The NPR1 gene plays a pivotal role in systemic acquired resistance in plants. Its overexpression in Arabidopsis and rice results in increased disease resistance and elevated expression of pathogenesis-related (PR) genes. An NPR1 homolog, MpNPR1-1, was cloned from apple (Malus x domestica) and overexpressed in two important apple cultivars, Galaxy and M26. Apple leaf pieces were transformed with the MpNPR1 cDNA under the control of the inducible Pin2 or constitutive Cauliflower mosaic virus (CaMV)35S promoter using Agrobacterium tumefaciens. Overexpression of MpNPR1 mRNA was shown by reverse transcriptase-polymerase chain reaction. Activation of some PR genes (PR2, PR5, and PR8) was observed. Resistance to fire blight was evaluated in a growth chamber by inoculation of the shoot tips of our own rooted 30-cm-tall plants with virulent strain Ea273 of Erwinia amylovora. Transformed Galaxy lines overexpressing MpNPR1 had 32 to 40% of shoot length infected, compared with 80% in control Galaxy plants. Transformed M26 lines overexpressing MpNPR1 under the control of the CaMV35S promoter also showed a significant reduction of disease compared with control M26 plants. Some MpNPR-overexpressing Galaxy lines also exhibited increased resistance to two important fungal pathogens of apple, Venturia inaequalis and Gymnosporangium juniperi-virginianae. Selected transformed lines have been propagated for field trials for disease resistance and fruit quality.

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The Vf locus, originating from the crabapple species Malus floribunda 821, confers resistance to five races of the fungal pathogen Venturia inaequalis, the causal agent of apple scab disease. Previously, a cluster of four receptor-like genes, Vfa1, Vfa2, Vfa3, and Vfa4, was identified within the Vf locus. Because the amino-acid sequence of Vfa3 is truncated, it was deemed nonfunctional. In this study, each of the three full-length Vfa genes was introduced into a plant cloning vector, pCAMBIA2301, and used for Agrobacterium-mediated transformation of two apple cultivars, Galaxy and McIntosh, to assess functionality of these genes and to characterize their roles in resistance to V. inaequalis. Transformed apple lines carrying each of Vfa1, Vfa2, or Vfa4 were developed, analyzed for the presence of the transgene using polymerase chain reaction and Southern blotting, and assayed for resistance to apple scab following inoculation with V. inaequalis. Transformed lines expressing Vfa4 were found to be susceptible to apple scab, whereas those expressing either Vfa1 or Vfa2 exhibited partial resistance to apple scab. Based on Western blot analysis as well as microscopic analysis of plant resistance reactions, the roles of Vfa1 and Vfa2 in apple scab disease resistance response are discussed.

Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.