The Arabidopsis genome contains a family of NDR1/HIN1 - like ( NHL ) genes that show homology to the nonrace-specific disease resistance ( NDR1 ) and the tobacco ( Nicotiana tabacum ) harpin-induced ( HIN1 ) genes. NHL3 is a pathogen-responsive member of this NHL gene family that is potentially involved in defense. In independent transgenic NHL3 -overexpressing plant lines, a clear correlation between increased resistance to virulent Pseudomonas syringae pv. tomato DC3000 and enhanced NHL3 transcript levels was seen. These transgenic plants did not show enhanced pathogenesis-related gene expression or reactive oxygen species accumulation. Biochemical and localization experiments were performed to assist elucidation of how NHL3 may confer enhanced disease resistance. Gene constructs expressing amino-terminal c-myc-tagged or carboxyl-terminal hemagglutinin epitope (HA)-tagged NHL3 demonstrated membrane localization in transiently transformed tobacco leaves. Stable Arabidopsis transformants containing the NHL3-HA construct corroborated the findings observed in tobacco. The detected immunoreactive proteins were 10 kD larger than the calculated size and could be partially accounted for by the glycosylation state. However, the expected size was not attained with deglycosylation, suggesting possibly additional posttranslational modification. Detergent treatment, but not chemicals used to strip membrane-associated proteins, could displace the immunoreactive signal from microsomal fractions, showing that NHL3 is tightly membrane associated. Furthermore, immunofluorescence and immunogold labeling, coupled with two-phase partitioning techniques, revealed plasma membrane localization of NHL3-HA. This subcellular localization of NHL3 positions it at an initial contact site to pathogens and may be important in facilitating interception of pathogen-derived signals.
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