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

Abstract

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 byColletotrichum acutatum.Despite the impact ofC. acutatumon 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 ofC. acutatumon 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, includingCaGpiP1encoding a glycosylphosphatidyl-inisotol (GPI)-anchored protein,CaNRT2.1encoding a membrane-bound nitrate/nitrite transporter, andCaRQH1encoding a RecQ helicase protein. In addition, T-DNA is inserted upstream of theCaHP1gene encoding a hypothetical protein. Functional characterization ofCaGpiP1,CaNRT2.1, andCaHP1by targeted gene disruption and bioassays indicated thatCaNRT2.1is responsible for the growth-defective phenotype of B7. Both B7 andCaNRT2.1mutant strains cannot utilize nitrate as nitrogen sources, thus restraining the fungal growth on a minimal nutrient medium. In addition toCaNRT2.1, our results showed that CaGpiP1 is a cell wall-associated GPI-anchored protein. However, after investigating the functions ofCaGpiP1andCaHP1in fungal pathogenicity, growth, development and stress tolerance, we were unable to uncover the roles of these two genes inC. acutatum.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 ofC. acutatum.