Truncation of class IV chitinases from Arabidopsis by secreted fungal proteases

Authors: Naumann, Todd; Price, Neil

Submitted to: Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2012
Publication Date: 4/5/2012
Citation: Naumann, T.A., Price, N.P. 2012. Truncation of class IV chitinases from Arabidopsis by secreted fungal proteases. Molecular Plant Pathology. 13:38-42. DOI: 10.111/j.1364-3703.2012.00805.x.

Interpretive Summary: Fungi cause devastating damage to U.S. agriculture through crop loss and by contaminating the food and feed supply with harmful toxins. Plants produce proteins called chitinases that act as a natural defense against fungi. Previously, we demonstrated that fungi that cause corn ear rot produce proteins that inactivate corn chitinase, indicating that the fungi may use this approach to overcome natural plant defenses. In this research, we discovered that these fungi can also cleave similar chitinases from other plants. In addition, we found that a fungal pathogen of crops in the cabbage family (broccoli, cabbage, cauliflower, canola, radish) also makes a protein that inactivates plant chitinases. These results suggest that fungal inactivation of plant chitinases is a general method used by fungi to overcome plant defenses. By showing the general nature of this interaction, we have identified a target that can be exploited by chemists, breeders, and genetic engineers to improve disease resistance of economically important crops.

Technical Abstract: Plant class IV chitinases have a small, amino-terminal chitin binding domain and a larger chitinase domain. Previous work on Zea mays chitinases ChitA and ChitB showed that their chitin binding domains bind insoluble chitin, that their catalytic domains degrade short, soluble forms of chitin, and that the chitin-binding domain is removed by proteases secreted by fungal pathogens. Here we analyze four chitinases from the model dicot Arabidopsis thaliana. We show that some of their chitin-binding domains bind insoluble chitin and that their chitinase domains degrade short, soluble forms of chitin and catalyze transglycosylation. Moreover, the chitin binding domain of some can be removed by Fv-cmp, a fungalysin protease produced by Fusarium verticillioides. Recombinant chitinases were expressed in Pichia pastoris. The basic chitinases AtchitIV3 and AtchitIV4 were purified by chitin affinity. Acidic chitinases AtchitIV1 and AtchitIV5 did not bind insoluble chitin, and were purified by other methods. The recombinant chitinases all degraded GlcNAc oligomers with degree of polymerization (dp) 6. The four chitinases also produced dp5 without production of dp1, indicating transglycosylation activity. Incubation of chitinases with Fv-cmp resulted in truncation of AtchitIV3 and AtchitIV5 while incubation with secreted proteins from Alternaria brassicae, a pathogen of A. thaliana and brassica crops, led to trunction of AtchitIV3 and AtchitIV4. Our finding that both A. thaliana and Z. mays class IV chitinases are truncated by proteases secreted by specialized pathogens of each plant suggests that this is a general mechanism of fungal pathogenicity.