ATMT mutagenesis of Colletotrichum acutatum identified a nitrite utilization mutant caused by the deletion of a 19-kb DNA fragment

Chia-Chi Kuo 1 Yung-Chu Lin 1 Jia-Fang Ho 1 Ming-Che Shih 2 Miin-Huey Lee 1
1Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan
2Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

Chili pepper is one of the most important food additives used in spicy cuisines worldwide. However, the yield and quality can be highly destructed by anthracnose disease caused by Colletotrichum species. In this study, we identified a nitrate utilization mutant through T-DNA insertional mutagenesis. T-DNA inserted in the upstream of two genes (Ca-02433-HP and Ca-02434-GPI-HP) with no known function. Ca-02434-GPI-HP is a glycosylphosphatidyl-inisotol-anchored protein and could be delivered to cell membrane or cell wall; therefore, it may have function involved in nutrient uptake. Analysis with GFP-fusion and cell-wall-degrading enzyme digestions reveals that Ca-02434-GPI-HP is a cell-wall associated protein. Functional analysis of the two genes with gene disrupted mutants,indicating that they are not involved in nitrate utilization defect of B7, neither involved in the function of pathogenicity, growth, development and tolerance to various environmental stresses. After whole genome sequencing and PCR analysis, T-DNA insertion was found to cause large DNA fragment (19-kb) deletion in B7. Three genes are deleted including Ca-02435-NPT which encodes a Nitrite/Nitrite transporter. Gene disruption and complementation analysis demonstrated that Ca-02435-NTP is the factor causing nitrate utilization defect of B7.