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IRCHLB Proceedings Jan 2011: www.plantmanagementnetwork.org

11.4 Promoter Regulation of the -Glucuronidase (GUS) Gene and Antimicrobial Peptide
D4E1 in a Citrus Rootstock

Benyon, L.S., Stover, E., Bowman, K.D., McCollum, G., Niedz, R. U.S. Horticultural Research
Laboratory, USDA-ARS, Fort Pierce, FL, USA

Because agrochemicals and conventional breeding for disease resistance have not been sufficient
in controlling huanglongbing (HLB), alternative strategies for sustaining citrus crops have
attracted attention. Since no HLB resistance has been identified within cultivated citrus,
transgenic solutions to the disease have become a focus of breeding programs. Among these
solutions, use of antimicrobial peptides (AMPs) to enhance plant resistance is at the forefront.
Virtually all organisms possess an innate immune response involving AMPs that can counter a
microbial infection. At the USHRL, we have chosen to use synthetic or plant AMPs over those
of animal origin. The first of the AMPs selected was D4E1, a 17 amino acid synthetic AMP
which forms a sheet (Lucca et al., 1998) and is active against Agrobacterium tumefaciens in
poplar (Mentag et al., 2003). It is a highly active AMP with a minimum inhibitory concentration
(MIC) less than 1 M (Stover et al., 2008). We constructed a binary vector (pUSHRL) with an
expression cassette containing either the -glucuronidase (GUS) reporter gene or the D4E1 AMP
under the control of several promoters in the trifoliate rootstock US-802. Because Candidatus
Liberibacter, the gram-negative bacterium associated with HLB, infects only the phloem tissue
of the plants, it may be desirable to express potential transgenes specifically in the phloem or
companion cells. Thus, we opted to provide seven different promoters for our two genes. The
promoters included: 2x35S from the cauliflower mosaic virus, WDV from wheat dwarf
geminivirus, PR-1 the pathogenesis-related protein gene 1 promoter from tobacco, AtSS the
sucrose-H+ symporter gene promoter from Arabidopsis, SSyn the sucrose synthase promoter
from citrus, PP2 the phloem protein 2-like gene promoter from citrus, and 409S a truncated
polyubiquitin promoter from potato. The promoter activities include constitutive (2x35S; 409S),
inducible (PR-1), phloem-specific (AtSS, SSyn, and WDV), and Affymetrix citrus microchip
HLB up-regulated (PP2). Seedlings are being screened for transformation efficiency, localization
of expression, and level of transgene expression.

References

Lucca, A.J., de Bland, J.M., Grimm, C., Jacks, T.J., Cary, J.W., Jaynes, J.M., Cleveland, T.E.,
Walsh, T.J. 1998. Fungicidal properties, sterol binding, and proteolytic resistance of the
synthetic peptide D4E1. Canadian Journal of Microbiology 44:514-520.
Mentag, R., Luckevich, M., Morency, M.J., Seguin, A. 2003. Bacterial disease resistance of
transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1. Tree
Physiology 23:405-411.
Stover, E., Bowman, K., McCollum, G., Niedz, R. 2008. Developing transgenic solutions for
HLB resistant citrus at the US Horticultural Research Laboratory. Proceedings of the
International Research Conference on Huanglongbing, p. 374.