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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  Colletotrichum acutatum.  Despite the impact of  C. acutatum  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  C. acutatum  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  CaGpiP1  encoding a glycosylphosphatidyl-inisotol (GPI)-anchored protein,  CaNRT2.1  encoding a membrane-bound nitrate/nitrite transporter, and  CaRQH1  encoding a RecQ helicase protein. In addition, T-DNA is inserted upstream of the  CaHP1  gene encoding a hypothetical protein. Functional characterization of  CaGpiP1 ,  CaNRT2.1 , and  CaHP1  by targeted gene disruption and bioassays indicated that  CaNRT2.1  is responsible for the growth-defective phenotype of B7. Both B7 and  CaNRT2.1  mutant strains cannot utilize nitrate as nitrogen sources, thus restraining the fungal growth on a minimal nutrient medium. In addition to  CaNRT2.1 , our results showed that CaGpiP1 is a cell wall-associated GPI-anchored protein. However, after investigating the functions of  CaGpiP1  and  CaHP1  in fungal pathogenicity, growth, development and stress tolerance, we were unable to uncover the roles of these two genes in  C. 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 of  C. acutatum .

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