Members of the NPF3 Transporter Subfamily Encode Pathogen-Inducible Nitrate/Nitrite Transporters in Grapevine and Arabidopsis

Abstract

Vitis vinifera, the major grapevine species cultivated for wine production, is very susceptible to Erysiphe necator, the causal agent of powdery mildew (PM). This obligate biotrophic fungal pathogen attacks both leaf and berry, greatly affecting yield and quality. To investigate possible mechanisms of nutrient acquisition by successful biotrophs, we characterized a candidate NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY (NPF, formerly NRT1/PTR) member, grapevine NFP3.2, that was up-regulated in E. necator-inoculated susceptible V. vinifera Cabernet Sauvignon leaves, but not in resistant V. aestivalis Norton. Expression in Xenopus laevis oocytes and two-electrode voltage clamp measurements showed that VvNPF3.2 is a low-affinity transporter for both nitrate and nitrite and displays characteristics of NPF members from other plants. We also cloned the Arabidopsis ortholog, AtNPF3.1, and showed that AtNPF3.1 similarly transported nitrate and nitrite with low affinity. With an Arabidopsis triple mutant that is susceptible to E. necator, we found that AtNPF3.1 is up-regulated in the leaves of infected Arabidopsis similarly to VvNPF3.2 in susceptible grapevine leaves. Expression of the reporter β-glucuronidase (GUS) driven by the promoter of VvNPF3.2 or AtNPF3.1 in Arabidopsis indicated that both transporters are expressed in vascular tissue, with expression in major and minor veins, respectively. Interestingly, the promoter of VvNPF3.2 allowed induced expression of GUS in minor veins in PM-infected leaves. Our experiments lay the groundwork for investigating the manipulation of host nutrient distribution by biotrophic pathogens and characterizing physiological variables in the pathogenesis of this difficult to study grapevine disease.