Interactions among three distinct CesA proteins essential for cellulose synthesis

Abstract

In a screen to identify novel cellulose deficient mutants, three lines were shown to be allelic and define a novel complementation group, irregular xylem5 (irx5). IRX5 was cloned and encodes a member of the CesA family of cellulose synthase catalytic subunits (AtCesA4). irx5 plants have an identical phenotype to previously described mutations in two other members of this gene family (IRX1 and IRX3). IRX5, IRX3, and IRX1 are coexpressed in exactly the same cells, and all three proteins interact in detergent solubilized extracts, suggesting that three members of this gene family are required for cellulose synthesis in secondary cell walls. The association of IRX1 and IRX3 was reduced to undetectable levels in the absence of IRX5. Consequently, these data suggest that IRX5, IRX3, and IRX1 are all essential components of the cellulose synthesizing complex and the presence of all three subunits is required for the correct assembly of this complex.

Figure 1

Cross sections of wild-type and irx5-1 stems. Sections of wild type (A, C, E, and G) and irx5-1 (B, D, F, and H) were stained with Toluidine blue. xe, xylem elements; if, interfascicular cells. Arrowheads indicate irregular cell walls. (Bars in A–D represent 0.05 mm. Bars in E–H represent 0.01 mm.)

Figure 2

Specificity of IRX5 antibody. Protein gel blot of wild-type and irx5-1, irx5-2, and irx5-3 extracts. (A) Blot probed with anti-IRX5 antibody. Arrowhead denotes full-length IRX5; asterisks represent nonspecific cross-reacting bands. (B) Blot probed with anti-IRX1 antibody. (C) Blot probed with anti-IRX3 antibody. Molecular mass markers are shown at left in kDa.

Figure 3

Colocalization of IRX5, IRX1, and IRX3 as shown by tissue printing. Tissue prints of wild-type (A, C, and E) and irx5-1 (B, D, and F) stems probed with anti-IRX5 antibody (A and B), anti-IRX1 antibody (C and D), or anti-IRX3 antibody (E and F). X, xylem; IF, interfascicular region. Arrows indicate the same points in serial sections.

Figure 4

Copurification of IRX5, IRX3, and IRX1 as shown by protein gel blots. Total protein (TP), pooled washes (W), and eluate (EL) from NHisIRX3 probed with anti-IRX3, IRX5, IRX1, and RD28 (aquaporin) antibodies. Molecular mass markers are shown at left in kDa.

Figure 5

Association of IRX1 and IRX3 in wild type, irx5-1, and irx1-1. Detergent solubilized extracts from wild type, irx5-1, and irx1-1 immunoprecipitated with anti-IRX1 antibody (IP1), anti-IRX3 antibody (IP3), or anti-IRX5 antibody (IP5). TP, total protein; W, final wash. (A and C) Blots probed with anti-IRX1 antibodies. (B) Blots probed with anti-IRX3 antibodies.