Bacterial Food-borne Pathogens and Mycology Research Unit, National Center for Agriculture Utilization Research, Peoria, IL 61604, USA.
Mol Plant Pathol. 2011 May;12(4):365-72. doi: 10.1111/j.1364-3703.2010.00677.x. Epub 2010 Nov 25.
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.
在商业玉米中，chiA 基因至少有两个不同的等位基因，它们编码 ChitA 几丁质酶的同种型，这种蛋白质在发育中的种子中含量丰富。已知的两种同种型都被真菌病原体双极镰刀菌分泌的几丁质酶修饰蛋白（cmp）Bz-cmp 修饰。一种同种型（ChitA-B73）也被真菌病原体玉米赤霉分泌的蛋白 Stm-cmp 修饰，而另一种（ChitA-LH82）则具有抗性。这两种 ChitA 同种型具有六个差异或多态性（P1-P6）。为了确定几丁质结合域中的 P2 多态性是否导致对 Stm-cmp 修饰的抗性或敏感性，以及确定 Stm-cmp 和 Bz-cmp 是否为蛋白酶，构建了产生重组玉米 ChitA（rChitA）同种型和突变 rChitA 的酵母毕赤酵母异源表达菌株。从酵母培养物中纯化 rChitA 同种型和突变 rChitA，并将其用作 Stm-cmp 和 Bz-cmp 测定中的底物。与天然蛋白一样，Bz-cmp 修饰了 rChitA-LH82 和 rChitA-B73，而 Stm-cmp 仅修饰 rChitA-B73。突变 rChitA 中，将 P2 氨基酸改变为另一种同种型的氨基酸，导致 Stm-cmp 敏感性显著改变。未修饰和修饰的 rChitA-B73 的氨基末端测序表明，Stm-cmp 在 P2 丙氨酸的氨基末端侧切割肽键，而 Bz-cmp 在多甘氨酸铰链区域，即 P3 位点切割。结果表明，Stm-cmp 和 Bz-cmp 是蛋白酶，它们在氨基末端截断 ChitA 几丁质酶，但在不同的位点。两个位点都对应于两种同种型的多态性，表明 P2 和 P3 处的序列多样性是防止真菌蛋白酶截断的选择压力的结果。
