The resistant plant at the heart of the study, Capsicum annuum var. 61R (Ca61R), descends from a single seed of accession PI 271322. When its leaves are infiltrated with Xanthomonas euvesicatoria Race 6 (XeR6), the strain used throughout the study, the tissue responds with what the authors call non-hypersensitive resistance: the leaf develops a slight swelling and pale green discoloration but remains alive and photosynthetically active, with no necrosis and no leaf abscission. By contrast, the susceptible cultivar Feherozon (CaFo) shows yellowing and water-soaking within four to six days, complete necrosis within six to twelve days, and leaf drop within two weeks. Bacterial spot, caused by X. euvesicatoria, is a major disease of pepper and tomato worldwide, thriving in hot, humid conditions.

To clone the locus, the team crossed resistant Ca61R with susceptible CaFo and confirmed that susceptibility is dominant: all 31 F1 hybrids were susceptible, consistent with the recessive inheritance of resistance. An allelism test with the reference line Early CalWonder 60R, in which resistance is conditioned by a single homozygous recessive locus, produced resistant offspring in all 31 crosses, establishing that the Ca61R determinant is an allele of bs6. The researchers then generated more than 4000 F2 seeds and began mapping with molecular markers distributed across all 12 pepper linkage groups. Three markers on chromosome 6 showed linkage to the resistance phenotype, placing the locus on the proximal end of the chromosome in a region of roughly up to 212 megabases.

Fine mapping sharpened the picture considerably. Using seven new markers in the interval, the team first delimited the locus to a region of less than 1.4 megabases in 92 F2 plants, then genotyped markers flanking that interval in the full population of more than 4000 individuals. Ninety-eight plants carried discordant genotypes between the flanking markers and were analyzed further with newly developed internal markers. Recombination breakpoints in plants P6, P164, P1023 and P22 ultimately confined the locus to an interval between markers m1637 and m1642 spanning approximately 30 kilobases. The corresponding DNA regions from the susceptible and resistant parents, about 29.7 and 30.0 kilobases respectively, were sequenced and deposited in GenBank.

Sequence and transcript analysis of this fine-mapped interval revealed the surprising architecture of the susceptibility locus. Only one subregion was transcriptionally active, and within it the susceptible parent carries two genes that together encode three predicted proteins, named pBs6FoNPK, pBs6FoN and pBs6FoPK. The first is a single 375-amino-acid protein with both an N-terminal and a C-terminal pseudokinase domain; the second gene produces a single polycistronic transcript encoding two separate proteins, one resembling the N-terminal domain and one the pseudokinase domain. Similarity searches showed that pBs6FoNPK shares 35 percent sequence identity and 53 percent similarity with the ZRK4 pseudokinase of Arabidopsis thaliana, and all three proteins carry alterations in the glycine loop, VAIK, HRD, DFG and APE motifs characteristic of the RLCK XII-2 pseudokinase family, suggesting they lack kinase activity, although catalytic activity was not directly tested.

The resistant parent carries the mutated counterparts. In Ca61R, a single-nucleotide insertion causes a frameshift and premature stop codon that truncates pbs661npk, a second insertion similarly truncates pbs661n, and pbs661pk differs from its susceptible counterpart by just one amino acid, a serine-to-alanine substitution at position 133. All three mutant proteins are considered functionless. Because the second gene’s two open reading frames sit on one transcript, the authors tested whether the downstream protein is actually translated: a fusion of the upstream protein to the DsRed fluorescent reporter was expressed in Nicotiana benthamiana leaf cells, providing evidence that the downstream ORF can indeed be translated in vivo.

Functional tests then established the striking redundancy of the system. Using virus-induced gene silencing in the susceptible CaFo background, the researchers found that silencing either gene alone left plants fully susceptible, but simultaneous silencing of both genes, which eliminates all three proteins, converted the plants to resistance, with qPCR confirming that neither transcript remained detectable. The reverse experiment sealed the logic: when the susceptible alleles of Bs6FoNPK, Bs6FoN or Bs6FoPK were transiently expressed under their native promoters in resistant Ca61R leaves, susceptibility returned, with water-soaking, discolored tissue and necrotic lesions appearing within ten days of bacterial inoculation. An unrelated pseudokinase gene and an empty vector had no such effect.

Two additional lines of evidence reinforced the conclusion. The recombinant plant P1023F3, which carries functional versions of only the truncated maternal proteins fused with a paternal pBs6FoPK, was susceptible, indicating that a single functional protein among the three is enough to confer susceptibility. Similarly, the cultivar MicropepRed, whose pbs6MPK protein carries two amino acid substitutions and two deletions predicted to disable it while its other two proteins remain functional, was also susceptible. Taken together, the authors conclude that the three proteins act redundantly and interchangeably to promote effector-triggered susceptibility, and that loss of function of all three is required for the recessive resistance phenotype.

How the proteins promote susceptibility remains an open question. The authors hypothesize that when any of the three functional pseudokinases is present, an effector-triggered susceptibility program prevails over the plant’s immune responses, possibly by interfering with NBS-LRR receptor signaling that would otherwise activate effector-triggered immunity. When all three are disabled, as in Ca61R, that interference is lifted and resistance manifests. Notably, the resistance is non-hypersensitive: unlike classic resistance responses that kill infected cells rapidly, the inoculated leaf tissue stays viable. The authors note that no breakdown of bs5- or bs6-mediated resistance by X. euvesicatoria has been reported to date, and that recessive susceptibility genes like these are candidates for targeted gene editing in breeding durable, environmentally friendly resistance in pepper and related crops such as tomato.

Subject of Research: Map-based cloning of the recessive bs6 bacterial spot resistance locus in pepper, which encodes mutant alleles of three redundant pseudokinase susceptibility proteins.

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

Article References: Toth, Z., Toth, Z. G., Kalo, P., Kiss, G. B., & Szabo, Z. (2026). Map-based cloning of the susceptibility locus conditioning bs6 resistance in pepper (Capsicum annuum L.) encoding mutant alleles of pseudokinase proteins. Theoretical and Applied Genetics, 139(11), Article 308. https://doi.org/10.1007/s00122-026-05407-2

Image Credits: AI Generated

DOI: 10.1007/s00122-026-05407-2

Keywords: pepper, Capsicum annuum, bs6 locus, bacterial spot, Xanthomonas euvesicatoria, map-based cloning, pseudokinase, recessive resistance, susceptibility genes, VIGS, chromosome 6, plant breeding