AUTHOR=Kuo Chia-Chi , Lin Yung-Chu , Chen Li-Hung , Lin Meng-Yi , Shih Ming-Che , Lee Miin-Huey 

TITLE=CaNRT2.1 Is Required for Nitrate but Not Nitrite Uptake in Chili Pepper Pathogen Colletotrichum acutatum

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.613674

DOI=10.3389/fmicb.2020.613674

ISSN=1664-302X

ABSTRACT=<p>Chili peppers are an important food additive used in spicy cuisines worldwide. However, the yield and quality of chilis are threatened by anthracnose disease caused by <italic>Colletotrichum acutatum.</italic> Despite the impact of <italic>C. acutatum</italic> on chili production, the genes involved in fungal development and pathogenicity in this species have not been well characterized. In this study, through T-DNA insertional mutagenesis, we identified a mutant strain termed B7, which is defective for the growth of <italic>C. acutatum</italic> on a minimal nutrient medium. Our bioinformatics analysis revealed that a large fragment DNA (19.8 kb) is deleted from the B7 genome, thus resulting in the deletion of three genes, including <italic>CaGpiP1</italic> encoding a glycosylphosphatidyl-inisotol (GPI)-anchored protein, <italic>CaNRT2.1</italic> encoding a membrane-bound nitrate/nitrite transporter, and <italic>CaRQH1</italic> encoding a RecQ helicase protein. In addition, T-DNA is inserted upstream of the <italic>CaHP1</italic> gene encoding a hypothetical protein. Functional characterization of <italic>CaGpiP1</italic>, <italic>CaNRT2.1</italic>, and <italic>CaHP1</italic> by targeted gene disruption and bioassays indicated that <italic>CaNRT2.1</italic> is responsible for the growth-defective phenotype of B7. Both B7 and <italic>CaNRT2.1</italic> mutant strains cannot utilize nitrate as nitrogen sources, thus restraining the fungal growth on a minimal nutrient medium. In addition to <italic>CaNRT2.1</italic>, our results showed that CaGpiP1 is a cell wall-associated GPI-anchored protein. However, after investigating the functions of <italic>CaGpiP1</italic> and <italic>CaHP1</italic> in fungal pathogenicity, growth, development and stress tolerance, we were unable to uncover the roles of these two genes in <italic>C. acutatum.</italic> Collectively, in this study, our results identify the growth-defective strain B7 via T-DNA insertion and reveal the critical role of CaNRT2.1 in nitrate transportation for the fungal growth of <italic>C. acutatum</italic>.</p>