Characterization of a tomato protein that inhibits a xyloglucan‐specific endoglucanase

Abstract

SummaryA basic, 51 kDa protein was puri®ed from suspension-cultured tomato and shown to inhibit the hydrolytic activity of a xyloglucan-speci®c endoglucanase (XEG) from the fungus Aspergillus aculeatus. The tomato (Lycopersicon esculentum) protein, termed XEG inhibitor protein (XEGIP), inhibits XEG activity by forming a 1 : 1 protein:protein complex with a K i % 0.5 nM. To our knowledge, XEGIP is the ®rst reported proteinaceous inhibitor of any endo-b-1,4-glucanase, including the cellulases. The cDNA encoding XEGIP was cloned and sequenced. Database analysis revealed homology with carrot extracellular dermal glycoprotein (EDGP), which has a putative role in plant defense. XEGIP also has sequence similarity to ESTs from a broad range of plant species, suggesting that XEGIP-like genes are widely distributed in the plant kingdom. Although Southern analysis detected only a single XEGIP gene in tomato, at least ®ve other XEGIP-like tomato sequences have been identi®ed. Similar small families of XEGIP-like sequences are present in other plants, including Arabidopsis. XEGIP also has some sequence similarity to two previously characterized proteins, basic globulin 7S protein from soybean and conglutin c from lupin. Several amino acids in the XEGIP sequence, notably 8 of the 12 cysteines, are generally conserved in all the XEGIP-like proteins we have encountered, suggesting a fundamental structural similarity. Northern analysis revealed that XEGIP is widely expressed in tomato vegetative tissues and is present in expanding and maturing fruit, but is downregulated during ripening.

Structural Basis for Inhibition of Aspergillus niger Xylanase by Triticum aestivum Xylanase Inhibitor-I

“…In a superimposition of the structure of a catalytically inactive Bacillus circulans xylanase mutant complexed with xylobiose (PDB code 1bcx) (36) (in subsites Ϫ1 and Ϫ2) with the structure of the ANXI⅐TAXI-I complex (root mean square deviation of 0.6 Å for 181 xylanase C␣ atoms), five Leu 292 atoms (N, C ␣ , C ␤ , C ␥ , and C ␦1 ) coincide with C-5, O-5, C-1, C-2 and O-2 of the xylose in subsite Ϫ2 (Fig. 3B) TAXI-I displays significant sequence similarity with a carrot extracellular dermal glycoprotein (37), a tomato xyloglucanspecific endoglucanase inhibitor protein (38) and with TAXIlike proteins (16) identified in barley (HVXI), rye (SCXI-I to SCXI-IV), and durum wheat (TDXI-I and TDXI-II). In addition, screening of the available data bases revealed that multiple TAXI-I homologous genes also occur in soybean, rice, and Arabidopsis thaliana (16).…”

“…Based on a rapid increase of the carrot extracellular dermal glycoprotein mRNA level in response to wounding, it was suggested to be a plant defense protein (37). Recent characterization of tomato xyloglucan-specific endoglucanase inhibitor protein revealed a possible plant defense function as an inhibitor of a microbial glucanase of GH family 12 (38). Moreover, GH family 12 glucanases share a common ␤-sandwich fold with GH family 11 xylanases.…”

The role of phospholipase D in plant stress responses

“…Plant β-Dxylosidases have been suggested to be involved in cell-wall remodeling (Goujon et al, 2003). Surprisingly, the 51 kDa band that was more abundant in silenced cellsuspension culture medium was identified by the MSMS of 4 peptides as the xyloglucan-specific endoglucanase inhibitor protein XEGIP (total MASCOT score 113; accession number AY155579; Qin et al, 2003). This protein has been shown to form a complex with the xyloglucan-specific endoglucanase from Aspergillus aculeatus, inhibiting its glucanase activity.…”

Cell wall integrity

“…71 Depolymerization of xyloglucan has been proposed to play an important role during both cell wall expansion and pathogen invasion. [72][73][74] Endo-β-1,4-xylanases are key enzymes in the degradation of xylans and a number of endoxylanases are produced by microbial pathogens to break through the plant cell wall. Rice plants expressing a thermostable exogenous xylanase gene (ATX) exhibited upregulation of SOD, CAT and xylanase inhibitor (RIXI) genes, which could play roles in plant defense.…”