Fungal cellulase is an elicitor but its enzymatic activity is not required for its elicitor activity

Abstract

Plant-pathogenic fungi produce cellulases. However, little information is available on cellulase as an elicitor in plant-pathogen interactions. Here, an endocellulase (EG1) was isolated from Rhizoctonia solani. It contains a putative protein of 227 amino acids with a signal peptide and a family-45 glycosyl hydrolase domain. Its aspartic acid (Asp) residue at position 32 was changed to alanine (Ala), resulting in full loss of its catalytic activity. Wild-type and mutated forms of the endoglucanase were expressed in yeast and purified to homogeneity. The purified wild-type and mutant forms induced cell death in maize, tobacco and Arabidopsis leaves, and the transcription of three defence marker genes in maize and tobacco and 10 genes related to defence responses in maize. Moreover, they also induced the accumulation of reactive oxygen species (ROS), medium alkalinization, Ca(2+) accumulation and ethylene biosynthesis of suspension-cultured tobacco cells. Similarly, production of the EG1 wild-type and mutated forms in tobacco induced cell death using the Potato virus X (PVX) expression system. In vivo, expression of EG1 was also related to cell death during infection of maize by R. solani. These results provide direct evidence that the endoglucanase is an elicitor, but its enzymatic activity is not required for its elicitor activity.

Verticillium dahliae manipulates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate‐binding module 1

“…The cells were lysed by freezing and thawing, followed by sonication, and the recombinant protein was purified with elution buffer (column buffer supplemented with 10 mM maltose) on a 5-ml amylose column MBPTrap TM HP (GE Healthcare Life Sciences, Issaquah, WA, USA). Enzyme activities were measured as described previously using CMC (C4888; Sigma, St. Louis, MO, USA) as the substrate (Ma et al, 2015a).…”

“…The elicitor activity was detected by infiltration of recombinant wild-type and mutant proteins in N. benthamiana; the VdNLP1 and MBP were used as positive and negative controls respectively. Cytosolic Ca 21 was measured using the Ca 21 -sensitive fluorescent dye Fluo-4 acetoxymethyl ester (Fluo 4-AM) as described previously (Ma et al, 2015a). Nicotiana tabacum L. cv.…”

“…The ROS generation in plant leaves was detected using 3'3-diaminobenzidine (DAB) solution as described previously (Bindschedler et al, 2006). Intracellular generation of ROS was performed as described previously (Ma et al, 2015a). Briefly, 1-ml aliquots of BY-2 cells were treated with 100 nM recombinant proteins and incubated for 3 h at 258C and 150 rpm.…”

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae, which confers an endo mode of activity, affects bacterial virulence, but not the induction of immune responses, in rice

“…For example, ethylene‐inducing xylanase from T . reesei and cellulase from Rhizoctonia solani have been shown to be inducers of host defence responses without the requirement of any enzymatic activity (Enkerli et al ., ; Ma et al ., ; Ron and Avni, ).…”

The regulatory mechanism of fungal elicitor-induced secondary metabolite biosynthesis in medical plants

“…It has been reported that an endocellulase (EG1) isolated from Rhizoctonia solani contains a putative protein of 227 amino acids with a signal peptide and a family-45 glycosyl hydrolase domain. Its aspartic acid (Asp) residue at position 32 was changed to alanine (Ala), resulting in full loss of its catalytic activityhence the enzymatic activity of this endoglucanase is not required for its elicitor activity (Ma et al 2015).…”