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.

Fig 1. Recombinant TuGK viral vectors expressing phytoplasma effector genes in Nicotiana benthamiana plants.

A, Schematic diagram of pBD-TuGR, pBD-TuGK, pBD-TuGK-PHYL1, and pBD-TuGK-SAP54. The TuMV genes are indicated by white boxes. The green fluorescent protein gene (GFP) is indicated by a black box. The SAP54 of AYWB and PHYL1 of PnWB genes are indicated by gray boxes. Asterisks (*) indicate the Arg182Lys mutation (R182K) in HC-Pro. The 35S promoter and NOS terminator are indicated by black boxes. The left border (LB) and right border (RB) sequences are indicated. B, Viral infectivity assay of the recombinant viruses. GFP expression by TuGR, TuGK, TuGK-PHYL1, and TuGK-SAP54 in N. benthamiana plants. Images were acquired of N. benthamiana plants at 4 dpi with fluorescence microscopy at 400× magnification. Bar, 25 μm.

Fig 2. Detection of effector expression in TuGK-PHYL1- and TuGK-SAP54-infected plants.

Foreign gene expression in TuMV-infected Nicotiana benthamiana plants at 4 dpi (A) or 8 dpi (B). IL indicates an inoculated leaf, and SL indicates a systemic leaf. C, Foreign gene expression in TuMV-infected Arabidopsis dcl2-4/dcl4-1 double-mutants (dcl2/4). TuGR, a wild-type TuMV that expresses the green fluorescent protein (GFP) gene; TuGK, an HC-Pro Arg182Lys mutant of TuMV that expresses the GFP gene; TuGK-PHYL1, TuGK expressing the GFP-PHYL1 fusion gene; TuGK-SAP54, TuGK expressing the GFP-SAP54 fusion gene. The upper panels were detected using an 8,000× dilution of the GFP antibody. The lower panels were detected using a 10,000× dilution of TuMV coat protein (CP) antiserum. D, SAP54 and PHYL1 detection using specific antisera at 10,000× dilutions. The large subunit of ribulose-1,5-bisphosphate carboxylase (*) was used as a loading control.

Fig 3. Longitudinal section of the shoot apical meristem (SAM) of Nicotiana benthamiana plants.

The TuGK- and TuGK-PHYL1-infected SAM sections were evaluated by confocal microscopy. Complete stacks demonstrate that GFP (green), GFP-PHYL1 (green) and chlorophyll (red) were present in the SAM region. The SAM tissues were corrected at 4 dpi of TuGK- or TuGK-PHYL1-infected plants. Bar, 100 μm.

Fig 4. Leafy flower phenotypes of TuGK-PHYL1- and TuGK-SAP54-infected Arabidopsis plants.

A, The leafy flower phenotypes of SAP54 and PHYL1 plants. Bar, 0.2 cm. The leafy flower phenotypes of TuGK-SAP54- or TuGK-PHYL1-infected Col-0 (B) and dcl2/4 (C) plants. Mock indicates inoculation with buffer alone. The photographs were taken at 20 dpi. Bar, 0.2 cm.

Fig 5. Flower-related gene expression in TuGK-PHYL1-and TuGK-SAP54-infected Arabidopsis flower tissues.

The expression profiles of the AtAP3 (A, left panel), AtSVP (B, left panel), AtFT (C, left panel), and AtUBQ10 (D, left panel) genes in flower-tissues of Col-0 and SAP54 plants were evaluated by transcriptome deep sequencing. The reads per kilobase per million mapped reads (RPKM) values are used to indicate gene expression levels. The bars represent the SE of 6 AtUBQ10 isoforms (n = 6). Real-time RT-PCR evaluation of AtAP3 (A, right panel), AtSVP (B, right panel), AtFT (C, right panel), and AtUBQ10 (D, right panel) expression in various recombinant TuGK virus-infected dcl2-4/dcl4-1 (dcl2/4) plants at 20 dpi. SAP54 represents SAP54 plants. The bars represent the SE (n = 3). The relative expression levels were normalized to the AtUBQ10 level. The letters indicate significant differences among these relative expression levels of the RNA samples tested by Fisher’s least significant difference (LSD) method at ‘alpha’ = 0.05, with false discovery rate (FDR) correction after an analysis of variance (ANOVA).