Huanlongbing (HLB) has caused the loss of thousands of trees in Florida’s multi-billion dollar citrus industry. An effective, long-term strategy to controlling this disease will be by the incorporation of genetic resistance into commercial genotypes. Because conventional breeding is limited by the lack of natural resistance in citrus to HLB, genetic engineering is now considered a significant alternative to incorporating such characteristics. In fact, despite general concerns from the public against genetically modified organisms (GMOs), one National Academy report¹ stated that genetic engineering will be the way to fully exterminate HLB, while growers’ support of a transgenic approach for disease resistant traits also continues to rise. The primary transformation method of citrus typically uses Agrobacterium, in which explants are suspended with the bacterium and subsequently placed on selection media. After treatment, the explants produce shoots that can ultimately lead to stable transgenic plants. Due to the slow growth and lengthy maturation, this process takes several years to produce reproductive trees and must be optimized for each cultivar. Consequently, transformation efficiency is substantially less than other model systems. The commercialization of transgenic disease resistant cultivars is even slower due to regulations limiting GMOs worldwide. In order to decrease the dependence upon bacterial vectors and increase transformation efficiency, we have researched an alternative method for introducing nucleic acids into plants that does not involve Agrobacterium and instead uses cell penetrating peptides (CPPs). CPPs are short, positively charged amino acid sequences that bind to negatively charged molecules and subsequently translocate across cellular membranes. Most surprisingly plant cell walls can also be bypassed, as CPPs are currently used in plants in transient expression and gene silencing assays. Until now, CPPs have not been examined in citrus or other woody crops for stable transformation protocols. We have developed a method for the transient expression of reporter genes (GUS and GFP) using plasmid DNA and CPPs. Our data indicate that up to 50% of treated explants express GUS when CPPs are used alone. Several optimization steps have been tested and the expression efficiency can be increased up to 100% when CPPs are used in conjunction with a lipid transfection reagent. We have also produced hypocotyl segments which survived kanamycin selection. Some produced shoots that rooted and were planted in soil and have been maintained in a growth chamber. PCR and reporter gene analysis will confirm if stable integration has occurred. Our novel protocol could have far reaching effects for the successful integration of disease resistant GMOs in global markets by limiting the perceived negative effect of bacterial vectors.

We have produced a number of 'Carrizo' citrange (Citrus sinensis x Poncirus trifoliata) transformed with the Arabidopsis thaliana NPR1, a transcriptional co-activator that is key in the regulation of systemic acquired resistance (SAR) and the expression of pathogenesis related (PR) genes. Over-expression of this gene has been shown to induce broad spectrum disease resistance in several species. One of the limitations in obtaining genetically resistant citrus plants to HLB is how lengthy it is to propagate and evaluate the transgenic plants. Using grafting with infected budwood takes several months, is labor intensive and normally requires specialized greenhouse space which can be limited. We have developed a system to quickly screen AtNPR1 transgenic lines and determine if they exhibit an enhanced defense response to Candidatus Liberibacter asiaticus PAMPs. First, we used a synthetic peptide of L-flg22 (22 amino acid flagellin epitope derived from CLas) capable of triggering immunity in citrus. Second, using real time PCR, we determined changes in the expression levels of a battery of genes associated with defense in citrus in a time course of up to 72 hours after infiltration with L-flg22 and compared it with the expression in wild type plants. Certain lines consistently showed an enhanced defense response when exposed to L-flg22 thus identifying the ones with the most potential. The advantage of this method as a first step in the screening process is that is quick, controlled and does not require specialized greenhouse space. The selected lines are being further evaluated through graft inoculation for their tolerance to HLB.

‘Duncan’ Grapefruit (Citrus paradisi Macf.) and ‘Sun Chu Sha’ mandarin (C. reticulalta Blanco) represent two citrus genotypes that have different levels of tolerance to citrus greening or huanglongbing (HLB), a bacterial disease caused by Candidatus Liberibacter sp. In this study, the response of the two genotypes to the conserved 22 amino acid domain of the Liberibacter flagellin (Lflg22), a Pathogen-Associated Molecular Pattern (PAMP), were compared. The expression levels of citrus defense-associated genes including AZI1, EDS1, NDR1, SGT1, RAR1, PAL1, ICS1, PAL1, NPR1, NPR2, NPR3, PR1 and RdRp in response to Lflg22 were analyzed. The HLB moderately tolerant Sun Chu Sha showed a stronger response to Lflg22 than the HLB-sensitive grapefruit. These results suggest that differences in the levels of PAMP-triggered Immunity (PTI) between the two genotypes are associated with the observed levels of HLB tolerance. Interestingly, although the Ca. L. asiaticus flagellin gene has been shown to be functional, no flagellum has been observed in this bacterium.