Identification of a TuMV isolate (TuMV-ZR) from Pseudostellaria heterophylla and its development into a viral expression vector.

Pseudostellaria heterophylla (P. heterophylla) is a popular Chinese medicinal herb that is cultivated widely in China. Viral infection is commonly encountered during the production of P. heterophylla. To identify viruses causing P. heterophylla disease, sRNA and mRNA libraries were built for 2 sets of P. heterophylla plants, one set that was planted only once (FGP) and one that was planted three consecutive three times (TGP) in a field, using virus-free tuberous roots as reproductive materials. A comprehensive procedure, including assembling virus-derived sRNA (vsRNA), assessing and cloning the full-length viral genome, building an infectious cloning vector and constructing a virus-based expression vector, was performed to identify viruses infecting P. heterophylla. Ultimately, 48 contig-related viruses were mined from 6 sRNA and 6 mRNA P. heterophylla libraries. A 9762-bp fragment was predicted to be the complete genome of TuMV virus. This sequence was cloned from P. heterophylla, and its infectivity was evaluated using the virus-infection model plant Nicotiana benthamiana (N. benthamiana) and host plant P. heterophylla. The resulting 9839-bp viral genome was successfully obtained from P. heterophylla and identified as a new P. heterophylla TuMV-ZR isolate. Simultaneously, TuMV-ZR infectious clones were shown to effectively infect P. heterophylla. Furthermore, TuMV-ZR expression vectors were developed, and the ability of a TuMV-ZR-based vector to express foreign genes was determined by analysis with the reporter gene EGFP. TuMV-ZR-based vectors were found to continuously express foreign genes in different organs of P. heterophylla throughout the whole vegetative period. In addition, TuMV-ZR vectors carrying EGFP accumulated in the tuberous roots of P. heterophylla, confirming that tuberous roots are key targets for viral infection and transmission. This study revealed the core pathogenicity of P. heterophylla mosaic virus and developed a new TuMV-ZR-based expression tool that led to long-term protein expression in P. heterophylla, laying the foundation for the identification of the mechanisms of P. heterophylla infection with mosaic viruses and developing tools to express value proteins in the tuberous roots of the medicinal plant P. heterophylla.

Over-immunity mediated abnormal deposition of lignin arrests the normal enlargement of the root tubers of Rehmannia glutinosa under consecutive monoculture stress.

The rapid accumulation of lignin in the cell wall is one of important immune defense mechanism in response to adversity stress in plants. In this study, we found that the enlargement of the root tubers of Rehmannia glutinosa (R. glutinosa) is arrested under consecutive monoculture stress, and this process is accompanied by abnormal accumulation of lignin. Meanwhile, the function of key catalytic enzyme genes in lignin biosynthetic pathway under consecutive monoculture stress was systematically analyzed, of which roles of core genes were validated using reverse genetics. We elucidated that an abnormal deposition of lignin in R. glutinosa roots, induced by consecutive monoculture stress, and arrested the enlargement of root tubers. Additionally, by manipulating the key catalytic enzyme gene RgCCR6, we were able to alter lignin content of roots of R. glutinosa, thereby affecting tuber enlargement. We speculate that cell lignification is an important defense strategy in resistance against consecutive monoculture stress, but the overreacted defense hindered the normal enlargement of root tubers. The findings provide new insights for effectively improving yield reductions of root crops subjected to environmental stress.