We are still working to obtain stably transformed citrus containing the BS3 promoter with added TAL effector binding elements (4 or 14 EBE) fused to a defense response inducing gene. We have obtained three transformants with a 14 EBE construct driving either the AvrGf1 or AvrGf2 Xanthomonas effector gene in Carrizo citrange, a citrus variety more amenable to transformation. PCR-based analysis of gene expression demonstrated that these constructs were induced as expected upon infection with the virulent strain X. citri strain Xcc306, validating that the promoter works in stably-transformed citrus. Whereas expression of AvrGf1 or 2 genes in orange and grapefruit triggers a hypersensitive defense response, this reaction doesn’t occur in Carrizo and we are not able to assess resistance to X. citri in these plants. In Duncan grapefruit, our efforts to transform constructs with AvrGf1 and AvrGf2 transgenes, where an inducible hypersensitive response is expected, have led to the isolation of seven putative transformants. These were sequenced to determine whether the transgene construct was intact. We found that all seven had deletions in the area of the transgene comprising parts of the promoter region and Avr gene. We believe our difficulty in obtaining transgenic grapefruit is arising either because the construct may have a tendency to recombine in Agrobacterium or during the transformation process, or the promoter may be leaky at some point during the transformation process, even though we have shown that it is tightly regulated in transient assays in leaves. Therefore, we are taking further steps to assess the stability and background expression of the construct. To investigate construct stability, transgenic tobacco plants (Nicotiana tabacum) resulting from the constructs pCAMBIA2201, pCAMBIA2201:NosT:Bs3super::avrGF2, and pCAMBIA2201:NosT:Bs34box::avrGF2 have been generated to determine whether the constructs are stable in another system. If construct sequence optimization is necessary, it will be easier in the tobacco system. We are also carefully assessing the potential for background expression of the construct. Four Carrizo transgenics containing the 14EBE construct fused to GUS were obtained, and these will be verified for the presence of the intact transgene by PCR, followed by GUS staining to determine whether there is any unexpected expression in any tissues other than leaves. In addition, N. tabacum and N. benthamiana transgenic plants carrying the 14 EBE construct fused to GUS will be stained to determine whether GUS is expressed anywhere in the plant. If one of the added EBEs produces unwanted background expression, we will be better able to determine which one is problematic in this system. We will also attempt to transform Duncan grapefruit with a construct containing the Bs3 promoter without any added EBEs, fused to an Avr gene or to GUS, to assess whether the unaltered promoter produces any background expression. Work also continues in the tomato model system, where one transgenic line carrying the disease resistance construct showed a reduction in symptoms in initial tests. T2 plants are being generated for further study.