The Effects of Turnip Mosaic Virus Infections on the Deposition of Secondary Cell Walls and Developmental Defects in Arabidopsis Plants Are Virus-Strain Specific

Abstract

Two isolates of Turnip mosaic virus (UK 1 and JPN 1), representative of two different viral strains, induced differential alterations on secondary cell wall (SCW) development in Arabidopsis thaliana, suggesting cell-type specific effects of these viral infections. These potential effects were analyzed in inflorescence stems and flowers of infected plants, together with other possible cellular effects of the infections. Results obtained from macroscopic and histochemical analyses showed that infection with either virus significantly narrowed stem area, but defects in SCW were only found in JPN 1 infections. In flowers, reduced endothecium lignification was also found for JPN 1, while UK 1 infections induced severe floral cell and organ development alterations. A transcriptomic analysis focused on genes controlling and regulating SCW formation also showed notable differences between both viral isolates. UK 1 infections induced a general transcriptional decrease of most regulatory genes, whereas a more complex pattern of alterations was found in JPN 1 infections. The role of the previously identified viral determinant of most developmental alterations, the P3 protein, was also studied through the use of viral chimeras. No SCW alterations or creeping habit growth were found in infections by the chimeras, indicating that if the P3 viral protein is involved in the determination of these symptoms, it is not the only determinant. Finally, considerations as to the possibility of a taxonomical reappraisal of these TuMV viral strains are provided.

Keywords: developmental alterations; secondary cell wall; turnip mosaic virus; viral chimeras; viral strains.

Figure 1

Inflorescence stem areas of non-infected and TuMV-infected Arabidopsis plants, showing statistically significant differences (marked with and asterisk) between infected and non-infected plants (see text for experimental details). The statistical analysis was a student’s t-test comparing Mock and JPN 1, and Mock and UK 1 (p<0.05).

Figure 2

Lignin histochemical analyses of the secondary cell walls (SCWs) of the inflorescence stems from plants mock-inoculated (buffer) or inoculated with either TuMV strain (JPN 1 or UK (1). Top row shows lignin staining with phloroglucinol; middle row shows lignin intrinsic fluorescence upon ultraviolet excitation (UV light); and bottom row shows cellulose staining with Calcofluor. Bars indicate 100 μm. The cortex (co), xylem vessels (xv), and interfascicular fibers (if) are marked with letters. Note the lack of differentiation in the interfascicular fibers of JPN 1.

Figure 3

Xylan immunohistochemical analysis. The presence of xylan was revealed by its recognition by the monoclonal antibody LM10, followed by the reaction with a fluorescent secondary antibody. Xylan is only weakly detected in the xylem in TuMV strain JPN 1-infected plants (middle row) compared to buffer control (top) or strain UK 1 (bottom). In the third column, the fluorescence images are superimposed on the bright field shown in the second column. The cortex (co), xylem vessels (xv), interfascicular fibers (if), and pith (pi) are marked with letters.

Figure 4

Phenotypes of Arabidopsis plants inoculated with viral chimeras (Sánchez et al., 2015) between TuMV isolates UK 1 and JPN 1, centered in the P3 region of the viral genome. The chimeras used for inoculation are shown in the panels.

Figure 5

Visualization of lignin by UV (upper row) and xylan (monoclonal antibody LM 10, bottom row) in the inflorescence stem SCW of buffer-inoculated or TuMV chimeras centered in the P3 region of the viral genome. Bars indicate 100 μm. Xylem vessels (xv) and interfascicular fibers (if) are marked.

Figure 6

Transcriptomic analysis of selected genes involved in SCW biosynthesis after TuMV infection. The graph shows the results of RT-qPCR of selected genes, represented as relative gene expression in JPN-1-infected or UK-1-infected strains of TuMV, compared to mock-inoculated plants. Comparisons are pairwise of each virus-infected and mock-inoculated. Genes analyzed and color-coded bars are shown in the figure. Asterisk indicates a significant difference compared with wild type (^*p<0.05 by t-test). Error bars show standard deviation.

Figure 7

Phenotypes of flowers from control buffer-inoculated and two strains of TuMV-infected Arabidopsis plants. Scanning electron micrographs of flowers at anthesis (A-C) and flower anthers (D-F). Pollen grains are visible in D and E (arrows). Transversal sections of anthers from flowers at anthesis stained with toluidine blue (G-I). Non-infected mock plants show strong lignification of the endothecium. Endothecium cells showing absence of lignification are marked by asterisks in H. Abnormal pollen grains are marked by arrow heads. Scale bars indicate: 1mm in A-C and 100μm in D-I. Epidermis (Ep); endothecium (En); stomium (St); and septum (S).