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

Sin-Fen Hu,Wei-Yao Chen,Li-Yu Daisy Liu,Hsiao-Feng Lo,Shih-Shun Lin,Syuan-Fei Hong,Ting-Yu Tseng,Chiao-Yin Yang,Yu-Hsin Huang,Chan-Pin Lin,Keith R Davis

doi:10.1371/journal.pone.0130139

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.

Highlights

Aster yellow witches’-broom (AYWB) phytoplasma causes virescence and phyllody symptoms in host plants [1]
TuGK expresses phytoplasma effectors in planta pBD-TuGK was used to carry the PHYL1 or SAP54 gene (Fig 1A). Both effector genes were fused at the C-terminus of green fluorescent protein (GFP) and inserted between the NIb and coat protein (CP) genes with NIa protease cleavage sites (Fig 1A). pBD-TuGK-PHYL1 and pBD-TuGK-SAP54 were produced by the TuGK-PHYL1 and TuGK-SAP54 recombinant viruses in N. benthamiana plants after agro-infiltration and exhibited a GFP signal under fluorescence microscopy (Fig 1B)
Compared with plants infected with a wild-type Turnip mosaic virus (TuMV) that expresses GFP (TuGR), GFP fluorescence was lower in the TuGK, TuGK-PHYL1, and TuGK-SAP54-infected plants (Fig 1B)

Summary

Introduction

Aster yellow witches’-broom (AYWB) phytoplasma causes virescence and phyllody symptoms in host plants [1]. Maclean et al (2011) individually expressed several putative secreted AYWB phytoplasma proteins (SAPs) in Arabidopsis to identify the phytoplasma effector that induces these virescence and phyllody symptoms (hereafter leafy flower). The PHYL1 effector of Onion yellows phytoplasma, an effector orthologous to SAP54, results in a leafy flower phenotype in Arabidopsis expressing the PHYL1 gene [3], suggesting that SAP54 and PHYL1 play roles in leafy flower formation. Our pervious study indicated that Peanut witches’-broom (PnWB) phytoplasma occurs leafy flower symptoms in Catharanthus roseus plants [4], and its PHYL1 gene has been identified [5]. The transgenic Arabidopsis expressing SAP54 (SAP54 plants) have a seedless problem that restricted the further study in function of effectors. An alternative strategy for PHYL1/SAP54 gain-of-function in vivo that is independent from the transgenic approach is needed

Methods

Results

Discussion

Conclusion