Carl Jonas Jorge Spetz
Research Scientist
Authors
Xiaoyu Yuan Keya Xu Fang Yan Zhiyuan Liu Carl Jonas Jorge Spetz Huanbin Zhou Xiaojie Wang Huaibing Jin Xifeng Wang Yan LiuAbstract
Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is one of the causal agents of wheat viral disease, which severely impacts wheat production in most wheat-growing regions in the world. Currently, there is little information about natural resistance against WDV in common wheat germplasms. CRISPR/Cas9 technology is being utilized to manufacture transgenic plants resistant to different diseases. In the present study, we used the CRISPR/Cas9 system targeting overlapping regions of coat protein (CP) and movement protein (MP) (referred to as CP/MP) or large intergenic region (LIR) in the wheat variety ‘Fielder’ to develop resistance against WDV. WDV-inoculated T1 progenies expressing Cas9 and sgRNA for CP/MP and LIR showed complete resistance against WDV and no accumulation of viral DNA compared with control plants. Mutation analysis revealed that the CP/MP and LIR targeting sites have small indels in the corresponding Cas9-positive plants. Additionally, virus inhibition and indel mutations occurred in T2 homozygous lines. Together, our work gives efficient results of the engineering of CRISPR/Cas9-mediated WDV resistance in common wheat plants, and the specific sgRNAs identified in this study can be extended to utilize the CRISPR/Cas9 system to confer resistance to WDV in other cereal crops such as barley, oats, and rye.
Abstract
Sweet potato (Ipomoea batatas L. Lam.) is a major source of food in many parts of Ethiopia. In recent years, viral diseases have become the main threat to sweet potato production in Ethiopia. Previous virus survey studies carried out from 1986 to 2020 reported eight viruses infecting sweet potato in Ethiopia. Consequently, obtaining and multiplying virus-free planting materials have been difficult for farmers and commercial multipliers. This study was conducted to detect viruses infecting the five sweet potato varieties used as source plants and compare the virus elimination efficiency between meristem cultures from untreated and heat-treated mother plants and production of virus-free sweet-potato-planting materials. Seven common viruses were tested for, using grafting to Ipomoea setosa, enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT–PCR) before and after elimination procedures as screening and confirmatory methods. The sweet potato feathery mottle virus (SPFMV) elimination efficiencies of meristem cultures from untreated (grown at 25 ± 1 °C) and heat-treated (grown at 39 ± 1 °C) potted plants of sweet potato varieties were evaluated and compared. Sweet potato feathery mottle virus (SPFMV) was detected in 12 of the 15 source plants tested. Triple infections of SPFMV, sweet potato chlorotic stunt virus (SPCSV), and sweet potato virus C (SPVC) were detected in one of the fifteen plants. This study reports the detection of SPVC for the first time in sweet potato plants from Ethiopia. The cutting of meristems from heat-treated plants further increased the percentage of virus-free plantlets by ca 10% to ca 16%, depending on the plant variety. Elimination efficiency also seemed to vary among varieties: the greatest difference was observed for ‘Tola’, and the least difference was observed for ‘Guntute’. The present study provided protocols for detecting viruses and generating virus-free sweet-potato-planting materials in Ethiopia.
Authors
Xueqi Li Sujie Zhang Chenyang Wang Bin Ren Fang Yan Shaofang Li Carl Jonas Jorge Spetz Jinguang Huang Xueping Zhou Huanbin ZhouAbstract
In situ epitope tagging is crucial for probing gene expression, protein localization, and the dynamics of protein interactions within their natural cellular context. However, the practical application of this technique in plants presents considerable hurdles. Here, we comprehensively explored the potential of the CRISPR/Cas nuclease-mediated prime editing and different DNA repair pathways in epitope tagging of endogenous rice (Oryza sativa) genes. We found that a SpCas9 nuclease/microhomology-mediated end joining (MMEJ)-based prime editing (PE) strategy (termed NM-PE) facilitates more straightforward and efficient gene tagging compared to the conventional and other derivative PE methods. Furthermore, the PAM-flexible SpRY and ScCas9 nucleases-based prime editors have been engineered and implemented for the tagging of endogenous genes with diverse epitopes, significantly broadening the applicability of NM-PE in rice. Moreover, NM-PE has been successfully adopted in simultaneous tagging of the MAP kinase (MPK) genes OsMPK1 and OsMPK13 in rice plants with c-Myc and HA tags, respectively. Taken together, our results indicate great potential of the NM-PE toolkit in the targeted gene tagging for Rice Protein Tagging Project, gene function study and genetic improvement.