Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family Closteroviridae, designated grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular and pathological properties of this virus. Such knowledge gap is due to multiple factors, including unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we have tested four methods for inoculating tissue cultured grapevine plantlets with cDNA clones of GLRaV-3: i) vacuum agro-infiltration; ii) agro-pricking; iii) agro-drenching and iv) agro-injection. We have shown that vacuum agro-infiltration was the most effective among these methods. Further, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We have found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor, and dormancy treatment both had strong effects on the infection rates. To our best knowledge, the experimental protocol reported here is the most effective system for launching infection of grapevine using cDNA clones of grapevine viruses featuring up to 70% infection rate. This system has strong potential to facilitate grapevine virology research including fulfillment of Koch’s postulates for GLRD and other major virus diseases as well as the molecular, cellular, and pathological properties of GLRaVs and potentially other important grapevine viruses.

Objective: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in MeCP2, a transcription factor. MeCP2 mutations cause abnormal expression of downstream genes and eventually lead to brain dysfunction. The role of MeCP2 in brain neural development remains unclear. To further elucidate this role, a MeCP2-null rat model was created with the CRISPR/cas9 system. Method: A MeCP2-cas9 vector was constructed and then microinjected into fertilized rat ova in vitro. Two mutations by CRISPR/cas9 were confirmed to cause deletions in exon 2 of MeCP2 via DNA sequencing, and protein expression was measured by Western blotting. Transcriptome data for three brain tissues, the cerebellum, cerebral cortex and hippocampus, were obtained via next-generation sequencing. Results: MeCP2-null rats were successfully obtained, and preliminary analysis showed that the MeCP2-null rats exhibited motor dysfunction and anxious and depressed behaviour. In addition, differentially expressed genes (DEGs) were identified in the three MeCP2-null brain tissues compared to wild-type rat brain tissues. In the rat cerebellum, 388 downregulated DEGs were mainly involved in the calcium ion signalling pathway and the PI3K-Akt signalling pathway. In the cerebral cortex, 386 upregulated DEGs were primarily involved in intracellular signal transduction, protein phosphorylation and the MAPK signalling pathway. In the hippocampus, the DEGs were related to the MAPK signalling pathway. Conclusion: We constructed a MeCP2-null rat model with unique features with CRISPR/cas9 technology to study RTT and analysed DEGs in three rat brain tissues to highlight potential targets for the development of new medicines.