRA. We further demonstrated that CPWT had high RNA binding activity leading to translation inhibition in wheat germ systems, and CPWT was associated with SGS3 into punctate granules in vivo. Thus, we propose that the RNAs bound and protected by CPWT possibly serve as templates of RDR6/SGS3 complexes for siRNA amplification. Together, these findings suggest that the CMV CP acts as a central hub that modulates antiviral silencing and VSRs activity, and mediates viral self-attenuation and long-term symptom recovery.

Microscope-assisted endoscopic interlaminar ligation of spinal arteriovenous fistulas: technical note.

Spinal dural arteriovenous fistulas (SDAVFs) are the most common type of spinal arteriovenous malformations, and microsurgical ligation is the treatment modality most frequently used for these lesions. Developments in endoscopic techniques have made endoscopy an even less invasive alternative to routine microsurgical approaches in spine surgery, but endoscopic management of SDAVF or other intradural spinal lesions has not been reported to date. The authors describe the use of a microscope-assisted endoscopic interlaminar approach for the ligation of the proximal draining vein of an L-1 SDAVF in a 58-year-old man. A complete cure was confirmed by postoperative angiography. The postoperative course was uneventful, and short-term follow-up showed improvements in the patient's neurological function. The authors conclude that the endoscopic interlaminar approach with microscope assistance is a safe, minimally invasive, innovative technique for the surgical management of SDAVFs in selected patients.