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

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

doi:10.3389/fmicb.2020.613674

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 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.

Highlights

Chili peppers, belonging to the genus Capsicum, are one of the most important food additives
Effective Agrobacterium tumefaciensmediated transformation (ATMT) transgenic systems have been successfully established in many plant pathogenic fungi to analyze gene function by insertional mutagenesis (Michielse et al, 2005; Maruthachalam et al, 2008, 2011; Huser et al, 2009)
We investigated genes involved in the growth defect of transformant B7 and identified a large DNA fragment deletion at the T-DNA insertion site

Summary

Introduction

Chili peppers, belonging to the genus Capsicum, are one of the most important food additives. C. acutatum is the dominant pathogen in Taiwan, and infection by this pathogen often results in severe yield losses (Liao et al, 2012a). This pathogen can infect the fruit of chili peppers, bell peppers, and tomatoes, as well as mango leaves (Liao et al, 2012a). This pathogen forms an especially highly branched infection structure (HBPS) in the cuticle layer of chili pepper fruits during the infection (Liao et al, 2012b). Despite the impact of C. acutatum on various hosts, the factors involved in its pathogenicity, HBPS formation and nutrient uptake await to be illustrated

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Results

Conclusion