Mapping, cloning, and functional characterization of CsPBGD in leaf necrosis and its potential role in disease resistance in cucumber (Cucumis sativus L.).

Abstract

Key message: Map-based cloning revealed that the mutation in a highly conserved amino acid of the CsPBGD, which encodes porphobilinogen deaminase, causes the phenotype of leaf necrosis and enhanced resistance to powdery mildew and gray mold in cucumber. Lesion mimic mutants (LMMs) are valuable genetic resources for studying programmed cell death (PCD) and disease resistance. Although a number of genes controlling lesion mimic have been identified in model species, none have been mapped or cloned in cucumber. Here, we identified two cucumber mutants, C1173 and C2123, which exhibit leaf necrosis due to PCD. Genetic analysis revealed that these phenotypes are controlled by two semi-dominant loci, ln1 and ln2, respectively. Both mutants were heterozygous, as homozygous dominants were lethal (one caused cotyledon etiolation lethality; the other was unobtainable). Fine mapping placed the ln1 locus within a 54.1 kb region on chromosome 3. Further investigation revealed ln1 and ln2 were allelic mutations, with CsPBGD (CsaV3_3G031800), encoding porphobilinogen deaminase, identified as the candidate gene for both mutants. Mutations in CsPBGD resulted in amino acid substitutions, Ala314Val in ln1 and Arg197Lys in ln2, disrupting enzyme activity and altering H₂O₂ accumulation. CsPBGD expression was significantly reduced in various organs of ln1. VIGS of CsPBGD in both cucumber and tobacco successfully displayed the leaf necrosis phenotype. CsPBGD proteins from both mutants and wild type (WT) were localized in chloroplasts. The mutants exhibited significantly enhanced resistance to powdery mildew (Podosphaera xanthii) and gray mold disease (Botrytis cinerea). Further studies showed that CsPBGD expression in the mutant was significantly more downregulated than in WT after P. xanthii infection, alongside increased H₂O₂ accumulation. This study is the first to characterize and clone CsPBGD in cucumber, revealing its involvement in resistance to disease.