CCS52 and DEL1 genes are key components of the endocycle in nematode-induced feeding sites
Article first published online: 23 AUG 2012
© 2012 Institut National de la Recherche Agronomic. The Plant Journal © 2012 Blackwell Publishing Ltd
The Plant Journal
Volume 72, Issue 2, pages 185–198, October 2012
How to Cite
de Almeida Engler, J., Kyndt, T., Vieira, P., Van Cappelle, E., Boudolf, V., Sanchez, V., Escobar, C., De Veylder, L., Engler, G., Abad, P. and Gheysen, G. (2012), CCS52 and DEL1 genes are key components of the endocycle in nematode-induced feeding sites. The Plant Journal, 72: 185–198. doi: 10.1111/j.1365-313X.2012.05054.x
- Issue published online: 16 OCT 2012
- Article first published online: 23 AUG 2012
- Accepted manuscript online: 28 MAY 2012 08:50AM EST
- Received 9 November 2011; revised 13 April 2012; accepted 4 May 2012; published online 23 August 2012.
Figure S1. Analysis of CCS52 overexpressing and knockdown lines. (a) Expression levels of CCS52B in three overexpressing lines compared to wild-type Arabidopsis. (b) Expression levels of CCS52A1, CCS52A2 and CCS52B in Arabidopsis knockdown lines compared to wild-type. *Statistically significant differences were determined by the Student’s test (P < 0.01). (c) Flow cytometric measurements illustrating the ploidy of ±1000 nuclei of cotyledons of three hpCCS52 lines. Lower ploidy levels were seen in hpCCS52 lines compared to wild-type. (d) Flow cytometric analysis of CCS52B overexpressing lines. Endoreduplication index of ±1000 nuclei of roots of three CCS52B overexpressing lines. *Statistically significant differences were determined by the Student’s test (P < 0.01).
Figure S2. Effect of ectopic expression of CCS52B on trichomes and CCS52B and CCS52A2 in Arabidopsis root apex. (a) Leaves of plants overexpressing CCS52B in Arabidopsis contain trichomes with 4 to 5 branches (a) compared to wild-type (b) leaves showing only 3 branches. (c-e) Bright field images of longitudinal tissue sections stained with toluidine blue. (c) Root apex overexpressing CCS52B showing enlarged and misaligned cells, (d) root apex overexpressing CCS52A2 showing misaligned cells and (e) control wild-type. RAM, root apical meristem; EZ, elongation zone; Ep, epidermis; C, cortex. Bars = 50 µm.
Figure S3. Images of infected roots with root-knot (a) and cyst nematodes (b) in the CCS52BOE line, and (c-d) wild-type respectively. Asterisk, gall; n, nematode; S, syncytium. Bars = 100 µm.
Figure S4. Nuclei in M. incognita-induced galls and H. schachtii-induced syncytia in wild-type and transgenic Arabidopsis lines. Micrographs (a,a’; b,b’) were generated from serial confocal optical sections using 3D-shadow projection revealing nuclei (gray) distribution in giant cells. Micrographs (c-f) show DAPI stained nuclei. Micrographs (g,h) are projections of serial confocal optical sections revealing PI stained nuclei (red). Bars = 20 µm. (a,a’) Nuclei of giant cells 21DAI stained with PI overexpressing CCS52B showing large chromocenters, and (b,b’) of wild-type. (c) Fluorescence image of DAPI stained nuclei in a wild-type gall 21DAI, and (d) nuclei in a gall 21 DAI of a CCS52B knockdown line filled with small and more elongated nuclei. (e) Fluorescent image of DAPI stained nuclei in a wild-type syncytium 21DAI and a detail to the right side, and (f) nuclei in a syncytium of CCS52B knockdown line 21DAI showing small and more elongated nuclei and a detail to the right side. (g) Fluorescence image of PI stained cleared nuclei in a gall of wild-type roots 21DAI, and (h) in a gall of DEL1 overexpressing line 21DAI. Note the decreased nuclear size in giant cells and multiple small nuclei of the dividing neighbouring cells.
Figure S5. Nuclear sizes of giant cells of hpCCS52AB knockdown line, and CCS52B, CCS52A2 and DEL1 overexpressing lines. White bars represent galls 7DAI, gray bars 14DAI and black bars 21DAI. *Statistically significant differences were determined by the Student’s test (P < 0.05).
Table S1. Primer sequences used in (a) gene cloning of AtCCS52A and AtCCS52B (b) qRT-PCR of AtCCS52A1, AtCCS52A2, AtCCS52B and AtACT2.
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