Overexpression and evaluation of an antimicrobial peptide from Heuchera sanguinea (Hs-AFP1) for inhibition of fungal pathogens in transgenic tabacco

[摘要] ENGLISH ABSTRACT:Seed germination is the most vulnerable time in a plant's life cycle, since the thick protectiveseed coat ruptures and the moist and humid soil environment not only favours seedgermination, but also the growth and development of plant pathogens. Infection of plant seedsduring germination, however, is the exception rather than the rule. Plant seeds have- - -developed a--cemplex preformed defense mechanism that includes anttfungal agents thatdiffuseinto the surrounding environment to form a protective layer around the seed. Thisprotective layer prevents fungal and bacterial pathogens from infecting the young seedling.Over the last decade, scientists have studied the defense mechanisms of differentseeds in an effort to understand and ultimately to introduce and/or manipulate thesemechanisms in plants as part of the plant's endogenous disease resistance to pathogens.Various chemical compounds, peptides and proteins that showed strong in vitro activitiesagainst various fungi were isolated in these efforts. The mere demonstration of in vitro activityalone, however, is not sufficient to assign a defense role to these antifungal agents. Typically,mutant plants that have lost the ability to produce the antifungal agent, or mutants that areoverproducing the agent, have been used to correlate the mutant phenotype to either adecline or increase in disease resistance respectively. Genetic transformation and thesubsequent development of transgenic plants have made an unprecedented impact in thisregard, specifically in understanding the role of specific defense-related proteins and theirinteraction with plant pathogens.In this study, the antifungal peptide, Hs-AFP1, from Heuchera sanguinea, a plantdefensin, was evaluated in a heterologous in planta environment as a defense protein withpotential for engineering disease resistant crops. The in vitro assays performed with Hs-AFP1against Botrytis cinerea showed antifungal activities of 88% growth inhibition at aconcentration of 8 J,lg/ml of the purified peptide, while inducing a characteristichyperbranching effect on the Botrytis hyphae. Tobacco was subsequently transformed with aconstruct, pFAJ3068, expressing Hs-AFP1 under the strong constitutive 35S promoter. Thepeptide was targeted to the apoplastic region with the signal peptide from Mj-AMP2, anantimicrobial peptide from Mirabilis jalapa. Due to reports of peptide instability in transgenicplant systems, two additional constructs were prepared and transformed into tobacco toanticipate possible Hs-AFP1 instability in the heterologous tobacco environment. A putativepeptide stabilization construct, pHs-EXG1, consisted of a fusion between Hs-AFP1 and theantifungal exo-glucanase (encoded by EXG1) from Saccharomyces cerevisiae. A controlconstruct, pMj-EXG1, expressing EXG1 targeted to the apoplastic region with the Mj-AMP2signal peptide, was also prepared and transformed into tobacco to normalize the backgroundantifungal activity as a result of the exoglucanase in the fusion construct lines.Tobacco was successfully transformed with pFAJ3068, pHs-EXG1 and pMj-EXG1,resulting in transgenic tobacco lines designated THs, THE and TME respectively. Transgeneexpression was confirmed for the THs and THE transgenic lines. The translation of thesetranscripts into proteins was also confirmed with Western blot analysis. Moreover, theheterologous production of Hs-AFP1 in tobacco led to an increase in disease resistance toB. cinerea in the THs lines in comparison with the untransformed tobacco controls. Anincrease of up to 42% in disease resistance was observed in an in planta detached leafassay. Crude protein extracts from the THs lines were also analyzed in an in vitro quantitativefungal growth assay. This assay confirmed the results obtained with the disease resistanceassay, with crude protein extracts exhibiting up to 40% fungal growth inhibition. Theincubation of B. cinerea in the presence of crude protein extracts from THs lines resulted inhyperbranching of the fungal hyphae, which is characteristic of Hs-AFP1 activity.From these analyses it was clear that the heterologously expressed Hs-AFP1 wasquite stable in the transgenic environment. The fusion between Hs-AFP1 and EXG1 did notincrease the stability of Hs-AFP1, but rather led to a loss of the Hs-AFP1 activity. All theanalyses performed showed the THE lines to be reduced in their ability to inhibit fungalinfection in comparison to the THs line. Also, microscopic analysis of the effects of the crudeTHE extracts on B. cinerea growth showed no hyperbranching activity, again confirming theloss of peptide activity due to the fusion to EXG1. This is in agreement with previous work, inwhich sarcotoxin 1A was fused to a reporter gene and also lost activity.Although integration of the Mj-EXG1 expression cassette was confirmed, no mRNAlevels could be detected with Northern blot or RT-PCR analysis of the TME lines. These linesalso did not show any in vitro antifungal activities, probably indicating post-transcriptionalgene silencing. This silencing was overcome in the fusion constructs that were expressed inthe THE plant lines. These lines also showed EXG1 protein activity, as measured by~-glucosidase assays. Although the THE lines did not serve the functions originallyenvisaged, they fortuitously showed that a fusion strategy might stabilize glucanaseexpression in a transgenic environment. A variety of glucanases have been shown to beprone to gene silencing when overexpressed in a plant environment and the yeast glucanasecan now be added to that list if it is not present as a fusion protein.Overall, this study confirmed that Hs-AFP1 is involved in plant defense systems andprovided valuable information on the stability of small peptides in a heterologous environment.The positive results obtained with overexpressed Hs-AFP1 on fungal inhibition in this studymerits further investigations into the use of this peptide in the engineering of disease-resistantcrops.

[发布机构] Stellenbosch University