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Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi

  • Genetic Transformation and Hybridization
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Abstract

Three antimicrobial peptides exhibiting in vitro antifungal activity were expressed in Arabidopsis to compare their in planta activity. β-Purothionin, cecropin B, and phor21 were expressed under an endogenous promoter with moderate-level activity and excreted extracellularly. Expression of β-purothionin rendered the greatest antibacterial and antifungal resistance while cecropin B enhanced only antibacterial activity and phor21 did not improve antimicrobial resistance. The transgenic β-purothionin arrested fungal growth on leaf surfaces and infection of stomata. Leaf extracts from plants producing β-purothionin and cecropin B displayed membrane permeabilizing activity. The in planta antimicrobial activity of the tested peptides was consistent with previously reported in vitro experiments. The expression strategy allowed enhanced antifungal resistance without high-level transgene expression.

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Abbreviations

35S:

Enhanced CaVM 35S

CA:

A. thaliana chloroplast carbonic anhydrase

cfu:

Colony forming unit

GFP:

Green fluorescent protein

EGFP:

Enhanced green fluorescent protein

GUS:

β-Glucuronidase

GUSP:

β-Glucuronidase Plus

MU:

4-Methylumbelliferon

RSP:

Rice endochitinase signal peptide

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Acknowledgements

We thank Dr. S. Bartlett and Dr. R.Cooper (Louisiana State University) for providing pΔSal and pCP6. We thank Dr. R. Skadsen, USDA and Demegen, Inc. as well for providing pBlue-Thio12. The pathogens F. oxysporum, P. syringae, and R. solani were kindly provided by Dr. B. Cammue (Center of Microbial and Plant Genetics, Heverlee, Belgium), Dr. R. Innes (University of California), and Dr. C. Rush (Louisiana State University), respectively. This work was supported by Louisiana Agricultural Experimental Station, Louisiana State University

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Authors and Affiliations

  1. LSU AgCenter Biotechnology Laboratory, Louisiana State University, 115 Wilson Bldg., Baton Rouge, LA, 70803, USA

    S. V. Oard & F. M. Enright

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  1. S. V. Oard
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  2. F. M. Enright
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Correspondence to S. V. Oard.

Additional information

Communicated by D.A. Somers

Electronic supplementary material

Evaluation of plant fitness

Plant fitness of transformants expressing antimicrobial peptides was evaluated using scoring method. Plant height, leaf size, leaf shape, rosette fullness, and seed production were scored from 0 to 9 where 9 corresponds to the untransformed Col-0 phenotype. Because T0 plants should be selected on an antibiotic containing medium, Col-0 could not be used for their evaluation. To overcome this problem, we produced a positive control line (line PC) by transforming Col-0 with pCAMBIA1305.1 and selecting a transgenic line with high-level GUS activity and the untransformed Col-0 phenotype. All T0 plants were scored by comparing with PC line three and eighth weeks after plating on the selective medium. T1 and T2 plants for each line were grown in soil and compared directly with Col-1. Note that transgenic plants with low scores were found even in control transformations when plants were transformed with pCAMBIA1305.1. However, only healthy plants scoring 8 and 9 for all five criteria were selected for further analysis. Although the lines expressing β-purothionin displayed slower growth on MS media in comparison with PC line, they displayed normal growth in soil. Figure 1 demonstrates the PUR2 phenotype compared to the untransformed Col-0, and Figure 2 displays plants from several selected transgenic lines.

Fig 1s.

T2 homozygous PUR2 line. a. A 5-week-old plant. b. 5-week old plants (left tray) and two-month-old plants (right tray); untransformed Col-0 plants are indicated with labeling tags.

Fig 2s.

T2 homozygous lines expressing antimicrobial peptides. a. 5-week old plants of lines (1) PUR2, (2) CEC6, and (3) PHOR3; (C) untransformed Col-0 plant; (PC) PC plant. b. Two-month-old plants of the PUR2, CEC6, and PHOR3 lines; untransformed Col-0 plants are indicated with labeling tags.

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Oard, S.V., Enright, F.M. Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi. Plant Cell Rep 25, 561–572 (2006). https://doi.org/10.1007/s00299-005-0102-5

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  • DOI: https://doi.org/10.1007/s00299-005-0102-5

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