Development of a Mild Viral Expression System for Gain-Of-Function Study of Phytoplasma Effector In Planta

Abstract

PHYL1 and SAP54 are orthologs of pathogenic effectors of Aster yellow witches’-broom (AYWB) phytoplasma and Peanut witches’-broom (PnWB) phytoplasma, respectively. These effectors cause virescence and phyllody symptoms (hereafter leafy flower) in phytoplasma-infected plants. T0 lines of transgenic Arabidopsis expressing the PHYL1 or SAP54 genes (PHYL1 or SAP54 plants) show a leafy flower phenotype and result in seedless, suggesting that PHYL1 and SAP54 interfere with reproduction stage that restrict gain-of-function studies in the next generation of transgenic plants. Turnip mosaic virus (TuMV) mild strain (TuGK) has an Arg182Lys mutation in the helper-component proteinase (HC-ProR182K) that blocks suppression of the miRNA pathway and prevents symptom development in TuGK-infected plants. We exploited TuGK as a viral vector for gain-of-function studies of PHYL1 and SAP54 in Arabidopsis plants. TuGK-PHYL1- and TuGK-SAP54-infected Arabidopsis plants produced identical leafy flower phenotypes and similar gene expression profiles as PHYL1 and SAP54 plants. In addition, the leafy flower formation rate was enhanced in TuGK-PHYL1- or TuGK-SAP54-infected Arabidopsis plants that compared with the T0 lines of PHYL1 plants. These results provide more evidence and novel directions for further studying the mechanism of PHYL1/SAP54-mediated leafy flower development. In addition, the TuGK vector is a good alternative in transgenic plant approaches for rapid gene expression in gain-of-function studies.

A novel ilarvirus protein CP-RT is expressed via stop codon readthrough and suppresses RDR6-dependent RNA silencing

“…For cucumber mosaic virus, mutations in CP which increased the silencing suppression activity of 2b were found to change the virus accumulation pattern in SAM from transient to persistent and consequently abolished recovery in wild type N. benthamiana but not in RDR6 or SGS3 mutants (Zhang et al, 2017). A turnip mosaic virus variant with point mutations decreasing the VSR activity of HC-Pro was able to infect SAM while the wild type virus was excluded from SAM (Hu et al, 2015). It was also demonstrated that HC-Pro of another potyvirus, sugarcane mosaic virus, downregulates the accumulation of host RDR6 mRNA (Zhang et al, 2008).…”

A novel ilarvirus protein CP-RT is expressed via stop codon readthrough and suppresses RDR6-dependent RNA silencing

“…benthamiana but not in RDR6 or SGS3 mutants [ 125 ]. A turnip mosaic virus variant with point mutations decreasing the VSR activity of HC-Pro was able to infect SAM while the wild type virus was excluded from SAM [ 126 ]. It was also demonstrated that HC-Pro of another potyvirus, sugarcane mosaic virus, downregulates the accumulation of host RDR6 mRNA [ 127 ].…”

“…For cucumber mosaic virus, mutations in CP which increased the silencing suppression activity of 2b were found to change the virus accumulation pattern in SAM from transient to persistent and consequently abolished recovery in wild type N. benthamiana but not in RDR6 or SGS3 mutants [125]. A turnip mosaic virus variant with point mutations decreasing the VSR activity of HC-Pro was able to infect SAM while the wild type virus was excluded from SAM [126]. It was also demonstrated that HC-Pro of another potyvirus, sugarcane mosaic virus, downregulates the accumulation of host RDR6 mRNA [127].…”

Detection, Identification and Molecular Characterization of the 16SrII-V Subgroup Phytoplasma Strain Associated with Pisum sativum and Parthenium hysterophorus L.

“…After centrifugation at 13,000× g for 12 min, supernatants were obtained and proteins were separated using SDS-PAGE. Polyclonal antibodies against Imp and PHYL1 were used to monitor protein amounts [ 30 , 41 ]. Western blotting was performed using enhanced chemiluminescence western-blotting reagents (Amersham), and chemiluminescence signals were captured using ImageQuant LAS 4000 Mini (GE Healthcare).…”