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

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 β 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 μM (Stover et al., 2008). We constructed a binary vector (pUSHRL) with an expression cassette containing either the β-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.