Map-based cloning of the susceptibility locus conditioning bs6 resistance in pepper (Capsicum annuum L.) encoding mutant alleles of pseudokinase proteins

Key message The bs6 resistance gene against bacterial spot was identified by map-based cloning in pepper (C. annuum L.). Abstract The recessive bs6 61 locus of pepper Capsicum annuum L. var. 61R (Ca61R) conditions a non-hypersensitive resistance trait, characterized by a slightly swollen, pale green, photosynthetically active leaf tissue, following Xanthomonas euvesicatoria Race 6 (XeR6) infection. In contrast, C. annuum cv. Feherozon (CaFo) is susceptible to XeR6. Map-based cloning of the bs6 61 locus revealed that the bs6 61 locus encodes three predicted proteins, pbs6 61 npk, pbs6 61 n, pbs6 61 pk in Ca61R which are the counterpart of the three wild type predicted proteins, pBs6 Fo NPK, pBs6 Fo N and pBs6 Fo PK in CaFo, respectively. Sequence analysis revealed that the pBs6FoNPK protein showed 35% identity and 53% similarity to the ZRK4 pseudokinase (pAtZRK4) identified in Arabidopsis thaliana , which possesses N-terminal and C-terminal (pseudokinase) domains. The pBs6 Fo N and pBs6 Fo PK proteins resemble the N-terminal and C-terminal domains of the pBs6FoNPK protein, respectively, thus, they also contain pseudokinase domains/sequences. In Ca61R, pbs6 61 npk and pbs6 61 n are truncated counterparts of pBs6 Fo NPK and pBs6 Fo N, respectively, as a consequence of a single-nucleotide insertion that causes frameshifts and premature stop codons, whereas pbs6 61 pk differs from pBs6 Fo PK by a single amino acid, a serine to alanine substitution at position 133. These three proteins are considered functionless. The ORF of pBs6 Fo N, pBs6 Fo PK and pbs6 61 n, pbs6 61 pk, respectively, are encoded by a single transcript in both parental plants. For this reason the translation of pBs6 Fo PK was ambiguous, however, the expression of pBs6 Fo N-dsRed fusion protein demonstrated that pBs6 Fo PK in fact can be translated. The three wild type proteins (pBs6 Fo NPK, pBs6 Fo N and pBs6 Fo PK) were independently responsible for ETS (effector-triggered susceptibility), because all three genes had to be in mutant configuration to manifest the resistant phenotype, in addition, any one of the three was able to reverse the resistant phenotype to susceptibility. Functional analysis using virus-induced gene silencing and transient genetic complementation demonstrated that the three CaFo proteins contribute redundantly to XeR6 susceptibility. Silencing either Bs6 Fo NPK, Bs6 Fo N and Bs6 Fo PK alone did not alter susceptibility, whereas simultaneous silencing of these genes conferred resistance. Conversely, the individual expression of pBs6 Fo NPK, pBs6 Fo N, and pBs6 Fo PK, in the resistant Ca61R background each restored susceptibility. These results demonstrate that the three Bs6 proteins act redundantly to promote XeR6 susceptibility and that loss of function of all three corresponding proteins was required for the recessive resistance phenotype conferred by bs6 61 .