Modification of recombinant maize ChitA chitinase by fungal chitinase-modifying proteins

In commercial maize, there are at least two different alleles of the chiA gene that encode alloforms of ChitA chitinase, a protein that is abundant in developing seed. Both known alloforms are modified by Bz-cmp, a chitinase-modifying protein (cmp) secreted by the fungal pathogen Bipolaris zeicola. One alloform (ChitA-B73) is also modified by Stm-cmp, a protein secreted by the fungal pathogen Stenocarpella maydis, whereas the other (ChitA-LH82) is resistant. The two ChitA alloforms possess six differences or polymorphisms (P1-P6). To determine whether the P2 polymorphism in the chitin-binding domain is responsible for resistance or susceptibility to modification by Stm-cmp, and to determine whether Stm-cmp and Bz-cmp are proteases, heterologous expression strains of the yeast Pichia pastoris that produce recombinant maize ChitA (rChitA) alloforms and mutant rChitAs were created. rChitA alloforms and mutant rChitAs were purified from yeast cultures and used as substrates in assays with Stm-cmp and Bz-cmp. As with native protein, Bz-cmp modified both rChitA-LH82 and rChitA-B73, whereas Stm-cmp modified rChitA-B73 only. Mutant rChitAs, in which the P2 amino acids were changed to those of the other alloform, resulted in a significant exchange in Stm-cmp susceptibility. Amino-terminal sequencing of unmodified and modified rChitA-B73 demonstrated that Stm-cmp cleaves the peptide bond on the amino-terminal side of the P2 alanine, whereas Bz-cmp cleaves in the poly-glycine hinge region, the site of P3. The results demonstrate that Stm-cmp and Bz-cmp are proteases that truncate ChitA chitinase at the amino terminus, but at different sites. Both sites correspond to polymorphisms in the two alloforms, suggesting that the sequence diversity at P2 and P3 is the result of selective pressure to prevent truncation by fungal proteases.