Abstract
Host plant protease inhibitors offer resistance to proteases from invading pathogens. Trypsin inhibitors (TIs), in particular, serve as protective agents against insect and pathogen attacks. In this study, we designed a pair of degenerate primers based on highly conserved motifs at the N- and C-termini of the TI from tartary buckwheat (Fagopyrum tataricum; Ft) to clone the central portion. Genomic walking was performed to isolate the 5′ and 3′ flanking regions of FtTI. We demonstrated the successful PCR amplification of a 644 bp portion of FtTI. The full-length DNA of FtTI contains a complete open reading frame of 264 bp, encoding 87 amino acids with a mass of approximately 9.5 kDa. The FtTI protein sequence was 49% identical and 67% similar to potato protease inhibitors. Site-directed mutagenesis identified the residues, Asp67 and Arg68, as crucial for the inhibitory activity of the FtTI. Recombinant and mutant FtTI inhibited both the hyphal growth and spore germination of Alternaria solani. The calculated 50% inhibitory concentrations of FtTI ranged from 5–100 μg mL−1 for spore germination and 1–50 μg mL−1 for fungal growth. Thus, recombinant FtTI may function in host resistance against a variety of fungal plant pathogens.
Highlights
Fungal pathogens have a major influence on agricultural crop yields and are a very serious threat to global food security
To reveal the molecular mechanism associated with the inhibitory effect of FtTI toward animal proteases and proteases present in plant-pathogenic microorganisms, the nucleotide sequences of 86 protein residues and three mutants of FtTI were artificially synthesized by PCR amplification and independently expressed by fusion in E. coli DE3
Degenerate PCR primers (Table 1) were designed to amplify an internal fragment of FtTI based on the conserved regions of several protein sequences of known protease inhibitors obtained from the NCBI Protein Sequence Database
Summary
Fungal pathogens have a major influence on agricultural crop yields and are a very serious threat to global food security. The most used and effective control method is the application of synthetic chemical fungicides They are generally used in excess, causing environmental contamination and human health problems, and can even lead to the emergence of pathogen-resistant strains [2]. In many cases, degenerate primers designed using multiple sequence alignments are unsuccessful, owing to the poor conservation among the amino acid sequences being studied. The CODEHOP method has been applied to characterize novel genes and distinguish unknown plant protease inhibitors It has been useful for remotely relevant targets and with a finite template in complicated mixtures. To reveal the molecular mechanism associated with the inhibitory effect of FtTI toward animal proteases and proteases present in plant-pathogenic microorganisms, the nucleotide sequences of 86 protein residues and three mutants of FtTI were artificially synthesized by PCR amplification and independently expressed by fusion in E. coli DE3. The recombinant protein was purified by one-step affinity chromatography and its activity against plant-pathogenic fungi was evaluated
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