Submitted to: Molecular Plant Pathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/22/2010
Publication Date: 4/4/2011
Citation: Naumann, T.A. 2011. Modification of recombinant maize ChitA chitinase by fungal chitinase-modifying proteins. Molecular Plant Pathology. 12(4):365-372. Interpretive Summary: In this research, we characterized functional properties of a corn defense protein called ChitA. ChitA is present in developing kernels but is changed into a different form by proteins that are secreted by fungal pathogens during ear rot. ChitA proteins from different types of maize may be either susceptible or resistant to this change. This research discovered that the nature of the fungal-induced change involved cutting the ChitA protein at specific locations through a process called proteolysis. It was further shown that resistance to proteolyisis is largely the result of a single amino acid change in the ChitA protein. This information will be useful to corn breeders who seek to produce maize hybrids that are resistant to fungal disease, which will consequently reduce levels of fungal-produced toxins and produce better yields, making the US corn crop safer and more profitable.
Technical Abstract: 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 protein 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 while the other (ChitA-LH82) is resistant. The two ChitA alloforms have six differences or polymorphisms (P1-P6). To determine if the P2 polymorphism in the chitin binding domain is responsible for resistance or susceptibility to modification by Stm-cmp, and to determine if Stm-cmp and Bz-cmp are proteases, I created heterologous expression strains of the yeast Pichia pastoris that produce recombinant maize ChitA (rChitA) alloforms and mutant rChitAs. 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 while Stm-cmp only modified rChitA-B73. Mutant rChitAs in which the P2 amino acid was changed to that of the other alloform resulted in a significant reversal 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 while Bz-cmp cleaves in the poly-glycine hinge region, 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 with polymorphisms in the two alloforms, suggesting that the sequence diversity at P2 and P3 are the result of selective pressure to prevent truncation by fungal proteases.